Streptomyces murinus and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to streptomyces murinus and application thereof. The bacterial strain is Streptomyces murinus FLC2-09, and the bacterial strain is the Streptomyces murinus FLC2-09. The strain has a relatively high prevention effect on panax notoginseng root rot and can promote the growth of panax notoginseng. Therefore, the streptomyces murinus FLC2-09 provided by the invention can promote the biological prevention and control development of the panax notoginseng root rot, provides an applicable actinomycete strain resource, and has a wide application prospect in the aspect of panax notoginseng planting.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a Streptomyces murinus and its application. Background Art

[0002] Panax notoginseng Panax notoginseng (Burk) F.H.Chen is a traditional Chinese herbal medicine with a long history and high medicinal and economic value. The organic planting mode of Panax notoginseng under forest reduces the harm of pests and diseases, but the root rot of Panax notoginseng still occurs, and in some planting areas, the disease incidence is even very serious. The root rot of Panax notoginseng is the most serious disease during the growth process of Panax notoginseng, with an annual incidence rate of 5% - 20%, and up to 70% in severe cases. After the plant is infected, the underground part rots, and the above-ground part withers yellow or withers green. The cause of the root rot of Panax notoginseng is complex, and environmental conditions, autotoxicity of plants, pathogens, rhizosphere microecology, cultivation management measures, etc. may all cause the occurrence of the root rot of Panax notoginseng. The types of pathogens of the root rot of Panax notoginseng are diverse, including fungi, bacteria, and nematodes, with fungi being the main ones.

[0003] The cause of the root rot of Panax notoginseng is complex, and it is not easy to detect when the underground part is diseased, while it is often irreparable when the above-ground part is diseased. At present, the prevention and control of the root rot of Panax notoginseng mainly include physical control, chemical control, agricultural control, and biological control.

[0004] The most common method of physical control is to expose the soil to sunlight to reduce the number of pathogens in the soil, thereby reducing the occurrence of root rot. Panax notoginseng seeds can also be soaked in hot water to kill the surface pathogens. In addition, acidic soil is conducive to the growth of pathogens, and adjusting the soil pH can also reduce the incidence of the root rot of Panax notoginseng. In addition, using microwave rays, gamma rays, etc. can reduce the number of pathogenic microorganisms in the soil and reduce the saponin content in the soil without changing the physical and chemical properties of the soil, but these measures are difficult to operate, costly, and not suitable for the popularization and application of agricultural production.

[0005] In terms of chemical control, 300 - 500 times liquid of 20% thiophanate-methyl wettable powder, 300 times liquid of metalaxyl-manganese zinc, 500 times liquid of dexon can be used for root irrigation before or at the initial stage of the disease. 70% thiophanate-methyl, root health, 25% carbendazim, and 50% procymidone can also be used. In addition, using fumigants for treatment is also a common means for preventing and controlling the root rot of Panax notoginseng. Chloropicrin fumigation reduces the number of harmful microorganisms in the soil and can reduce the occurrence of the root rot of Panax notoginseng. Allyl isothiocyanate (AITC) can control the root rot of Panax notoginseng caused by F. solani. However, the long-term use of pesticides will cause pathogens to develop drug resistance, and at the same time cause pesticide residues and environmental pollution, affecting the quality of Panax notoginseng and human health, and also leading to changes in the soil microbial community structure, which is not conducive to the sustainable development of agriculture.

[0006] The main measures of agricultural control include crop rotation, strengthening field management, and breeding excellent varieties, etc. Tang et al. confirmed that after ten years of vegetable rotation in the soil of continuous cropping of Panax notoginseng, the abundance of pathogenic fungi such as Cylindrocarpon in the genus Cylindrocarpon in the soil decreased, and the abundance of beneficial bacteria such as Bacillus in the genus Bacillus increased, thus reducing the soil pathogenicity. However, the control measures of crop rotation are not easy to implement, with high economic costs, long rotation years, long time-consuming, and are easily affected by climate and terrain, etc.; even after 10 - 20 years of rotation, the pathogenic bacteria in the soil cannot be completely eliminated. Another study found that alternating micro-sprinkler irrigation and organic fertilizer can promote root growth by improving the soil environment and soil microorganisms, thereby reducing the occurrence of root rot of Panax notoginseng. Appropriate soil moisture management is an effective way to prevent the occurrence of root rot. In addition, the quality of seedlings is closely related to the occurrence of root rot. Sowing high-quality disease-free seedlings can greatly inhibit the occurrence of root rot. Strictly controlling the quality of seedlings in production can reduce the yield loss caused by root rot.

[0007] The method of biological control has no pollution to the environment and is safe for humans and livestock, and is increasingly valued by people. At present, a variety of microorganisms with control effects on root rot of Panax notoginseng have been discovered, including fungi, bacteria, and actinomycetes. Trichoderma 1126 and T5-1 can reduce the incidence of root rot of Panax notoginseng and at the same time promote the growth of Panax notoginseng. Bacillus velezensis B19 isolated from the soil of Panax notoginseng planting can inhibit the pathogenic fungi of root rot of Panax notoginseng. After treating the continuous cropping soil with this strain, the incidence of root rot of Panax notoginseng decreased significantly. The acarbose streptomycin isolated from the fermentation broth of Amycolatopsis Amycolatopsis can inhibit a variety of pathogenic bacteria of root rot of Panax notoginseng. Actinomycetes Actinomycetes are a class of Gram-positive prokaryotes widely existing in nature, with a large number and a wide variety, and have great potential for development and application. The active substances isolated from the metabolites of actinomycetes have played an important role in various fields such as industry, medicine, food, and agriculture, promoting the development of human economy and health. In agricultural production, actinomycetes can control a variety of agricultural pests. There is also research on actinomycetes to control root rot of Panax notoginseng. 8 strains of actinomycetes obtained from Panax notoginseng plants have Fusarium solani good antibacterial activity. Streptomyces assiniensis Streptomyces yatensis in actinomycetes can produce cyclic peptide secondary metabolites that antagonize root rot of Panax notoginseng. In addition, the roles of actinomycetes in controlling agricultural pests, removing weeds, degrading pesticides, and promoting plant growth have all been reported. In summary, actinomycetes are very potential biological control resources in the field of plant protection. Using biological control methods avoids the adverse effects brought by chemical pesticides, and is durable, safe, and effective, meeting the concept of sustainable development and being an important direction for the future development of green agriculture.

[0008] At present, the functions of the obtained microbial strains are single, there are few highly efficient strains, and many strains only have indoor plate control efficacy data and lack application data. These strains may lose their functions after entering the environment, hindering the practical application of microbial strain control. In the research and application of biocontrol strains for Panax notoginseng, there are even fewer research reports on actinomycetes, and there are no commercialized strains. Summary of the Invention

[0009] To solve the above technical problems, the present invention provides a strain of Streptomyces murinus Streptomycesmurinus which is Streptomyces murinus FLC2-09, and its preservation number is 30938.

[0010] This strain was isolated from the rhizosphere soil of Panax notoginseng under the forest. This strain was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on June 14, 2024. The address of the depositary institution: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, Taxonomic name: Streptomyces murinus Streptomycesmurinus and the deposit number is 30938.

[0011] This strain has a high control efficacy against root rot of Panax notoginseng and can promote the growth of Panax notoginseng.

[0012] The 16S rDNA sequence of the Streptomyces murinus FLC2-09 is shown in SEQ ID No.1.

[0013] Furthermore, the present invention provides a microbial inoculum containing the Streptomyces murinus FLC2-09 described above.

[0014] Preferably, the microbial inoculum is a fermentation broth or a freeze-dried powder of the fermentation broth containing the Streptomyces murinus FLC2-09.

[0015] Preferably, the freeze-dried powder of the fermentation broth further includes a freeze-drying protectant.

[0016] Furthermore, the present invention provides a product containing the Streptomyces murinus FLC2-09 described above; the product is an antibacterial agent, a plant growth promoter, a fertilizer or a fertilizer additive.

[0017] Furthermore, the present invention provides the application of the Streptomyces murinus FLC2-09, the microbial inoculum or the product in inhibiting Fusarium oxysporum.

[0018] Furthermore, the present invention provides the application of the Streptomyces murinus FLC2-09, the microbial inoculum or the product in preventing and controlling root rot of Panax notoginseng.

[0019] Furthermore, the present invention provides the application of the Streptomyces murinus FLC2-09, the microbial inoculum or the product in promoting the growth of Panax notoginseng plants.

[0020] Preferably, in the application, the growth of Panax notoginseng plants is promoted by improving at least one of the plant height, main root length, number of lateral roots, and fresh weight of the plants.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: Streptomyces murinus FLC2-09 provided by the present invention can preferably inhibit the mycelial growth of Fusarium oxysporum in the plate confrontation experiment. Fusarium oxysporummy The inhibition effect reaches 74.19%. In the in vitro root control efficacy test, the control effect on root rot of Panax notoginseng reaches 84.62%. In the pot control efficacy test, the inhibition effect on root rot of Panax notoginseng is 63.90%. At the same time, Streptomyces murinus FLC2-09 provided by the present invention can promote the growth of Panax notoginseng seedlings in terms of plant height, main root length, number of lateral roots, and fresh weight of the plants. Therefore, Streptomyces murinus FLC2-09 provided by the present invention can promote the development of biological control of root rot of Panax notoginseng, provide available actinomycete strain resources, and has broad application prospects in the cultivation of Panax notoginseng. Description of the Drawings

[0022] Figure 1 are the morphological characteristics and scanning electron micrographs of Streptomyces murinus FLC2-09 on PDA medium; wherein, A is a photograph of the morphological characteristics on PDA medium, and B is a scanning electron micrograph.

[0023] Figure 2 is a phylogenetic tree constructed from 16S rDNA sequences.

[0024] Figure 3 are the plate confrontation results of Streptomyces murinus FLC2-09 against Fusarium oxysporum; wherein, A is a blank control, and B is the inhibition effect of FLC2-09 on Fusarium oxysporum.

[0025] Figure 4 are the in vitro root control efficacy of Streptomyces murinus FLC2-09 against Fusarium oxysporum; wherein, A is a control inoculated only with Fusarium oxysporum, B is a blank control, and C is inoculated with both Fusarium oxysporum and Streptomyces murinus FLC2-09.

[0026] Figure 5 are the pot control efficacy of Streptomyces murinus FLC2-09 against Fusarium oxysporum; wherein, A is a blank control; B is inoculated with both Fusarium oxysporum and Streptomyces murinus FLC2-09; C is inoculated only with Fusarium oxysporum.

[0027] Figure 6It is a physical diagram of the growth of Panax notoginseng seedlings under different treatments. Among them, A is the pot experiment treatment of Panax notoginseng seedlings, and B is the growth of Panax notoginseng under different treatments 120 days after sowing. In the figure, A1~A3 and B1~B3 are sterile water control, culture solution control, and Streptomyces griseoruber FLC2-09 treatment in sequence.

[0028] Figure 7 It is a column chart of the growth of Panax notoginseng seedlings under different treatments. Different letters indicate significant differences at the P<0.05 level. Specific implementation manners

[0029] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without making creative efforts based on the embodiments in the present invention belong to the scope of protection of the present invention.

[0030] For those not specifying specific technologies or conditions in the embodiments, they are all conventional methods or are carried out according to the technologies or conditions described in the literature in this field, or according to the product specifications. For those reagents, instruments, etc. not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0031] Example 1 Isolation and purification of Streptomyces griseoruber In this example, actinomycetes were isolated and cultured from four base soil samples by the soil suspension dilution coating method using 8 kinds of media from the soil of the understory Panax notoginseng base. Streptomyces griseoruber FLC2-09 was screened from them. As Figure 1 shown in A, the substrate mycelium on the PDA medium is light yellow, the aerial mycelium is grayish brown, and no soluble pigment is produced. It is observed by scanning electron microscopy that the aerial hyphae form long spore chain filaments, and the sporangium is olive-shaped, as Figure 1 shown in B. Based on the above morphological characteristics, this strain was identified as Streptomyces.

[0032] After identification, this strain is Gram-positive; gelatin liquefaction, starch hydrolysis, MR reaction, urease reaction, and catalase reaction are positive; VP reaction is negative; it can utilize a variety of carbon sources such as fructose, xylose, glucose, sucrose, arginine, inositol, trehalose, sorbitol, and mannitol.

[0033] Furthermore, according to the instructions of the High-Efficiency Plant Genomic DNA Extraction Kit (TrellefTMHl-Pure Plant Genomic DNA Klt), the DNA of the strain was extracted. Using the extracted DNA genome as a template, with the bacterial universal primers 27F (5’-AGAGTTTGATCATGGCTCAG-3’, SEQ ID No.2) and 1492R (5’-GGTTACCTTACGACTT-3’, SEQ ID No.3), PCR amplification was carried out. The PCR amplification products were detected by electrophoresis on a 1.0% agarose gel, and then the PCR amplification products were sequenced. After the sequencing was completed, the gene sequences were subjected to similarity searches in the NCBI database; the gene sequences of typical strains with relatively high homology were selected as reference sequences, and using the MEGA11 software, a phylogenetic tree was constructed by the neighbor-joining method to determine the taxonomic status of the test strains. The phylogenetic tree is as shown in Figure 2 shown. Combining molecular, morphological, physiological and biochemical characteristics, Streptomyces murinus FLC2-09 was identified as Streptomyces murinus Streptomyces murinus , and the 16S rDNA sequence of Streptomyces murinus FLC2-09 is as shown in SEQ ID No.1.

[0034] Example 2 Inhibitory effect on Fusarium oxysporum, the main pathogen of Panax notoginseng root rot In this example, the test Fusarium oxysporum Fusarium oxysporum was provided by the Biocontrol Laboratory of the College of Plant Protection, Yunnan Agricultural University. The specific method is as follows: The inhibitory activity of Streptomyces murinus FLC2-09 against the mycelial growth of the test Fusarium oxysporum was determined by the plate confrontation culture method. A 5 mm pathogen mycelial disc was placed in the center of a PDA plate, and Streptomyces murinus FLC2-09 was placed 2.5 cm to the left and right of it. A blank control was set, and after incubating in an incubator at 28°C for 7 d, the growth radius of the pathogen colony was measured, and the inhibition rate was calculated.

[0035] Among them, the formula of the PDA medium is: 200 g of potato, 20 g of glucose, 18 g of agar powder, and 1000 mL of water. The results of the plate confrontation are as shown in Figure 3 shown, and the inhibition rate of Streptomyces murinus FLC2-09 against the mycelial growth of Fusarium oxysporum is 74.19%.

[0036] Example 3 In vitro control effect on Panax notoginseng root rot In this example, the control effect of Streptomyces murinus FLC2-09 on Panax notoginseng root rot was determined. The specific steps are as follows: Streptomyces murinus FLC2-09 was cultured in PDA medium at 28 °C with shaking at 160 r / min for 14 d and reserved. The surface of healthy two-year-old Panax notoginseng roots was disinfected, punctured with a sterile needle to create wounds, immersed in the actinomycete bacterial solution for 20 min and then taken out, placed in a petri dish lined with moist sterile filter paper. After 24 h, pathogen bacterial cakes were inoculated by puncturing holes in the Panax notoginseng roots, and blank controls, controls inoculated only with Streptomyces murinus FLC2-09, and controls inoculated only with the pathogen were set. Both the control group and the treatment group had 8 replicates. After culturing in the dark at a constant temperature of 26 °C for 15 d, the disease incidence of Panax notoginseng roots in each treatment was observed, and the disease index and control effect were evaluated.

[0037] The test results were as Figure 4 shown. There were no symptoms of rot or blackening on the surface and cut surface of the Panax notoginseng roots in the blank control and the control inoculated only with Streptomyces murinus FLC2-09. The Panax notoginseng roots in the control inoculated only with Fusarium oxysporum rotted severely, and the disease index reached 81.25. In the treatment inoculated with both Streptomyces murinus FLC2-09 and Fusarium oxysporum, rotting occurred, but to a lesser extent, the disease index was 12.50, and the control effect reached 84.62%. The test data are shown in Table 1.

[0038] Table 1 In vitro control effect of Streptomyces murinus FLC2-09 on root rot of Panax notoginseng

[0039] Pot control effect of Example 4 on root rot of Panax notoginseng In this example, the pot control effect of Streptomyces murinus FLC2-09 on root rot of Panax notoginseng was determined, and the specific steps were as follows: Five Streptomyces murinus FLC2-09 bacterial cakes were prepared with a puncher with an inner diameter of 5 mm, placed in 150 mL of PDB culture solution, cultured at 28 °C with shaking at 160 r / min for 14 d, and the fermentation broth was taken out and reserved. Healthy two-year-old Panax notoginseng plants were transplanted into small pots, with 3 plants per pot. After the Panax notoginseng plants survived, the test fermentation broth was inoculated onto the roots of the Panax notoginseng plants, 5 mL / plant; after 24 h, the pathogen bacterial solution (Fusarium oxysporum bacterial solution) with a spore amount of 10 6 spores / mL was poured onto the roots of the Panax notoginseng plants, and the roots were pretreated by acupuncture. Controls of inoculating only sterile water and only the pathogen were set. Each pot had 3 Panax notoginseng plants, and each treatment and control had 15 pots. The potted plants were placed in a shaded greenhouse. After 40 d, the disease incidence was observed, and the disease index and control effect were calculated.

[0040] The test results were as follows: The pot control effect of Streptomyces murinus FLC2-09 on Fusarium oxysporum was as Figure 5As shown, the disease index of the blank control treatment was 12.22%, the disease index of the treatment with only pathogen inoculation was 84.44%, the disease index of the treatment with both pathogen and Streptomyces murinus inoculation was 30.48%, and the control effect of Streptomyces murinus FLC2-09 in Panax notoginseng potted plants reached 63.90%, indicating that Streptomyces murinus FLC2-09 has good biocontrol potential.

[0041] Effect of Example 5 on the growth of Panax notoginseng seedlings In this example, the effect of Streptomyces murinus FLC2-09 on the growth of Panax notoginseng seedlings was determined, and the specific steps were as follows: Streptomyces murinus FLC2-09 was shaken in PDA culture medium for 14 d and reserved. Panax notoginseng seeds were soaked in 5% sodium hypochlorite for 5 min for surface disinfection. After being rinsed clean, they were sown in small pots (diameter 135 mm × height 105 mm). The soil in the pots was a mixed soil of sterilized soil and vermiculite with a mass ratio of 2:1. Sterile water control and culture medium control were set. 30 seeds were sown in each small pot, and there were 5 pots for each treatment and control. The corresponding treatment liquids were watered daily to keep the soil moist. After 120 d, the plant height, fresh weight of the plant, main root length, and number of lateral roots of Panax notoginseng seedlings were measured. After cultivating for 120 d, the growth conditions of Panax notoginseng seedlings in each treatment were as Figure 6 shown. The plant height, fresh weight of the plant, main root length, number of lateral roots, etc. of each treatment were measured, and the measurement results were as Figure 7 shown.

[0042] It can be seen that the plant height, main root length, number of lateral roots, and fresh weight of the plant in the treatment with Streptomyces murinus FLC2-09 were all higher than those in the culture medium control and the sterile water control. Among them, there were significant differences in the plant height, main root length, and number of lateral roots in the FLC2-09 treatment compared with the sterile water control and the culture medium control, but the promoting effect on the fresh weight of the plant was not significant. In summary, Streptomyces murinus FLC2-09 can promote the growth of Panax notoginseng plants in terms of plant height, main root length, and number of lateral roots.

[0043] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements for some of the technical features. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A Streptomyces murinus Streptomyces murinus , characterized in that It is Streptomyces murinus FLC2-09, and its preservation number is 30938.

2. Streptomyces murinus according to claim 1 Streptomyces murinus , characterized in that The 16S rDNA sequence of the Streptomyces murinus FLC2-09 is shown in SEQ ID No.

1.

3. A microbial inoculant, characterized in that, It contains Streptomyces murinus FLC2-09 described in claim 1 or 2.

4. The microbial inoculant according to claim 3, characterized in that, The microbial inoculum is a fermentation broth or freeze-dried powder of the fermentation broth containing the Streptomyces murinus FLC2-09.

5. The microbial inoculum according to claim 4, wherein The freeze-dried powder of the fermentation broth also includes a lyoprotectant.

6. A product, characterized in that, It contains Streptomyces murinus FLC2-09 described in claim 1 or 2; the product is an antibacterial agent, a plant growth promoter, a fertilizer or a fertilizer additive.

8. Application of Streptomyces murinus FLC2-09 described in claim 1 or 2, the microbial inoculum described in any one of claims 3 to 5, or the product described in claim 6 in inhibiting Fusarium oxysporum.

9. Application of Streptomyces murinus FLC2-09 described in claim 1 or 2, the microbial inoculum described in any one of claims 3 to 5, or the product described in claim 6 in preventing and controlling Panax notoginseng root rot.

10. Application of Streptomyces murinus FLC2-09 described in claim 1 or 2, the microbial inoculum described in any one of claims 3 to 5, or the product described in claim 6 in promoting the growth of Panax notoginseng plants.

10. The application according to claim 9, wherein Promote the growth of Panax notoginseng plants by increasing at least one of the plant height, main root length, number of lateral roots, and fresh weight of Panax notoginseng plants.

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