Streptomyces mori d2-3 and application thereof in inhibiting and promoting growth of areca

Abstract

The application belongs to the field of agricultural microorganism technology, and provides mulberry Streptomyces D2-3 and application thereof in areca fungus inhibition and growth promotion. The mulberry Streptomyces D2-3 is isolated from areca garden soil in Hainan Province, and the preservation number is CGMCC No. 38729. Through plate confrontation method and fermentation liquid rescreening, the strain D2-3 with strong antagonistic activity to areca pathogenic fungus C. heterocronemum is screened, and the inhibition rate of the fermentation liquid to C. heterocronemum reaches 99.76%. The strain has the abilities of dissolving phosphorus, producing iron carrier and secreting beta-1,3-glucanase, can significantly promote the growth of areca, improve the rhizosphere soil microbial community structure and increase the abundance of actinomycete flora. The D2-3 or the fermentation liquid thereof can be prepared into a microbial preparation for inhibiting areca pathogenic bacteria and promoting the growth of areca, is safe, efficient, environment-friendly, suitable for green prevention and control and healthy cultivation of areca diseases, and has important economic value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to Streptomyces mulberryis D2-3 and its application in inhibiting bacteria and promoting growth in areca nut. Background Technology

[0002] Areca catechu L. is an important economic crop in tropical regions of my country, widely cultivated in Hainan, Yunnan, and other areas. With the expansion of planting area and the increase in continuous cropping years, areca nut diseases have become increasingly prominent. Among them, root rot and stem base rot caused by Thieviopsis paradoxa are particularly serious, leading to stunted plant growth, reduced yields, and even complete plant death, becoming a key obstacle to the sustainable development of the areca nut industry.

[0003] Currently, the control of areca nut diseases still relies mainly on chemical pesticides. Long-term application not only easily leads to increased pathogen resistance and decreased efficacy, but also causes problems such as pesticide residues, environmental pollution, and soil microbial imbalance, which is inconsistent with the development direction of green agriculture and ecological control. Microbial control has advantages such as safety, high efficiency, environmental friendliness, and low likelihood of developing resistance, making it an important alternative to chemical pesticides.

[0004] Actinomycetes (especially Streptomyces) are an important group of agricultural microbial resources, widely distributed in the soil environment. They can secrete antibiotics, hydrolytic enzymes, and growth-promoting substances, possessing both antibacterial and disease-resistant functions as well as growth-promoting functions. Existing research shows that some Streptomyces can exert strong antagonistic effects against various plant pathogenic fungi and promote crop growth through mechanisms such as phosphorus solubilization, secretion of plant hormones, and improvement of rhizosphere microecology. However, highly efficient antagonistic actinomycete resources against the areca-specific pathogen *Leuconostoc mesenteroides* remain relatively scarce, and research on superior strains with both high antibacterial activity and significant growth-promoting effects is still insufficient.

[0005] Therefore, screening and developing a superior actinomycete that has a strong inhibitory effect on Areca catechu and can promote the growth of Areca catechu is of great practical significance for building a green prevention and control technology system for Areca catechu diseases and promoting the healthy and sustainable development of the Areca catechu industry. Summary of the Invention

[0006] The purpose of this invention is to provide *Streptomyces mulberryii* D2-3 and its application in the inhibition of fungi and promotion of growth in areca nut. Through screening, a superior actinomycete strain (D2-3) with highly efficient antagonistic activity against the areca nut pathogen *Leuconostoc mesenteroides* and growth-promoting function was obtained. Safe, efficient, and environmentally friendly microbial preparations were developed to solve the technical problems of excessive reliance on chemical pesticides for existing areca nut diseases, easy development of drug resistance in pathogens, pesticide residues and soil microecological imbalance, as well as insufficient antibacterial activity and insignificant growth-promoting effects of existing biocontrol strains.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: a mulberry streptomyces D2-3, wherein D2-3 is isolated from soil and its accession number is CGMCC No.38729.

[0008] The present invention further provides the application of the above-mentioned Streptomyces mulberryii D2-3.

[0009] Preferably, D2-3 is used to prepare a formulation that inhibits areca nut pathogens and promotes areca nut growth.

[0010] Preferably, the areca nut pathogen is *Rhizoctonia solani*, and the preparation has an inhibition rate of not less than 99% against *Rhizoctonia solani*.

[0011] Preferably, the formulation promotes growth by increasing the abundance of actinomycete flora, secreting β-1,3-glucanase, producing siderophores, and lysing phosphorus.

[0012] Preferably, the active ingredient of the preparation is D2-3 or the fermentation broth of D2-3.

[0013] Preferably, the fermentation broth is prepared as follows: the activated strain is placed in 60 mL of seed fermentation medium and shaken for 72 h to obtain seed liquid; the seed liquid is inoculated into the basic fermentation medium at an inoculation rate of 10% and shaken for 4 days to obtain fermentation broth of D2-3; the shaking conditions are: 28 ℃, 180 r / min.

[0014] Preferably, the seed fermentation medium comprises the following components: 20 g soluble starch, 1 g KNO3, 0.5 g NaCl, 0.5 g K2HPO4•3H2O, 0.5 g MgSO4•7H2O, 0.01 g FeSO4•7H2O, 15-20 g agar, 1000 mL distilled water, and pH 7.2-7.4.

[0015] Preferably, the basic fermentation medium has the following components: 10 g millet, 10 g glucose, 3 g peptone, 2.5 g sodium chloride, 2 g calcium carbonate, 1000 mL distilled water, and pH 7.2-7.4.

[0016] Preferably, the formulation includes various acceptable dosage forms, which are one or more of granules, powders, or liquids.

[0017] The beneficial effects of this invention are:

[0018] 1. Excellent antibacterial effect: The fermentation broth of strain D2-3 showed a mycelial growth inhibition rate of up to 99.76% against the fungus Leuconostoc melanocytoxin, the pathogen of areca root rot. The antibacterial effect is comparable to that of the chemical pesticide imazalil manganese salt, and is significantly better than conventional biocontrol strains and agents such as tridemorph. It can effectively block pathogen infection and effectively control areca root rot and stem base rot.

[0019] 2. Also possesses growth-promoting functions: Strain D2-3 can secrete β-1,3-glucanase, which targets and degrades the cell wall of pathogenic fungi, exerting an antibacterial effect; it also produces siderophores to chelate Fe in the environment. 3+ It provides iron nutrition to plants and simultaneously inhibits rhizosphere pathogens (iron competition), thus promoting growth and preventing disease. It also has phosphorus-solubilizing capabilities, activating insoluble phosphorus in the soil, increasing the content of available phosphorus in the rhizosphere, and promoting root development and nutrient absorption in areca nut trees. Pot experiments showed that applying D2-3 fermented liquid significantly increased the dry weight of areca nut seedlings, resulting in vigorous plant growth, dark green leaves, and a significant growth-promoting effect.

[0020] 3. Improves rhizosphere microecology and has high environmental safety: Strain D2-3 can stably colonize the rhizosphere soil of Areca catechu, increase the abundance of Actinobacteria, optimize the soil microbial community structure, and enhance the soil's disease resistance. Compared with chemical pesticides, this strain and its fermentation broth have no pesticide residues, no environmental pollution, and are less likely to induce drug resistance in pathogens, which meets the needs of green agriculture and ecological control development. Attached Figure Description

[0021] Figure 1 This is a diagram illustrating the inhibitory effect of the fermentation broth on Rhizoctonia solani in this invention.

[0022] Figure 2 This describes the morphological and culture characteristics of strain D2-3 in this invention;

[0023] Figure 3 This is a diagram showing the experimental results of the physiological and biochemical characteristics of strain D2-3 in this invention;

[0024] Figure 4 This is a phylogenetic tree of strain D2-3 and Streptomyces strains in this invention;

[0025] Figure 5 This is the antagonistic growth-promoting effect of D2-3 potted plants in this invention;

[0026] Figure 6 This invention describes the enrichment of soil microorganisms after applying D2-3 fermentation broth (CK (water control); Trt3 (D2-3 fermentation broth applied); Trt4 (blank fermentation broth without D2-3 application)).

[0027] Figure 7This invention shows the secretion of β-,3-glucanase by D2-3 (left), the phosphorus solubilization effect (middle), and the siderogenic carrier (right). Detailed Implementation

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] Example 1: Screening and Identification of D2-3

[0032] 1. Materials and Methods

[0033] Soil samples were collected from 10 areca nut plantations in Wenchang City, Qionghai City, Wanning City, Ding'an County and Tunchang County, Hainan Province, at a depth of 5-10 cm.

[0034] The target pathogen was Thieviopsis paradoxa, a pathogenic fungus of palm trees, which was obtained from the Plant Protection Research Center of the Coconut Research Institute, Chinese Academy of Tropical Agricultural Sciences.

[0035] Test reagents: 50% imazalil manganese salt wettable powder (produced by Bayer Crop Science); 750 g / L tridemorpholine emulsifiable concentrate (produced by Shanghai Shengnong Biochemical Products Co., Ltd.).

[0036] 2. Isolation and purification of actinomycetes

[0037] Separation was performed using the agar plate dilution method. 10 g of soil sample was weighed and added to a 250 mL Erlenmeyer flask containing 90 mL of sterile water. The sample was shaken at 28 ℃ and 180 r / min for 30 min. The supernatant was then diluted using a 10-fold serial dilution method to a final concentration of 10. -4 Diluent. Use a pipette to draw 100 µL of the solution. -4Add the diluent to Gao's No. 1 medium (see Example 2) containing 150 mg / L potassium dichromate solution. Spread the medium evenly with a sterile spreader until the diluent is dry to prevent water film formation and contamination. After even spreading, seal the petri dishes with plastic wrap and incubate upside down in a 28 ℃ artificial climate chamber for 10 days, repeating 3 times. Pick a single colony with a sterile inoculation loop, transfer it to modified Gao's No. 1 medium, streak it, and purify it by subculturing 2-3 times. Then, inoculate it onto modified Gao's No. 1 slant medium and incubate for 7 days. Store at 4 ℃ for later use.

[0038] 3. Screening of antagonistic actinomycetes

[0039] Using *Rhizoctonia solani* as the target bacterium, the antagonistic activity of isolated strains was initially and subsequently screened using the plate confrontation method. The specific procedures are as follows:

[0040] Initial screening: A 0.6 cm diameter *Lysimachia oryzae* mycelium block was inoculated in the center of a PDA plate (9 cm), and four different actinomycete mycelium discs were inoculated at the edge using a cross-inoculation method. The culture medium plate inoculated only with *Lysimachia oryzae* served as a control. After culturing in an artificial climate chamber at 28°C for 2 days, the antibacterial effect of the actinomycetes was observed, and antagonistic strains with antibacterial activity were selected for secondary screening.

[0041] Secondary screening: The antagonistic strains obtained from the initial screening were inoculated into Gao's No. 1 liquid medium and fermented in a shaker at 28 ℃ for 7 days at 180 r / min. The fermentation broth was centrifuged at 8000 r / min for 20 min, and the supernatant was collected and sterilized by filtering through a 0.22 µm bacterial filter. After the PDA medium cooled to approximately 60 ℃, 2 mL of fermentation broth was added to every 18 mL of medium, mixed well, and poured into plates to prepare toxic plates. Sterile water was added as a control. After solidification, 0.6 mm diameter *Leuconostoc mesenteroides* mycelial cakes were inoculated and cultured in a 28 ℃ incubator. Each strain was replicated three times. After the control plate was fully colonized, the colony diameter was measured using the cross-crossing method. The antibacterial effect was calculated using the following formula:

[0042] .

[0043] 4. Identification

[0044] 4.1 Morphological identification

[0045] (1) Observation of the culture characteristics of D2-3

[0046] After incubation at 28 ℃, observe the growth status of the strain, aerial mycelia, substrate mycelia, and the color of soluble pigments.

[0047] (2) The morphological characteristics of actinomycetes were observed under a microscope using the imprint method. A fungal block was punched with a 1 cm diameter punch and placed on a glass slide with the fungal side facing up. Another clean slide was quickly passed over a flame 2-3 times and then placed on the fungal moss. The imprint side was placed facing up and quickly passed over the flame 2-3 times to fix it. The morphological characteristics of the actinomycetes were then observed under a microscope.

[0048] 4.2 Molecular Identification

[0049] The 16S rDNA sequence of the strain was amplified by PCR using universal bacterial primers (27F: 5'-AGAGTTTGATCMTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The reaction system and reaction conditions are shown in Tables 1 and 2.

[0050] Table 1 PCR reaction system

[0051]

[0052] Table 2 PCR reaction conditions

[0053]

[0054] Note: The process of denaturation, annealing, and extension is repeated 35 times.

[0055] After the PCR reaction, 5 µL of the amplification product was subjected to agarose gel electrophoresis (1%). Products with distinct bands (approximately 1500 bp in size) were sent to the Guangzhou Sequencing Department of Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequences were submitted to the GenBank database for comparison with published valid strains. Appropriate strains were selected, and multiple sequence alignment was performed using MEGA6 software. A phylogenetic tree was constructed using the Neighbor-Jioning method.

[0056] 5. Results Analysis

[0057] 5.1 Isolation of Actinomycetes and Screening of Antagonistic Bacteria

[0058] 120 actinomycetes were isolated and purified from the tested soil samples. 22 antagonistic strains showing inhibitory effects against *Leuconostoc mesenteroides* were initially screened for further screening. Five actinomycete fermentation broths showed an inhibition rate greater than 70% against the mycelial growth of *Leuconostoc mesenteroides*, with strain D2-3 exhibiting an inhibition rate of 99.76%. Figure 1 This strain showed a significantly higher inhibition rate than other strains, and was identified as the dominant antagonistic strain, which was then classified and identified. The inhibition results are shown in Table 3.

[0059] Table 3. Inhibitory effect of actinomycete fermentation broth on *Lymnocladus mirabilis*

[0060]

[0061] Note: Different lowercase letters in the table indicate significant differences at the 0.05 level.

[0062] 5.2 Identification Results

[0063] Morphological characteristics: When strain D2-3 was cultured on Gao's No. 1 medium at 28 ℃ for 10 days, the colonies were round, dry, and opaque with neat edges. Both aerial and substrate hyphae were white, without the production of any soluble pigments. Under an optical microscope, the hyphae showed radial growth from the center outwards, mostly linear with a few curves. The aerial hyphae were well-developed, branched, and broke off upon maturity, differentiating into sporophytes. Figure 2 ).

[0064] Physiological and biochemical characteristics ( Figure 3 Strains D2-3 cannot liquefy gelatin, thus testing negative; they can peptone milk, thus testing positive; when Lugol's iodine solution is added around the colony, the surrounding area turns blue, indicating that amylase is not produced, thus testing negative; they cannot hydrolyze cellulose; in the hydrogen sulfide production experiment, no melanin was produced, indicating that hydrogen sulfide is not produced; in the nitrate reduction experiment, the colony immediately turns deep red after the addition of nitrate reducing reagent, indicating that nitrate can be reduced, thus testing positive. They can utilize nitrogen sources such as aspartic acid and tryptophan, but cannot utilize arginine. None of the carbon sources tested in this experiment can be utilized. See Table 4 for detailed physiological and biochemical characteristics.

[0065] Table 4 Physiological and biochemical characteristics of strain D2-3

[0066]

[0067] Note: In the table, "+" indicates a positive result or that the product can be used; "-" indicates a negative result or that the product cannot be used.

[0068] Based on experimental observations of the morphology, culture characteristics, and physiological and biochemical properties of strain D2-3, it was preliminarily identified as Streptomyces.

[0069] 5.3 16S rDNA Sequence Analysis

[0070] The 16S rNDA gene of strain D2-3 was sequenced to obtain a 1024 bp nucleic acid sequence, which was submitted to GenBank and obtained accession number ON786711. Homology comparison revealed that the 16S rNDA sequence of strain D2-3 was more than 99% similar to that of Streptomyces samsunensis (accession number: MK530504) in GenBank, and the two were located on the same independent branch in the phylogenetic tree. Figure 4Based on morphological and physiological-biochemical characteristics, strain D2-3 was ultimately identified as *Streptomyces samsunensis*, a species of *Streptomyces*.

[0071] 5.4 Preservation

[0072] The strain (D2-3) was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 18, 2026, and the activity test result showed that it was viable. The accession number is CGMCC No. 38729.

[0073] Example 2 Evaluation of the antagonistic effect of D2-3 on growth promotion

[0074] 1. Materials and Methods

[0075] (1) Culture medium

[0076] PDA medium: 200 g potato, 20 g glucose, 15-20 g agar powder, 1000 mL distilled water.

[0077] Gao's No. 1 synthetic medium: soluble starch 20 g, KNO3 1 g, NaCl 0.5 g, K2HPO4•3H2O 0.5 g, MgSO4•7H2O 0.5 g, FeSO4•7H2O 0.01 g, agar 15-20 g, distilled water 1000 mL, pH 7.2-7.4.

[0078] Seed fermentation medium: Gao's No. 1 synthetic medium.

[0079] Basic fermentation medium: 10 g millet, 10 g glucose, 3 g peptone, 2.5 g sodium chloride, 2 g calcium carbonate, 1000 mL distilled water, pH 7.2-7.4.

[0080] (2) Preparation of fermentation broth

[0081] Select the activated strain into a 150 mL Erlenmeyer flask containing 60 mL of seed fermentation medium, and culture at 28 ℃ and 180 r / min for 72 h to prepare the seed liquid; then inoculate it into the basic fermentation medium at a 10% inoculum and culture at 28 ℃ and 180 r / min for 4 days to obtain the fermentation broth for later use.

[0082] (3) Pot experiment on antagonistic growth-promoting effects

[0083] One- to two-year-old potted seedlings were selected, and the fermented liquid was diluted 5 times for root irrigation, once every 15 days, for a total of 4 applications. A control group consisting of seedlings not inoculated with fermented liquid and water was used. Each treatment consisted of 15 areca nut seedlings. The day after the first application of fermented liquid, *Leuconostoc mesenteroides* mycelium blocks were inoculated using the leaf wound inoculation method, and the mixture was kept moist with absorbent cotton for 24 hours. Thirty days after the last application, the disease incidence in areca nut seedlings was observed, and the dry weight of the areca nut seedlings and soil microbial diversity were measured.

[0084] (4) Preliminary exploration of the mechanism of growth promotion

[0085] D2-3 was inoculated into PVK medium (Beijing Cooler Technology Co., Ltd.) and β-1,3-glucanase detection medium (Beijing Cooler Technology Co., Ltd.), prepared and used according to the instructions for use of the medium, and cultured at 28 ℃ for 7 days. The phosphorus solubilization capacity, siderophore production capacity and β-1,3-glucanase production capacity of D2-3 were then detected.

[0086] 2. Results Analysis

[0087] (1) Antagonistic effect on growth promotion

[0088] The experimental results showed that three months after applying the fermentation solution, there were differences in the dry weight of plants under different treatments. The dry weight of plants treated with the fermentation solution was greater than that of plants treated with the untreated fermentation solution and the water control (Table 5). Plants in the untreated and water control groups all developed lesions, while plants treated with the fermentation solution did not develop the disease and exhibited a darker green color. Figure 5 The above results indicate that D2-3 promotes the growth of areca nut and also has a preventive effect against Rhizoctonia solani disease.

[0089] Table 5. Effects of antagonistic bacterial fermentation broth on the growth promotion of areca seedlings.

[0090]

[0091] (2) The impact of D2-3 on soil microbial diversity

[0092] The diversity of microorganisms in soils under different treatments was investigated using 16S rRNA gene (or fungal ITS, prokaryotic, and functional gene) sequencing technology. Based on LEfSe multi-level species hierarchical tree diagram analysis, significant differences were found in the microbial community structure between the clean water group (Ck), the fermentation broth group (Trt3), and the uninoculated fermentation broth group (Trt4). Further compositional and differential analyses revealed significant enrichment of *Bryobacteraceae*, *Bryobacterales*, and *Acidiferrimicrobium* in the clean water group; significant enrichment of *Hyphomicrobiales*, *Xanthobacteraceae*, and *Hyphomicrobiale* in the uninoculated fermentation broth group; and significant enrichment of actinomycete phyla (especially *Mycobacteriales* and *Streptomycetaceae*) in the fermentation broth group, confirming the colonization of exogenous growth-promoting actinomycetes in the soil. Figure 6 Compared to CK (no enrichment) and Trt4 (enrichment of non-actinomycetes), the colonization effect was attributed to live bacteria rather than the culture medium. This indicates that the growth-promoting actinomycetes successfully colonized in the soil, and the microbial community was significantly biased towards actinomycete enrichment, while the control group did not exhibit this phenomenon. This provides theoretical support for the application of actinomycete fertilizers.

[0093] (3) Preliminary exploration of the growth-promoting mechanism

[0094] D2-3 possesses the ability to solubilize phosphorus, produce siderophores, and secrete β-1,3-glucanase, exhibiting multi-pathway growth promotion and biocontrol potential. Figure 7The core antibacterial mechanism of β-1,3-glucanase lies in its targeted degradation of key structural components of the cell wall of pathogenic fungi. Existing research indicates that the main structural polysaccharides of fungal cell walls are interwoven with β-1,3-glucan, chitin, and β-1,6-glucan. β-1,3-glucanase hydrolyzes β-1,3-glucan chains, disrupting the integrity and mechanical strength of the cell wall, leading to cell wall rupture, leakage of contents, and ultimately inhibition of hyphal growth or cell death. β-1,3-glucanase secreted by actinomycetes (such as the cucumber endophyte gCLA4) has significant in vitro inhibitory effects on various plant pathogenic fungi (such as *Botryosphaeria dothidea* and *Sclerotinia sclerotiorum*). The direct contribution of phosphorus solubilization is a significant increase in the concentration of available phosphorus in the rhizosphere, meeting the plant's phosphorus nutrient requirements. Sufficient phosphorus supply greatly stimulates root growth, increasing root length, root weight, and root hair density, thereby expanding the nutrient absorption area. This not only facilitates phosphorus absorption but also synergistically promotes the absorption of other elements such as nitrogen and potassium. Studies have confirmed that the presence of nitrogen (such as ammonium ions) can synergistically promote the dissolution of certain forms of calcium phosphorus precipitates (such as DCPD), revealing the mutually promoting effect of nitrogen and phosphorus absorption. Fe in the iron-carrier chelating environment... 3+ It provides iron nutrition to plants and inhibits rhizosphere pathogens (iron competition), thus having both growth-promoting and disease-preventing effects.

[0095] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A type of *Streptomyces mulberryii* D2-3, characterized in that, The D2-3 was isolated from the soil and its accession number is CGMCC No. 38729.

2. The application of *Streptomyces mulberryii* D2-3 according to claim 1, characterized in that: The D2-3 is used to prepare a formulation that inhibits areca nut pathogens and promotes areca nut growth.

3. The application according to claim 2, characterized in that, The areca nut pathogen is *Rhizoctonia solani*, and the preparation has an inhibition rate of not less than 99% against *Rhizoctonia solani*.

4. The application according to claim 2, characterized in that, The formulation promotes growth by increasing the abundance of actinomycete flora, secreting β-1,3-glucanase, producing siderophores, and lysing phosphorus.

5. The application according to claim 2, characterized in that, The active ingredient of the preparation is D2-3 or the fermentation broth of D2-3.

6. The application according to claim 5, characterized in that, The fermentation broth was prepared as follows: the activated strain was placed in 60 mL of seed fermentation medium and shaken for 72 h to obtain seed liquid; the seed liquid was inoculated into the basic fermentation medium at an inoculation rate of 10% and shaken for 4 days to obtain fermentation broth of D2-3; the shaking conditions were: 28 ℃, 180 r / min.

7. The application according to claim 6, characterized in that, The seed fermentation medium consists of the following components: 20 g soluble starch, 1 g KNO3, 0.5 g NaCl, 0.5 g K2HPO4•3H2O, 0.5 g MgSO4•7H2O, 0.01 g FeSO4•7H2O, 15-20 g agar, 1000 mL distilled water, and pH 7.2-7.

4.

8. The application according to claim 6, characterized in that, The basic fermentation medium consists of the following components: 10g millet, 10g glucose, 3g peptone, 2.5g sodium chloride, 2g calcium carbonate, 1000mL distilled water, and pH 7.2-7.

4.

9. The application according to claim 2, characterized in that, The formulation includes various acceptable dosage forms, which are one or more of granules, powders or liquids.

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