A type of Streptomyces whitworthii and its applications

Abstract

This invention provides a *Streptomyces albidoflavus* WP-Qing 1, which was deposited on June 28, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27730. The 16S rRNA gene sequence of this *Streptomyces albidoflavus* is shown in SEQ ID NO: 1; the rpoB gene of this *Streptomyces albidoflavus* is shown in SEQ ID NO: 2. The *Streptomyces albidoflavus* WP-Qing 1 provided by this invention exhibits strong salt and alkali tolerance and can be used to dissolve inorganic phosphorus, produce indoleacetic acid, heptaphosphate, extracellular proteases, cellulases, and β-1,3-glucanase; it can also be used to antagonize plant pathogens. Therefore, this *Streptomyces albidoflavus* WP-Qing 1 has great application potential.

Description

Technical Field

[0001] This invention relates to the field of Streptomyces technology, specifically to a microyellow Streptomyces and its applications. Background Technology

[0002] Streptomyces are a group of Gram-positive bacteria widely used in bioengineering, drug development, and agriculture. They grow in soil, forming branched, hyphal-like structures. Streptomyces secrete enzymes to degrade organic matter and also secrete antibiotics to inhibit the growth of other bacteria.

[0003] Streptomyces possesses extensive genomic and metabolic diversity, making it an ideal source for developing new drugs. By studying the genome, metabolic pathways, and regulatory mechanisms of Streptomyces, new natural products and bioactive molecules can be continuously discovered, providing important resources for drug development.

[0004] In addition, Streptomyces can be used for soil improvement and crop protection. They improve soil quality and provide nutrients by decomposing organic matter and fixing nitrogen, thus enhancing plant disease resistance. Therefore, Streptomyces are widely used in agriculture, playing a positive role in improving crop yield and quality.

[0005] However, current research on Streptomyces is still insufficient, and many Streptomyces and their applications remain to be discovered. Summary of the Invention

[0006] The purpose of this invention is to provide a Streptomyces microcyticus and its applications.

[0007] The technical solution of the present invention is as follows:

[0008] A Streptomyces albidoflavus WP-Qing 1 species was deposited on June 28, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27730.

[0009] Correspondingly, a Streptomyces albidoflavus WP-Qing 1, the 16S rRNA gene sequence of which is shown in SEQ ID NO: 1.

[0010] Correspondingly, a Streptomyces albidoflavus WP-Qing1, whose rpoB gene is shown in SEQ ID NO: 2.

[0011] Correspondingly, a Streptomyces albidoflavus WP-Qing 1 strain exhibits strong salt and alkali tolerance, capable of growing normally on NA plates with a salt content (NaCl) of 5% and in a strongly alkaline environment (7.0 ≤ pH ≤ 11.0).

[0012] An application of *Streptomyces microcytogenes*, based on the above-mentioned *Streptomyces microcytogenes*, for dissolving inorganic phosphorus.

[0013] Phosphorus is an essential macronutrient for plant growth, but the available phosphorus (soluble phosphorus) in soil is usually very low. Furthermore, phosphorus from fertilizers applied to the soil is easily fixed. Therefore, improving the solubility of inorganic phosphorus is crucial for enhancing crop utilization of insoluble inorganic phosphorus in the soil. The *Streptomyces microcyticus* strain of this invention possesses a strong ability to dissolve inorganic phosphorus. Quantitative analysis showed that after inoculating WP-Qing 1 into a culture solution containing insoluble inorganic phosphorus, the soluble phosphorus content in the culture solution was ≥200 mg / L after a certain period of cultivation. This significantly exceeds the minimum standard requirement of 70 mg / L in NY / T1847-2010 (General Technical Requirements for Quality Evaluation of Microbial Fertilizer Production Strains), indicating great application potential.

[0014] An application of *Streptomyces microcytogenes*, based on the above-mentioned *Streptomyces microcytogenes*, for the production of indoleacetic acid.

[0015] Indoleacetic acid (IAA) is an important plant growth regulator with multiple functions, including promoting plant growth, inducing root formation, increasing survival rate and yield, and enhancing stress resistance. The *Streptomyces microcyticum* strain of this invention exhibits a strong ability to produce IAA; quantitative analysis shows that it produces ≥8.0 μg / mL of IAA. -1 This far exceeds the 5.0 μg·mL specified in NY / T 1847-2010. -1 It meets the minimum standard requirements and has great application potential.

[0016] An application of *Streptomyces microcyticum*, based on the above-mentioned *Streptomyces microcyticum*, for the production of ferrophiles.

[0017] Microbial-produced hematoxylins have a dual function of promoting plant growth and controlling plant diseases. The *Streptomyces microcyticus* strain of this invention has a strong hematoxylin-producing capacity; quantitative analysis shows that its hematoxylin-producing capacity reaches the +++ level, with an As / Ar ratio between 0.4 and 0.6, demonstrating its potential for application in promoting crop growth and controlling diseases.

[0018] An application of *Streptomyces microcytogenes*, based on the above-mentioned *Streptomyces microcytogenes*, for the production of extracellular proteases.

[0019] An application of *Streptomyces microcytogenes*, based on the above-mentioned *Streptomyces microcytogenes*, for the production of cellulase.

[0020] Extracellular proteases and cellulases produced by soil microorganisms can promote the decomposition of organic materials, convert macromolecular organic matter into small-molecule nutrients that are easily absorbed by plants, and prevent plant diseases. The *Streptomyces microcyticus* strain of this invention has the ability to produce extracellular proteases and cellulases, and can produce very obvious transparent zones on qualitative detection plates, showing potential in the application of microbial fertilizers.

[0021] An application of *Streptomyces microcytogenes*, based on the above-mentioned *Streptomyces microcytogenes*, for the production of β-1,3-glucanase.

[0022] β-1,3-glucanase can degrade the cell walls of plant pathogens and induce plant defense responses, playing an important role in plant disease resistance. The *Streptomyces microcyticus* strain of this invention possesses the ability to produce β-1,3-glucanase, generating distinct discoloration zones on qualitative detection plates, demonstrating its potential for controlling fungal plant diseases.

[0023] An application of *Streptomyces microcyticum*, based on the aforementioned *Streptomyces microcyticum*, for antagonizing plant pathogens.

[0024] The *Streptomyces microcyticus* of this invention can produce iron-loving enzymes, proteases, cellulases, and β-1,3-glucanase. These metabolites all have the function of preventing and controlling plant diseases. When these metabolites are applied alone or in combination, they exert a highly efficient and broad-spectrum antagonistic potential (ability) against plant pathogens.

[0025] Plant pathogens include fungi and bacteria.

[0026] The beneficial effects of this invention are as follows: This invention provides a novel *Streptomyces microcyticum* WP-Qing 1 with strong salt and alkali tolerance. This *Streptomyces microcyticum* WP-Qing 1 can grow normally on NA plates with a salt content (NaCl) of 5% and a strongly alkaline environment (7.0 ≤ pH ≤ 11.0). It can be used to dissolve inorganic phosphorus; it can be used to produce indoleacetic acid, heptaphosphate, extracellular protease, cellulase, and β-1,3-glucanase; and it can also be used to antagonize plant pathogens. Therefore, this *Streptomyces microcyticum* WP-Qing 1 has great application potential. Attached Figure Description

[0027] Figure 1 This is a tandem phylogenetic tree of the 16S rRNA-rpoB gene of Streoptomyces albidoflavus WP-Qing 1 in Example 1 of this application;

[0028] Figure 2 This is a colony morphology diagram of WP-Qing1 on NA medium plates in Example 1 of this application;

[0029] Figure 3It is the colony morphological feature diagram of WP-Qing 1 on the Gao's No. 1 medium plate in Example 1 of this application;

[0030] Figure 4 It is the microscopic morphological diagram of the spore chains and spores of WP-Qing 1 in Example 1 of this application;

[0031] Figure 5 It is the growth situation diagram of WP-Qing 1 on the NA plate with a salt content (NaCl) of 5% in Example 1 of this application;

[0032] Figure 6 It is the growth situation diagram of WP-Qing 1 on the NA plate containing alkali (NaOH) and with 7.0 ≤ pH value ≤ 11.0 in Example 1 of this application;

[0033] Figure 7 It is the growth situation diagram of WP-Qing 1 on the inorganic phosphorus detection plate in Example 2 of this application;

[0034] Figure 8 It is the measurement result diagram of the ability of WP-Qing 1 to dissolve inorganic phosphorus by colorimetry in Example 2 of this application;

[0035] Figure 9 It is the measurement result diagram of the ability of WP-Qing 1 to produce IAA by colorimetry in Example 3 of this application;

[0036] Figure 10 It is the growth situation diagram of WP-Qing 1 on the CAS detection plate in Example 4 of this application;

[0037] Figure 11 It is the measurement result diagram of the ability of WP-Qing 1 to produce siderophores by colorimetry in Example 4 of this application;

[0038] Figure 12 It is the growth situation diagram of WP-Qing 1 on the protease detection plate in Example 5 of this application;

[0039] Figure 13 It is the growth situation diagram of WP-Qing 1 on the cellulase activity measurement plate in Example 6 of this application;

[0040] Figure 14 It is the growth situation diagram of WP-Qing 1 on the aniline blue dextran agar medium plate in Example 7 of this application;

[0041] Figure 15 It is the growth situation diagram of WP-Qing 1 antagonizing the growth of Botryosphaeria sp. (Botryosphaeria piricola) on the PDA plate in Example 8 of this application;

[0042] Figure 16This is a diagram illustrating the antagonistic effect of WP-Qing 1 on the growth of Botrytis cinerea on a PDA plate in Example 8 of this application.

[0043] Figure 17 This is a diagram showing the growth of WP-Qing 1 antagonizing Alternaria sp. on a PDA plate in Example 8 of this application;

[0044] Figure 18 This is a diagram illustrating the antagonistic effect of WP-Qing 1 on the growth of Fusarium oxysporum on a PDA plate in Example 8 of this application;

[0045] Figure 19 This is a diagram showing the growth of WP-Qing 1 antagonizing Valsa sp. (black rot fungus) on a PDA plate in Example 8 of this application;

[0046] Figure 20 This is a diagram illustrating the growth of WP-Qing 1 antagonizing Fusarium oxysporum f.sp. niveum (a watermelon-specific variant of Fusarium oxysporum) on a PDA plate in Example 8 of this application;

[0047] Figure 21 This is a diagram illustrating the antagonistic effect of WP-Qing 1 on the growth of Pseudomonas syringaepv. actinidiae (a variant of Actinidia syringa) on NA plates in Example 8 of this application.

[0048] Figure 22 This is a diagram illustrating the antagonistic effect of WP-Qing 1 on the growth of Rhizoctonia cerealis VanderHoeven on a PDA plate in Example 8 of this application;

[0049] Figure 23 This is a diagram illustrating the antagonistic effect of WP-Qing 1 on the growth of Fuasarum graminearum on a PDA plate in Example 8 of this application.

[0050] Figure 24 This is a diagram illustrating the antagonistic effect of WP-Qing 1 on the growth of Fusarium proliferatum on a PDA plate in Example 8 of this application.

[0051] Figure 25 This is a diagram illustrating the antagonistic effect of WP-Qing 1 on the growth of Fusarium verticillioides on a PDA plate in Example 8 of this application.

[0052] Figure 26This is a diagram illustrating the antagonistic effect of WP-Qing 1 on the growth of Fusarium solani on a PDA plate in Example 8 of this application;

[0053] Figure 27 This is a bar chart showing the wheat plant height of the control and each treatment in the experimental results of Experiment Example 1 of this application;

[0054] Figure 28 This is a bar chart showing the wheat root length of the control and each treatment in the experimental results of Experiment Example 1 of this application;

[0055] Figure 29 This is a bar chart showing the wheat root count of the control and each treatment in the experimental results of Experiment Example 1 of this application;

[0056] Figure 30 This is a bar chart showing the fresh weight of wheat in the control and treatment groups in the experimental results of Example 1 of this application;

[0057] Figure 31 This is a bar chart showing the dry weight of wheat in the control and treatments in the experimental results of Experiment Example 1 of this application;

[0058] Figure 32 This is a growth status diagram of wheat at CK, T4, T5, and T6 in Experiment Example 1 of this application;

[0059] Figure 33 This is a bar chart showing the wheat plant height of the control and each treatment in the experimental results of Experiment Example 2 of this application;

[0060] Figure 34 This is a bar chart showing the wheat root length of the control and each treatment in the experimental results of Experiment Example 2 of this application;

[0061] Figure 35 This is a bar chart showing the wheat root count of the control and each treatment in the experimental results of Experiment Example 2 of this application;

[0062] Figure 36 This is a bar chart showing the fresh weight of wheat in the control and treatments in the experimental results of Experiment Example 2 of this application;

[0063] Figure 37 This is a bar chart showing the dry weight of wheat in the control and treatment of the experimental results in Example 2 of this application;

[0064] Figure 38 This is a growth status diagram of wheat at CK, T10, T11, and T12 in Experiment Example 2 of this application;

[0065] Figure 39 This is a diagram of the maize growth status in Experiment Example 3 of this application. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0067] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms, and “multiple” generally includes at least two unless the context clearly indicates otherwise.

[0068] It should be understood that although the terms first, second, third, etc., may be used to describe... in the embodiments of the present invention, these... should not be limited to these terms. These terms are only used to distinguish... For example, first... may also be referred to as second... without departing from the scope of the embodiments of the present invention, and similarly, second... may also be referred to as first...

[0069] Example 1: This example describes a Streoptomyces albidoflavus WP-Qing 1, which was deposited at the China General Microbiological Culture Collection Center on June 28, 2023, with the accession number CGMCC No. 27730.

[0070] In this embodiment of the application, the 16S rRNA gene sequence of Streoptomyces albidoflavus WP-Qing 1 is shown in SEQ ID NO: 1.

[0071] The sequence of SEQ ID NO:1 is shown below.

[0072]

[0073] In this embodiment, the rpoB gene of *Streptomyces albidoflavus* WP-Qing 1 is shown in SEQ ID NO: 2. The tandem phylogenetic tree of the 16S rRNA-rpoB gene of *Streptomyces albidoflavus* WP-Qing 1 is as follows: Figure 1 As shown in the figure. The rpoB gene refers to the gene encoding the β subunit of bacterial RNA polymerase.

[0074] The sequence of SEQ ID NO:2 is shown below.

[0075] .

[0076] In this embodiment of the application, the method for isolating Streoptomyces albidoflavus WP-Qing 1 is as follows.

[0077] Wheat seedlings were dug up with roots intact. Large clumps of soil and impurities were shaken off the roots. Under aseptic conditions, 10g of wheat roots (with a small amount of rhizosphere soil) were cut and placed in a 500ml Erlenmeyer flask containing 100ml of sterile water and glass beads. The flask was shaken at 120rpm for 30 minutes to obtain a suspension. The suspension was then serially diluted with sterile water, and 10g of each diluted solution was taken. -3 10 -4 Two dilutions of the suspension, 0.1 ml each, were spread onto inorganic phosphorus plates (Inorganic phosphorus plate medium: glucose 10.0 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO4). 4. 0.3g of 7H2O, 0.036g of FeSO4·7H2O, 0.03g of MnSO4·H2O, 2.0g of Ca3(PO4)2, 0.5g of yeast extract, 15-20g of agar, and distilled water to a final volume of 1000mL (pH 6.8-7.2). Each dilution was tested in triplicate. The plated agar plates were incubated at 30℃ for 3-5 days. Strands producing a clear zone were repeatedly streaked with inorganic phosphorus plates for purification, resulting in a pure strain.

[0078] In this embodiment of the application, the colony characteristics of Streoptomyces albidoflavus WP-Qing 1 are as follows.

[0079] The culture medium was incubated for 72 hours on NA medium plates (3g beef extract, 10g peptone, 5g NaCl, 15-20g agar, 1000mL pure water, pH 7.0-7.2, sterilized at 121℃ for 20min). The colonies were white, medium-sized, concentrically shaped, slightly darker in the center, with relatively neat and flat edges. The surface was dry and powdery, and easily scraped off. A light yellow pigment was produced on the back of the medium. The culture was carried out on Gao's No. 1 medium (soluble starch 20g, KNO3 0.1g, K2HPO4 0.05g, MgSO4·7H2O 0.05g, NaCl 0.05g, FeSO4·7H2O 0.001g, pure water 1000ml, pH 7.2-7.4, sterilized at 121℃ for 20min). The colony morphology was similar to that on NA medium, but the colonies were slightly larger, with concentric circles and radial bristles at the edges. A bluish-gray pigment was produced on the reverse side of the medium. The colony morphology of WP-Qing 1 on NA medium plates is as follows: Figure 2 As shown; the colony morphology of WP-Qing1 on Gao's No. 1 agar plates is as follows. Figure 3 As shown.

[0080] In the embodiments of this application, the culture characteristics of Streptomyces albidoflavus WP-Qing 1 in other culture media are shown in the table below.

[0081] Table 1. Culture characteristics of Streptomyces albidoflavus WP-Qing 1

[0082] Culture characteristics Aerial mycelium Intrabasal hyphae Soluble pigments Czapek's medium Douzhihuang Douzhihuang none Glucose-asparagine culture medium none Lychee White none Glycerol Asparagine Culture Medium White Douzhihuang none Inorganic salt starch culture medium Douzhihuang brown none ISP-2 culture medium Douzhihuang chestnut brown none Oat flour culture medium Douzhihuang Mustard none Gao's No. 1 culture medium White Light brown none Santal culture medium Douzhihuang dark brown none

[0083] In this embodiment, the spore filaments of *Streptomyces albidoflavus* WP-Qing 1 are straight, flexible, hook-shaped, or spiral-shaped; the spores are round or elliptical; their microscopic morphology is as follows: Figure 4 As shown.

[0084] In this embodiment of the application, some physiological and biochemical characteristics of Streptomyces albidoflavus WP-Qing 1 are shown in the table below.

[0085] Table 2. Partial physiological and biochemical characteristics of Streptomyces albidoflavus WP-Qing 1

[0086]

[0087] In this embodiment of the application, the salt and alkali resistance characteristics of Streoptomyces albidoflavus WP-Qing 1 are as follows.

[0088] Streptomyces albidoflavus WP-Qing 1 can grow normally on NA plates with a salt concentration (NaCl) of 5%, and the growth is as follows. Figure 5 As shown.

[0089] Streptomyces albidoflavus WP-Qing 1 can grow normally on NA plates containing alkali (NaOH) with a pH value of 7.0 ≤ pH ≤ 11.0. The growth pattern is as follows: Figure 6 The content shown in the green box is as follows.

[0090] Example 2: This example describes the application of Streptomyces albidoflavus WP-Qing 1, based on Streptomyces albidoflavus WP-Qing 1 from Example 1, for dissolving inorganic phosphorus.

[0091] When *Streptomyces albidoflavus* WP-Qing 1 was inoculated onto an inorganic phosphorus detection plate, a clear transparent zone was produced on the plate. Figure 7As shown. The ability of WP-Qing 1 to dissolve inorganic phosphorus was quantitatively determined, and the result showed that the ability of WP-Qing 1 to dissolve inorganic phosphorus was 220 mg / L.

[0092] In this embodiment, the formulation of the inorganic phosphorus detection plate culture medium is as follows: glucose 10.0g, NaCl 0.3g, (NH4)2SO4 0.5g, KCl 0.3g, MgSO4 4. 0.3g of 7H2O, 0.036g of FeSO4·7H2O, 0.03g of MnSO4·H2O, 2.0g of Ca3(PO4)2, 0.5g of yeast extract, 15-20g of agar, and distilled water to a final volume of 1000mL. The pH of the plate culture medium should be 6.8-7.2 to detect inorganic phosphorus.

[0093] In this embodiment, the method for quantitatively detecting the ability of WP-Qing 1 to dissolve inorganic phosphorus is the molybdenum antimony colorimetric method (NY / T 1847-2010 Appendix A), and the standard curve is: Y = 0.509X - 0.003r : 0.9996, where Y represents OD. 700 Value, where X represents the concentration of soluble phosphorus, mg / L, and the colorimetric results are as follows: Figure 8 As shown.

[0094] Example 3: This example describes the application of Streptomyces albidoflavus WP-Qing 1, based on Streptomyces albidoflavus WP-Qing 1 from Example 1, for the production of indoleacetic acid (IAA).

[0095] In this embodiment, the Salkowski colorimetric method is used for the quantitative determination of IAA. A standard curve is plotted: 200 μg / mL of analytical grade indoleacetic acid (IAA) is prepared. -1 The IAA standard solution was serially diluted to 0, 5, 10, 15, 20, 25, and 30 μg / mL. -1 For each concentration of IAA solution, take 2 ml of each solution and add 4 ml of Salkowski colorimetric reagent (10 ml of 0.5 mol / L FeCl3 and 500 ml of 35% perchloric acid, mixed before use and stored away from light). Shake well and react in the dark at 30°C for 30 min. Measure the colorimetric value of the developed reaction solution at a wavelength of 530 nm and plot a standard curve. The colorimetric results are as follows. Figure 9 As shown.

[0096] The standard curve obtained from the above process is y = 0.0318x + 0.0188, where y represents OD. 530 x represents the IAA concentration, in μg·mL -1 Correlation coefficient R 2 =0.9983.

[0097] The process for quantitatively determining the IAA production capacity of strain WP-Qing1 in this application embodiment is as follows: strain WP-Qing1 is inoculated into LB culture medium (LB culture medium: 10g tryptone, 5g yeast extract, 10g NaCl, 1000ml distilled water, pH 7.0), and incubated at 28℃ and 180r·min. -1 After shaking culture for 4 days, the supernatant was obtained by centrifugation at 10000 rpm for 10 min. 2 ml of the supernatant was taken, and 4 ml of Salkowski colorimetric reagent was added. The mixture was shaken well and incubated in the dark at 30℃ for 30 min. The colorimetric solution was then measured at 530 nm to obtain the OD value. The IAA content in the fermentation broth was calculated based on the IAA standard curve. The results showed that strain WP-Qing1 produced 8.833 μg·mL of IAA. -1 .

[0098] Example 4: This example describes the application of Streptomyces albidoflavus, based on Streptomyces albidoflavus WP-Qing 1 from Example 1, for the production of ferrophilic compounds.

[0099] In this embodiment of the application, WP-Qing1 is inoculated onto a CAS detection plate to form a distinct color-changing zone, demonstrating its ability to produce ferrophiles. The color-changing zone is as follows: Figure 10 As shown.

[0100] This application embodiment uses the color-changing zone method to qualitatively detect the ability of WP-Qing1 to produce ferophiles, including: inoculating the microbial bacteria activated by slant onto CAS detection medium plates, culturing at 28-30℃ for 3-5 days and then observing; those colonies that can produce a color-changing zone around them can produce ferophiles.

[0101] The preparation method for the CAS detection medium is as follows: Solution A (CAS staining solution) is prepared by dissolving 60.5 mg of Chromium Azurite S in 50 ml of deionized water, 10 ml of ferric iron solution (1 mmol / L FeCl3·6H2O, 10 mmol / L hydrochloric acid as solvent), and 72.9 mg of CTAB in 40 ml of deionized water. The above three solutions are mixed and brought to a final volume of 100 mL, then sterilized at 121°C for 20 min. Solution B is prepared by using 0.1 mol / L phosphate buffer solution with pH...

[0102] 6.8. Each 100mL contains 2.427g of Na₂HPO₄·12H₂O, 0.5905g of NaH₂PO₄·2H₂O, and 0.5905g of KH₂PO₄.

[0103] 0.075g of NH4Cl, 0.250g of NaCl, and 0.125g of sodium chloride were sterilized at 121°C for 20 minutes and diluted 10-fold before use. C medium preparation: Each 100ml of deionized water contains 1ml of 20% sucrose solution, 3ml of 10% acid-hydrolyzed casein, 100μL of 1mmol / L CaCl2, 2mL of 1mmol / L MgSO4, and 1.8g of agar. Sterilize at 121°C for 20 minutes. Before use, slowly add 5ml each of phosphate B solution and CAS staining solution to each 100ml of C medium at approximately 60°C, mix well, and the resulting CAS blue detection medium is obtained. All solutions were prepared with deionized water.

[0104] This application embodiment quantitatively determines the heparin-producing capacity of WP-Qing1, including: culturing WP-Qing1 in modified MSA medium for 96 hours; the siderophore concentration in the fermentation broth was SU = 58.10%, As / Ar = 0.4189, and the siderophore-producing capacity was rated as +++. Colorimetric results are as follows... Figure 11 As shown.

[0105] Specifically, the quantitative determination process described above is as follows: The bacteria to be tested are inoculated into modified MSA liquid medium and cultured at 28-30℃ on a shaker (150-180 r / min) for 3-5 days; the bacterial suspension is centrifuged at 8000 rpm for 15 min, and the supernatant is mixed with an equal volume (1:1) of CAS detection solution [60.5 mg Chromium Azurite S dissolved in 50 ml deionized water + 10 ml ferric solution (1 mmol / L FeCl3·6H2O, 10 mmol / L hydrochloric acid as solvent) + ...

[0106] 72.9 mg CTAB was dissolved in 40 ml of deionized water. The above three solutions were mixed and brought to a final volume of 100 mL, then sterilized at 121 °C for 20 min. After thorough mixing, the sample was allowed to stand at room temperature for 1 h. The absorbance (As) at 680 nm was measured. Double-distilled water was used as a control for zeroing. The absorbance (Ar) of the supernatant of the uninoculated modified MSA liquid medium was measured using the same method as a reference (Modified MSA liquid medium: 20.0 g / L sucrose, 2.0 g / L aspartic acid, 0.5 g / L K2HPO4, and MgSO4·7H2O, pH 7.2-7.5, sterilized at 121 °C for 20 min). The siderophore concentration was expressed as siderophore activity units (SU), SU = [(Ar-As) / Ar] × 100%, and each treatment was repeated 3 times. Bacterial siderophore production capacity is classified into As / Ar ratios ranging from 1.0 to 0, with each 0.2 increment adding a "+". Generally, bacteria with high siderophore production capacity (+++) have an As / Ar ratio below 0.5.

[0107] Example 5: This example describes the application of Streptomyces albidoflavus, based on Streptomyces albidoflavus WP-Qing 1 from Example 1, for the production of extracellular proteases.

[0108] In this embodiment of the application, the qualitative detection method for the protease activity of WP-Qing1 is as follows: microbial spots are inoculated onto a protease detection plate and cultured at 28-30℃ for 3-5 days. Colonies that produce a clear, transparent ring around their periphery are considered to be capable of producing protease. For example... Figure 12 As shown, WP-Qing1 produces a clear zone on the protease detection plate, proving that WP-Qing1 has the ability to produce extracellular proteases.

[0109] The formulation of the protease detection medium is as follows: A: 8g of skim milk powder dissolved in 300mL of water and sterilized at 115℃ for 10min; B: 8g of agar added to water to a final volume of 300mL and sterilized at 121℃ for 20min; A and B are sterilized separately and then mixed together.

[0110] Example 6: This example describes the application of Streptomyces albidoflavus WP-Qing 1, based on Streptomyces albidoflavus WP-Qing 1 from Example 1, for the production of cellulase.

[0111] In this embodiment of the application, the method for detecting cellulase activity of WP-Qing1 is as follows: Microbial inoculum is inoculated onto a cellulase activity assay plate and cultured at 28-30℃ for 3-5 days. Then, it is stained with 1 g / L Congo red for 1 hour, the stain is discarded, and the plate is then soaked in 1 mol / L NaCl for 1 hour, discarded, and observed. The presence of a clear zone indicates the production of cellulase. Figure 13 As shown, WP-Qing1 produces a clear zone on the cellulase detection plate, proving that WP-Qing1 has the ability to produce cellulase.

[0112] The formula for the cellulase activity assay medium is as follows: 10g peptone, 10g yeast extract, 10g sodium carboxymethyl cellulose, 5g sodium chloride, 1g potassium dihydrogen phosphate, and 18g agar, with a pH of 7.0.

[0113] Example 7: This example describes the application of Streptomyces albidoflavus, based on Streptomyces albidoflavus WP-Qing 1 from Example 1, for the production of β-1,3-glucanase.

[0114] In this embodiment of the application, the method for detecting the β-1,3-glucanase activity of WP-Qing1 is as follows: Microbial colonies are inoculated onto aniline blue dextran agar plates and cultured at 28-30℃ for 3-5 days. The color change around the colonies is observed; colonies that produce a colorless hydrolysis clear zone on the plate are capable of producing β-1,3-glucanase. Figure 14 As shown, WP-Qing1 produces a clear zone on the dextranase detection plate, proving that WP-Qing1 has the ability to produce dextranase.

[0115] The culture medium for detecting β-1,3-glucanase activity was formulated as follows: 1.0g glucose, 4.0g glucan, 1.0g K2HPO4, 3.0g Na2HPO4, 0.06g aniline blue, 0.5g FeSO4·7H2O, 12g agar powder, and 1000mL distilled water, with a pH of 6.5.

[0116] Example 8: This example describes the application of Streptomyces albidoflavus, based on Streptomyces albidoflavus WP-Qing 1 from Example 1, for antagonizing plant pathogens.

[0117] In this embodiment, the plate confrontation method was used to determine that WP-Qing 1 has antagonistic effects against multiple plant pathogens and exhibits a broad antibacterial spectrum. This indicates that the fermentation broth of WP-Qing 1 can significantly inhibit the growth of twelve plant pathogens. Specific results are as follows: Figures 15-26 As shown. Among them, Figure 15 The effect of WP-Qing 1 antagonizing the growth of Botryosphaeria sp. on PDA plates is shown. Figure 16 The effect of WP-Qing 1 on the antagonism of Botrytis cinerea growth on PDA plates is shown. Figure 17 The experiment demonstrates the antagonistic effect of WP-Qing 1 on the growth of Alternaria sp. on PDA plates. Figure 18 The experiment demonstrates the antagonistic effect of WP-Qing 1 on the growth of Fusarium oxysporum on PDA plates. Figure 19 The effect of WP-Qing 1 antagonizing the growth of Valsa sp. (black rot fungus) on PDA plates is shown. Figure 20 The results show the antagonistic effect of WP-Qing 1 on the growth of Fusarium oxysporum f.sp.niveum (watermelon-specific Fusarium oxysporum) on PDA plates; Figure 21 This demonstrates the antagonistic effect of WP-Qing 1 on the growth of Pseudomonas syringae pv. actinidiae (a kiwi fruit variant of Pseudomonas syringae) on NA plates. Figure 22The experiment demonstrates the antagonistic effect of WP-Qing 1 on the growth of Rhizoctonia cerealis Vander Hoeven on PDA plates. Figure 23 The experiment demonstrates the antagonistic effect of WP-Qing 1 on the growth of Fuasarum graminearum on PDA plates. Figure 24 This demonstrates the antagonistic effect of WP-Qing 1 on the growth of Fusarium proliferatum on PDA plates; Figure 25 The experiment demonstrates the antagonistic effect of WP-Qing 1 on the growth of Fusarium verticillioides on PDA plates. Figure 26 The growth of WP-Qing 1 antagonizing Fusarium solani on PDA plates is shown; Figures 15-26 It can be seen that WP-Qing1 fermentation liquid can significantly inhibit the growth of the above twelve plant pathogens, thereby achieving the effect of preventing and controlling a variety of plant diseases.

[0118] The twelve plant pathogens are listed in Table 3 below.

[0119] Table 3. Plant Pathogens and Pathogenic Types

[0120]

[0121] In this embodiment of the application, the flat plate confrontation method includes:

[0122] (1) When the plant pathogen is a fungus, the test strain is inoculated into NB culture medium and cultured at 28-30℃ and 180rpm for 48h to obtain antagonistic bacterial solution; the test pathogen is inoculated into PDA plates and cultured at 28-30℃ for 3-5 days. After the hyphae cover the plate, the fungal discs are punched into 5mm diameter discs using a sterile punch; a sterile PDA plate is taken, the pathogenic fungal discs are placed in the center of the PDA plate, and 4mm diameter sterile filter paper is used to pick up the antagonistic bacterial solution and evenly place it at 4 points 2.5cm away from the center of the plate; the plates inoculated with pathogen and antagonistic bacteria are placed in a constant temperature incubator and cultured at 28-30℃ for 3-5 days to observe whether an inhibition band is produced. Whether an inhibition band is produced and the width of the inhibition band can qualitatively evaluate the ability of the strain to antagonize the pathogen.

[0123] (2) When the plant pathogen is bacteria, the antagonistic strain to be tested and the pathogenic bacteria are inoculated separately into NB culture medium and cultured on a shaker at 180 rpm for 24-48 h at 28-35℃ to obtain antagonistic bacterial solution and pathogenic bacterial solution respectively. Take a sterile NA plate, and under aseptic conditions, add 0.2 ml of pathogenic bacterial solution to each plate and spread it evenly with a sterile spatula. Place sterile Oxford cups evenly into the plates with pathogenic bacterial solution and press them firmly with sterile forceps. Inject 0.2 ml of antagonistic bacterial solution into the Oxford cups. Place the plates containing antagonistic bacteria and pathogenic bacteria into a constant temperature incubator at 28-35℃ for 24-72 h and observe whether inhibition zones are formed. The formation of inhibition zones, especially large ones, indicates that the strain has a strong antagonistic ability against the pathogenic bacteria.

[0124] Experimental Example 1: This experimental example records the effect of WP-Qing 1 on dissolving inorganic phosphorus to promote wheat seed germination and seedling growth. The specific details are as follows.

[0125] (1) Experimental objective: To verify that WP-Qing1 has the ability to dissolve inorganic phosphorus and can promote wheat seed germination and seedling growth.

[0126] (2) The experimental design scheme is shown in the table below.

[0127] Table 4 Experimental Design Table for Experiment Example 1

[0128]

[0129] (3) Experimental steps:

[0130] After activating strain WP-Qing1 with Gao's No. 1 slant, it was inoculated with inorganic phosphorus culture medium (10.0g glucose, 0.5g (NH4)2SO4, 0.3g NaCl, 0.3g KCl, 0.3g MgSO4·7H2O, 0.036g FeSO4·7H2O, 0.03g MnSO4·H2O, 2.0g Ca3(PO4)2, 0.5g yeast extract, and distilled water to a final volume of 1000mL, pH 6.8-7.2). The culture was incubated at 30℃ and 180rpm for 5 days to obtain WP-Qing1 inorganic phosphorus culture medium (number T4). During the experiment, the WP-Qing1 inorganic phosphorus culture medium was diluted 5 times and 10 times with sterile pure water to obtain 5-fold dilution (number T5) and 10-fold dilution (number T6). T1 is a sterile inorganic phosphorus culture medium without inoculation. T2 and T3 are 5 times and 10 times its sterile water dilution, respectively. T1 is also shaken at 30°C and 180 rpm for 5 days.

[0131] After disinfecting wheat seeds with 75% alcohol for 30 seconds, soak them in sterile pure water for 8 hours. After removing the soaked seeds and draining them slightly, place them in a dish, cover them with a damp towel, and germinate them at room temperature for 12 hours until the seeds show white sprouts.

[0132] Take a 9cm diameter glass petri dish and line the bottom with a layer of sterile filter paper. Add 5ml of each treatment solution (CK, T1-T6) from the experimental design table to each petri dish and moisten the filter paper evenly. Make sure the filter paper is in close contact with the bottom of the petri dish and there are no air bubbles between the filter paper and the bottom of the petri dish. Place 25 wheat seeds with the white part showing evenly in each petri dish, with the groin facing down. Each treatment has 4 replicates.

[0133] During the seed germination stage, dark culture was used, with petri dishes covered with a damp towel and placed at 25-30℃. After 48 hours of culture, the germinated wheat petri dishes were transferred to an artificial climate chamber, where the following culture parameters were set: day / night duration: 12h / 12h, day / night temperature: 25℃ / 22℃, humidity: 60%, day / night light intensity: 1000LX / 000LX, and culture continued for another 72 hours. During this period, observations were made daily, and 5ml of sterile pure water was added to each petri dish to ensure that the filter paper inside the dish did not dry out. At the end of the experiment, the plant height, root length, root number, and biomass of wheat seedlings were measured. Twenty-five seedlings were measured for each treatment, and the data were averaged.

[0134] (4) Experimental results:

[0135] WP-Qing 1 can dissolve inorganic phosphorus and effectively promote the growth of wheat seedlings. For example... Figure 32 As shown, the growth-promoting effect of WP-Qing 1 inorganic phosphorus culture solution diluted 10 times was the most obvious.

[0136] like Figure 27 , Figure 28 , Figure 29 , Figure 30 and Figure 31 As shown, compared with the water control, the 10-fold dilution of WP-Qing 1 inorganic phosphorus culture solution increased the plant height of wheat by 14.56%, root length by 32.84%, root number by 39.82%, fresh weight by 28.22%, and dry weight by 9.20%.

[0137] Compared with a 10-fold dilution of uninoculated inorganic phosphorus culture medium, a 10-fold dilution of WP-Qing 1 inorganic phosphorus culture medium increased wheat plant height by 8.55%, root length by 10.97%, root number by 26.60%, fresh weight by 5.23%, and dry weight by 4.77%.

[0138] Experiment Example 2: This experiment describes the effect of WP-Qing 1 producing indoleacetic acid (IAA) on promoting wheat seed germination and seedling growth. The specific details are as follows.

[0139] (1) Experimental objective: To verify that WP-Qing1 produces IAA, which can promote wheat seed germination and seedling growth.

[0140] (2) The experimental design scheme is shown in Table 5 below.

[0141] Table 5 Experimental Design Table for Experiment Example 2

[0142]

[0143] (3) Experimental steps:

[0144] After activating strain WP-Qing1 with Gao's No. 1 slant, it was inoculated into peptone aqueous culture medium (10g tryptone, 5g NaCl, 1000ml distilled water, pH 7.6) and cultured at 30℃ and 180rpm for 5 days to obtain WP-Qing1 peptone aqueous culture medium (numbered T10). During the experiment, the WP-Qing1 peptone aqueous culture medium was diluted 5 times and 10 times with sterile pure water to obtain 5-fold dilution (numbered T11) and 10-fold dilution (numbered T12) of uncultured WP-Qing1 peptone culture medium. T7 was sterile peptone aqueous culture medium without inoculation, and T8 and T9 were 5-fold and 10-fold sterile water dilutions, respectively. T7 was also shaken at 30℃ and 180rpm for 5 days.

[0145] After disinfecting wheat seeds with 75% alcohol for 30 seconds, soak them in sterile pure water for 8 hours. After removing the soaked seeds and draining them slightly, place them in a dish, cover them with a damp towel, and germinate them at room temperature for 12 hours until the seeds show white sprouts.

[0146] Take a 9cm diameter glass petri dish and line the bottom with a layer of sterile filter paper. Add 5ml of each treatment solution (CK, T7-T11) from the experimental design table to each petri dish and moisten the filter paper evenly. Make sure the filter paper is in close contact with the bottom of the petri dish and there are no air bubbles between the filter paper and the bottom of the petri dish. Place 25 wheat seeds with the white part showing evenly in each petri dish, with the groin facing down. Each treatment has 4 replicates.

[0147] During the seed germination stage, dark culture was used, with petri dishes covered with a damp towel and placed at 25-30℃. After 48 hours of culture, the germinated wheat petri dishes were transferred to an artificial climate chamber, where the following culture parameters were set: day / night duration: 12h / 12h, day / night temperature: 25℃ / 22℃, humidity: 60%, day / night light intensity: 1000LX / 000LX, and culture continued for another 72 hours. During this period, observations were made daily, and 5ml of sterile pure water was added to each petri dish to ensure that the filter paper inside the dish did not dry out. At the end of the experiment, the plant height, root length, root number, and biomass of wheat seedlings were measured. Twenty-five seedlings were measured for each treatment, and the data were averaged.

[0148] (4) Experimental results:

[0149] WP-Qing1 can produce IAA, and its fermentation liquid can effectively promote the growth of wheat seedlings.

[0150] like Figure 38 As shown, the growth-promoting effect was most obvious with a 10-fold dilution of WP-Qing1 peptone aqueous culture solution.

[0151] As Figure 33 、 Figure 34 、 Figure 35 、 Figure 36 and Figure 37 shown, compared with the water control, the 10-fold diluted solution of WP-Qing 1 peptone culture broth increased the plant height of wheat by 7.34%, the root length by 9.86%, the number of roots by 5.35%, the fresh weight by 25.19%, and the dry weight by 10.17%.

[0152] Compared with the 10-fold diluted solution of peptone water culture broth without inoculation, the 10-fold diluted solution of WP-Qing 1 peptone water culture broth increased the plant height of wheat by 5.94%, the root length by 25.61%, the number of roots by 5.08%, the fresh weight by 8.68%, and the dry weight by 7.75%.

[0153] Test Example 3: This test example records the indoor control efficacy test of WP-Qing 1 against corn stalk rot. The specific content is as follows.

[0154] Corn seeds: Zhengdan 958. Soak the corn seeds in 1% sodium hypochlorite for 10 min, wash them 3 times with sterilized pure water, and germinate them in a constant temperature incubator at 25°C until the seeds show white buds for standby.

[0155] Tested biocontrol agents: Inoculate the activated WP-Qing 1 strain into NB culture broth, culture it on a shaker at 30°C and 180 rpm for 48 h to obtain the original solution of the antagonistic agent, numbered WP-Qing 1×1, with a spore concentration of 3.2×10 9 cfu / ml; During the test, dilute the original solution of the antagonistic agent with sterilized pure water by 5 times and 10 times respectively to obtain its 5-fold diluted solution and 10-fold diluted solution, numbered WP-Qing 1×5 and WP-Qing 1×10 respectively.

[0156] Tested pathogenic bacteria: Fusarium proliferatum, which can cause corn stalk rot. Inoculate the tested pathogenic bacteria into a 9-cm diameter PDA plate and culture it at 28 - 30°C for 3 - 5 d until the mycelium covers the plate for standby.

[0157] The culture nutrient solution uses 1 / 4 Hoagland nutrient solution.

[0158] (2) Test methods and steps

[0159] The test is set with a control (CK - soak corn seeds with sterile pure water), treatment 1 (T1 - soak corn seeds with WP-Qing 1×1 agent), treatment 2 (T2 - soak corn seeds with WP-Qing 1×5 agent), and treatment 3 (T3 - soak corn seeds with WP-Qing 1×10 agent).

[0160] Emerging corn seeds were soaked for 3 hours in the corresponding treatment solutions (CK, T1, T2, and T3), and then sown on plates containing established pathogens, with 15 seeds per plate and two replicates per treatment. The plates were placed in a climate chamber and initially incubated in the dark at 25°C. After the first leaf appeared, the climate chamber parameters were set as follows: day / night duration: 12h / 12h; day / night temperature: 25°C / 22°C; humidity: 60%; day / night light intensity: 1000 LX / 000 LX, for a total of 25 days. On day 6, treatments T1, T2, and T3 were supplemented with their respective biocontrol solutions (5 ml per plate), while CK was supplemented with an equal volume of sterile water. On days 8 and 16, each plate was supplemented with 5 ml of 1 / 4 Hoagland nutrient solution. For the remaining days, each plate was supplemented with the same amount of sterile water daily to keep the culture medium moist.

[0161] Investigation: On the third day of cultivation, when the sprouts reached 1cm in length, the germination rate of the corn was investigated; after 25 days of cultivation, the height of the corn plants and the disease status were investigated.

[0162] The formula for calculating the protective effect is as follows:

[0163] Incidence rate % = (Number of infected plants / Total number of plants surveyed) × 100%;

[0164] Prevention and control effect = {(control incidence rate - treatment incidence rate) / control incidence rate} × 100%;

[0165] (3) The test results are shown in Table 6.

[0166] Table 6 shows the indoor control efficacy of WP-Qing 1 fermentation broth against corn stalk base rot.

[0167]

[0168]

[0169] Experimental conclusion: WP-Qing1 fermentation liquid can effectively prevent corn stalk rot. The control effect and corn plant height indicate that different concentrations of WP-Qing1 fermentation liquid have varying efficacy against corn stalk rot. Corn plant height and disease status are as follows... Figure 39 As shown, the 5-fold dilution of WP-Qing 1 fermentation broth showed the best control efficacy, reaching 50.6%, with an average corn plant height of 21.94 cm, which was 95.2% higher than the control group (CK). The 10-fold dilution was the second most effective, with a control efficacy of 40.9%. The average corn plant height reached 18.07 cm, which was 60.8% higher than the control group (CK).

Claims

1. A type of Streptomyces microcytogenes ( Streoptomyces albidoflavus WP - Green 1, characterized in that: The Streptomyces microcyticum was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 28, 2023, with accession number CGMCC No. 27730.

2. The *Streptomyces whitlowensis* WP-Green 1 as described in claim 1, characterized in that, The 16S rRNA gene sequence of *Streptomyces microcyticus* WP-Qing 1 is shown in SEQ ID NO:

1.

3. The *Streptomyces whitworthii* WP-Green 1 as described in claim 1, characterized in that, The rpoB gene of *Streptomyces microcyticus* WP-Qing 1 is shown in SEQ ID NO:

2.

4. An application based on *Streptomyces microcyticum* WP-Qing 1 according to any one of claims 1 to 3, characterized in that, Used to dissolve inorganic phosphorus.

5. An application of *Streptomyces microcyticum* WP-Green 1 according to any one of claims 1 to 3, characterized in that, Used to produce indoleacetic acid.

6. An application based on *Streptomyces microcyticum* WP-Green 1 according to any one of claims 1 to 3, characterized in that, Used to produce ferrophosphate.

7. An application based on *Streptomyces microcyticum* WP-Qing 1 according to any one of claims 1 to 3, characterized in that, Used to produce extracellular proteases.

8. An application based on *Streptomyces microcyticum* WP-Green 1 according to any one of claims 1 to 3, characterized in that, Used to produce cellulase.

9. An application of *Streptomyces microcyticum* WP-Qing 1 according to any one of claims 1 to 3, characterized in that, Used to produce β-1,3-glucanase.

10. An application of *Streptomyces microcyticum* WP-Qing 1 according to any one of claims 1 to 3, characterized in that, It is used to antagonize *Pyrocystis pyriformis*, *Botrytis cinerea*, *Alternaria alternata*, *Fusarium oxysporum*, *Pseudomonas syringae* (a variant of *Actinidia kiwifruit*), *Rhizoctonia graminearum*, *Fusarium graminearum*, *Fusarium laminarum*, *Fusarium verticillatum*, and / or *Fusarium solani*.

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