Streptomyces, fermentation broth, and use of both

Abstract

Provided are Streptomyces and a use thereof. The Streptomyces is named Streptomyces linyiensis, which was preserved in China General Microbiological Culture Collection Center (CGMCC) on 28 September 2022, and has a preservation number of CGMCC No. 25834. A Streptomyces fermentation broth can be used to significantly improve the abundance of nitrogen-fixing microorganisms in crop rhizosphere soil, improve the nitrogen-fixing enzyme activity of the crop rhizosphere soil, improve the nitrogen-fixing capability of the nitrogen-fixing microorganisms in the crop rhizosphere soil, and promote the absorption of nitrogen nutrients by crops, and can be used for preparing bacterial agents, microbial fertilizers or biopesticides having corresponding functions.

Description

Streptomyces, fermentation liquid and application thereof

[0001] Priority information

[0002] This disclosure claims priority to and the benefits of patent application 202311211459.1 filed on September 19, 2023 with the State Intellectual Property Office of China, and incorporates the entire text of which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of microbial technology. Specifically, the present disclosure relates to a Streptomyces strain that can increase the abundance of nitrogen-fixing microorganisms in crop rhizosphere soil, increase the nitrogenase activity in crop rhizosphere soil, improve the nitrogen-fixing ability of nitrogen-fixing microorganisms in crop rhizosphere soil, and promote the absorption of nitrogen nutrients by crops, and its application. Background Art

[0004] Soil microorganisms are a general term for bacteria, fungi, actinomycetes, and algae that live in the soil. They are tiny, usually measured in micrometers or nanometers. Usually, there are 10 6 ~10 9 Their types and numbers vary with the soil-forming environment and soil depth. They carry out oxidation, nitrification, ammonification, nitrogen fixation, sulfidation and other processes in the soil, promoting the decomposition of soil organic matter and the transformation of nutrients.

[0005] Biological nitrogen fixation is one of the most important ecological processes on Earth. In farmland ecosystems, approximately 24% of the nitrogen in total crop biomass comes from non-symbiotic nitrogen fixation by microorganisms. The rhizosphere is the most microbially active zone in farmland soil, and the nitrogen-fixing microbial community in the rhizosphere is closely related to crop growth. However, the long-term application of large amounts of fertilizers and organic matter has significantly reduced the nitrogen fixation activity of farmland soil microorganisms. In recent years, research on soil nitrogen-fixing microorganisms has primarily focused on the nitrogen-fixing bacterial community and the factors that influence it. However, little research has been conducted on the mechanisms that regulate nitrogen fixation by rhizosphere microorganisms.

[0006] Nitrogen-fixing microorganisms refer to microorganisms that can convert free nitrogen in the air into nitrogen-containing compounds. They are divided into autotrophic nitrogen-fixing bacteria and symbiotic nitrogen-fixing bacteria. Autotrophic nitrogen-fixing bacteria generally refer to strains that do not coexist with plants and have nitrogen-fixing functions, including Actinomycetes, Streptomyces, Micromonas, and Luteomonas; symbiotic nitrogen-fixing bacteria mainly include Rhizobium, Mesorhizobium, Sinorhizobium, and Frankia.

[0007] Nitrogen-fixing microorganisms play a vital role in crop growth, particularly in legumes, providing a significant amount of nitrogen for crop growth. Improving the abundance and nitrogen-fixing capacity of nitrogen-fixing bacteria in rhizosphere soils, thereby increasing crop yields and reducing fertilizer use, holds great promise for development.

[0008] Summary of the Invention

[0009] The present disclosure aims to solve one of the technical problems in the related art to at least a certain extent. To this end, one purpose of the present disclosure is to provide a Streptomyces strain, whose function can increase the abundance of microorganisms with nitrogen-fixing function in the rhizosphere soil of crops and increase the nitrogenase activity in the rhizosphere soil of crops. The metabolites of the Streptomyces strain can be used to increase the abundance of nitrogen-fixing bacteria such as Actinomycetes, Streptomyces, Rhizobium, Mesorhizobium, Luteomonas, and Micromonas in the soil, increase nitrogenase activity, increase the abundance of indigenous nitrogen-fixing microorganisms, increase the absorption of nitrogen nutrients by crops, improve the soil microecological environment, and have the possibility of being developed into soil functional fertilizers or pesticides.

[0010] To this end, the present disclosure provides a microorganism on the one hand, which is Streptomyces linyiensis, which was deposited in the General Microbiology Center of the China Culture Collection Administration on September 28, 2022, with the deposit number CGMCC No. 25834, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] The Streptomyces provided by the present invention is a pure strain obtained from the soil of vegetable fields in Shandong Province by using the soil continuous gradient dilution method combined with the plate streak separation method. Through tomato field trials, cucumber field trials, and cabbage field trials, it has been verified that it can effectively increase the abundance of nitrogen-fixing microorganisms in the root soil of crops, for example, it can increase the abundance of nitrogen-fixing microorganisms such as Actinomyces, Streptomyces, Rhizobium, Mesorhizobium, and Micromonas. It is used in the preparation of microbial agents, composite agents, microbial fertilizers or biological pesticides, which can increase the nitrogenase activity of rhizosphere soil, improve the soil microecological environment, promote crop growth, and increase crop yield.

[0012] According to an embodiment of the present disclosure, the microorganism has a 16S rDNA sequence as shown in SEQ ID NO: 1.

[0013] A second aspect of the present disclosure provides a fermentation broth, which is fermented by the microorganism described in the first aspect.

[0014] A third aspect of the present disclosure provides a microbial agent, which includes the microorganism described in the first aspect of the present disclosure or the fermentation broth described in the second aspect.

[0015] According to an embodiment of the present disclosure, the microbial agent is in the form of dry powder, and the effective viable bacteria count of Streptomyces linyiensis per gram of the microbial agent is at least 2×10 9 CFU.

[0016] According to an embodiment of the present disclosure, the microbial agent is obtained by the following steps:

[0017] Fermenting and culturing the Streptomyces linyiensis to obtain a fermentation product;

[0018] The fermentation product is spray-dried and pulverized to obtain the microbial agent.

[0019] A fourth aspect of the present disclosure provides a composite bacterial agent, which includes the microbial agent described in the third aspect.

[0020] According to an embodiment of the present disclosure, the composite microbial agent includes a first microbial agent and a second microbial agent, the first microbial agent is the microbial agent described in the third aspect, and the second microbial agent includes at least one selected from Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus gelatinosa, Streptomyces freundii, and Streptomyces tenuifolius.

[0021] According to an embodiment of the present disclosure, the composite microbial agent includes 10-50 parts by weight of the first microbial agent and 5-20 parts by weight of the second microbial agent;

[0022] The second microbial agent comprises 0-5 parts by weight of Streptomyces tenuis, 0-10 parts by weight of Bacillus gelatinosa, 0-5 parts by weight of Bacillus subtilis, 0-5 parts by weight of Bacillus amyloliquefaciens, and 0-5 parts by weight of Streptomyces freundii.

[0023] According to the embodiment of the present disclosure, the effective viable bacteria count of Streptomyces linyiensis per gram of the composite bacterial agent is at least 2×10 9 CFU, and at least one selected from the following:

[0024] The effective viable count of Streptomyces tenuifolius is at least 5×10 8 CFU;

[0025] The effective viable count of Bacillus jelly is at least 5×10 8 CFU;

[0026] The effective viable cell count of Bacillus subtilis is at least 5×10 8 CFU;

[0027] The effective viable count of Bacillus amyloliquefaciens is at least 5×10 8 CFU;

[0028] The effective viable cell count of Streptomyces flexneri is at least 5×10 8 CFU.

[0029] The fifth aspect of the present disclosure provides the use of the microorganisms described in the first aspect, the fermentation liquid described in the second aspect, the microbial agent described in the third aspect, or the composite agent described in the fourth aspect in the preparation of microbial fertilizers or biopesticides. The Streptomyces and microbial agents and composite agents containing the same provided by the present disclosure can increase the abundance of nitrogen-fixing microorganisms in the rhizosphere soil of crops, increase the nitrogenase activity in the rhizosphere soil of crops, and use them in the preparation of microbial fertilizers and / or biopesticides to improve the soil microecological environment, promote crop growth, and increase crop yields.

[0030] A sixth aspect of the present disclosure provides a microbial fertilizer, which includes the Streptomyces described in the first aspect, the fermentation broth described in the second aspect, the microbial agent described in the third aspect, or the composite agent described in the fourth aspect.

[0031] According to an embodiment of the present disclosure, the microbial fertilizer is solid fertilizer or liquid fertilizer.

[0032] According to an embodiment of the present disclosure, the microbial fertilizer is a solid fertilizer, and the number of effective living microorganisms contained in each gram of the microbial fertilizer is at least 2×10 9 CFU.

[0033] According to an embodiment of the present disclosure, the microbial fertilizer is a liquid fertilizer, and the number of effective living microorganisms contained in each liter of the microbial fertilizer is at least 2×10 9 CFU.

[0034] According to an embodiment of the present disclosure, the microbial fertilizer further includes a basic fertilizer, and the basic fertilizer includes at least one selected from compound fertilizers, water-soluble fertilizers, organic fertilizers, and foliar fertilizers.

[0035] A seventh aspect of the present disclosure provides a biological pesticide, comprising the Streptomyces described in the first aspect, the fermentation broth described in the second aspect, the microbial agent described in the third aspect, or the composite agent described in the fourth aspect.

[0036] According to the embodiment of the present disclosure, the number of effective live microorganisms contained in each liter of the biopesticide is at least 2×10 9 CFU.

[0037] In an eighth aspect, the present disclosure provides the use of the microorganism described in the first aspect, the fermentation liquid described in the second aspect, the microbial agent described in the third aspect, the composite agent described in the fourth aspect, the microbial fertilizer described in the sixth aspect, or the biological pesticide described in the seventh aspect in increasing the abundance of nitrogen-fixing microorganisms in the rhizosphere soil of crops, or increasing the nitrogenase activity in the rhizosphere soil of crops.

[0038] According to an embodiment of the present disclosure, the nitrogen-fixing microorganism includes at least one of the genera Actinomyces, Streptomyces, Rhizobium, Mesorhizobium, Micromonas, and Xanthomonas.

[0039] A ninth aspect of the present disclosure provides a method for increasing the abundance of nitrogen-fixing microorganisms in crop rhizosphere soil, comprising:

[0040] The microorganisms described in the first aspect, the fermentation liquid described in the second aspect, the microbial agent described in the third aspect, the composite agent described in the fourth aspect, the microbial fertilizer described in the sixth aspect, or the biological pesticide described in the seventh aspect are applied to crops.

[0041] According to an embodiment of the present disclosure, the crop includes at least one selected from corn, wheat, soybean, rice, potato, sorghum, cucumber, pepper, tomato, peanut, eggplant, and cotton.

[0042] A tenth aspect of the present disclosure provides a method for increasing nitrogenase activity in crop rhizosphere soil, comprising:

[0043] The microorganisms described in the first aspect, the fermentation liquid described in the second aspect, the microbial agent described in the third aspect, the composite agent described in the fourth aspect, the microbial fertilizer described in the sixth aspect, or the biological pesticide described in the seventh aspect are applied to crops.

[0044] According to an embodiment of the present disclosure, the crop includes at least one selected from corn, wheat, soybean, rice, potato, sorghum, cucumber, pepper, tomato, peanut, eggplant, and cotton.

[0045] The beneficial effects of the present disclosure over the prior art are as follows:

[0046] The Streptomyces provided herein has advantages such as stable properties, simple cultivation, significant effects, wide applicability, and no environmental pollution. Its use in the preparation of microbial agents, composite agents, microbial fertilizers, or biopesticides can increase the abundance of nitrogen-fixing microorganisms in crop rhizosphere soil, enhance nitrogenase activity in crop rhizosphere soil, improve the soil microecological environment, promote crop growth, and increase crop yields. It is particularly effective in crops such as tomatoes, cucumbers, and cabbage, thus facilitating crop cultivation and agricultural production, and possesses excellent application value.

[0047] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure.

[0048] Collection information:

[0049] Strain name: Streptomyces linyiensis

[0050] Date of deposit: September 28, 2022

[0051] Depository: General Microbiology Center of China Culture Collection Administration

[0052] Deposit number: CGMCC No.25834 BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0054] Figure 1 shows a photo of a colony of Streptomyces linyiense on a culture medium;

[0055] FIG2 shows a schematic diagram of the phylogenetic tree of Streptomyces linyiensis.

[0056] Detailed Description of the Invention

[0057] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0058] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. Throughout the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0059] According to an embodiment of the present disclosure, the first aspect of the present disclosure provides a microorganism, which is Streptomyces linyiensis, which was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on September 28, 2022, with a deposit number of CGMCC No. 25834. The strain was screened from the soil of vegetable fields in Shandong Province. After colony morphology observation, cell Gram staining, physiological and biochemical characteristics detection and 16S rDNA gene analysis, the strain was identified as a new species. According to the international naming rules, the strain was named Streptomyces linyiensis. The Streptomyces provided by the present disclosure can effectively increase the abundance of nitrogen-fixing microorganisms in the root soil of crops, for example, it can increase the abundance of actinomycetes, Streptomyces, Rhizobium, Mesorhizobium, Luteomonas, and Micromonas. Using it in the preparation of microbial agents, compound agents, microbial fertilizers or biological pesticides can increase the abundance of nitrogen-fixing microorganisms in the rhizosphere soil of crops, increase the activity of nitrogenase in the rhizosphere soil of crops, improve the soil microecological environment, promote crop growth and increase crop yields.

[0060] According to an embodiment of the present disclosure, the microorganism has a 16S rDNA sequence as shown in SEQ ID NO: 1.

[0061] According to an embodiment of the present disclosure, a second aspect of the present disclosure provides a fermentation broth, which is fermented by the microorganism described in the first aspect.

[0062] According to an embodiment of the present disclosure, a third aspect of the present disclosure provides a microbial agent, which includes the microorganism described in the first aspect of the present disclosure or the fermentation broth described in the second aspect.

[0063] According to an embodiment of the present disclosure, the microbial agent is in the form of dry powder, and the effective viable bacteria count of Streptomyces linyiensis per gram of the microbial agent is at least 2×10 9 CFU.

[0064] According to an embodiment of the present disclosure, the microbial agent is obtained by the following steps:

[0065] Fermenting and culturing the Streptomyces linyiensis to obtain a fermentation product;

[0066] The fermentation product is spray-dried and pulverized to obtain the microbial agent.

[0067] According to an embodiment of the present disclosure, a fourth aspect of the present disclosure provides a composite bacterial agent, which includes the microbial agent described in the third aspect.

[0068] According to an embodiment of the present disclosure, the composite microbial agent includes a first microbial agent and a second microbial agent, the first microbial agent is the microbial agent described in the third aspect, and the second microbial agent includes at least one selected from Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus gelatinosa, Streptomyces freundii, and Streptomyces tenuifolius.

[0069] According to an embodiment of the present disclosure, the composite microbial agent includes 10-50 parts by weight of the first microbial agent and 5-20 parts by weight of the second microbial agent;

[0070] The second microbial agent comprises 0-5 parts by weight of Streptomyces tenuis, 0-10 parts by weight of Bacillus gelatinosa, 0-5 parts by weight of Bacillus subtilis, 0-5 parts by weight of Bacillus amyloliquefaciens, and 0-5 parts by weight of Streptomyces freundii.

[0071] According to the embodiment of the present disclosure, the effective viable bacteria count of Streptomyces linyiensis per gram of the composite bacterial agent is at least 2×10 9 CFU, and at least one selected from the following:

[0072] The effective viable count of Streptomyces tenuifolius is at least 5×10 8 CFU;

[0073] The effective viable count of jelly-like spore bacilli is at least 5×10 8 CFU;

[0074] The effective viable cell count of Bacillus subtilis is at least 5×10 8 CFU;

[0075] The effective viable count of Bacillus amyloliquefaciens is at least 5×10 8 CFU;

[0076] The effective viable cell count of Streptomyces flexneri is at least 5×10 8 CFU.

[0077] According to an embodiment of the present disclosure, the fifth aspect of the present disclosure provides the use of the microorganism described in the first aspect, the fermentation broth described in the second aspect, the microbial agent described in the third aspect, or the composite agent described in the fourth aspect in the preparation of microbial fertilizers or biological pesticides.

[0078] According to an embodiment of the present disclosure, the sixth aspect of the present disclosure provides a microbial fertilizer, which includes the microorganism described in the first aspect, the fermentation liquid described in the second aspect, the microbial agent described in the third aspect, or the composite agent described in the fourth aspect.

[0079] According to an embodiment of the present disclosure, the microbial fertilizer is solid fertilizer or liquid fertilizer.

[0080] According to an embodiment of the present disclosure, the microbial fertilizer is a solid fertilizer, and the number of effective living microorganisms contained in each gram of the microbial fertilizer is at least 2×10 9 CFU.

[0081] According to an embodiment of the present disclosure, the microbial fertilizer is a liquid fertilizer, and the number of effective living microorganisms contained in each liter of the microbial fertilizer is at least 2×10 9 CFU.

[0082] According to an embodiment of the present disclosure, the microbial fertilizer further includes a basic fertilizer, and the basic fertilizer includes at least one selected from compound fertilizers, water-soluble fertilizers, organic fertilizers, and foliar fertilizers.

[0083] According to an embodiment of the present disclosure, the seventh aspect of the present disclosure provides a biological pesticide, comprising the Streptomyces described in the first aspect, the fermentation broth described in the second aspect, the microbial agent described in the third aspect, or the composite agent described in the fourth aspect.

[0084] According to the embodiment of the present disclosure, the number of effective live microorganisms contained in each liter of the biopesticide is at least 2×109 CFU.

[0085] According to an embodiment of the present disclosure, the eighth aspect of the present disclosure provides the use of the microorganism described in the first aspect, the fermentation liquid described in the second aspect, the microbial agent described in the third aspect, the composite agent described in the fourth aspect, the microbial fertilizer described in the sixth aspect, or the biological pesticide described in the seventh aspect in increasing the abundance of nitrogen-fixing microorganisms in the rhizosphere soil of crops, or increasing the nitrogenase activity in the rhizosphere soil of crops.

[0086] According to an embodiment of the present disclosure, the nitrogen-fixing microorganism includes at least one of the genera Actinomyces, Streptomyces, Rhizobium, Mesorhizobium, Micromonas, and Xanthomonas.

[0087] According to an embodiment of the present disclosure, a ninth aspect of the present disclosure provides a method for increasing the abundance of nitrogen-fixing microorganisms in crop rhizosphere soil, comprising:

[0088] The microorganisms described in the first aspect, the fermentation liquid described in the second aspect, the microbial agent described in the third aspect, the composite agent described in the fourth aspect, the microbial fertilizer described in the sixth aspect, or the biological pesticide described in the seventh aspect are applied to crops.

[0089] According to an embodiment of the present disclosure, the crop includes at least one selected from corn, wheat, soybean, rice, potato, sorghum, cucumber, pepper, tomato, peanut, eggplant, and cotton.

[0090] According to an embodiment of the present disclosure, a tenth aspect of the present disclosure provides a method for increasing nitrogenase activity in crop rhizosphere soil, comprising:

[0091] The microorganisms described in the first aspect, the fermentation liquid described in the second aspect, the microbial agent described in the third aspect, the composite agent described in the fourth aspect, the microbial fertilizer described in the sixth aspect, or the biological pesticide described in the seventh aspect are applied to crops.

[0092] According to an embodiment of the present disclosure, the crop includes at least one selected from corn, wheat, soybean, rice, potato, sorghum, cucumber, pepper, tomato, peanut, eggplant, and cotton.

[0093] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0094] Example 1 Isolation and purification of Streptomyces

[0095] (1) Strain isolation

[0096] Soil was collected from vegetable fields in Shandong Province and placed in sterile sample bags at 4°C for future use. The strains were isolated using the soil dilution plate method. The collected soil was mixed, 10 grams of soil was added to sterile water, the volume was adjusted to 100 ml, and the shaker was shaken for 30 minutes at 30°C and 180 rpm. After shaking, 1 ml of soil suspension was added to 9 ml of sterile water to prepare a concentration of 10 -2 After mixing, use a sterile pipette to draw 10 -2 Add 9 ml of sterile water to 1 ml of soil suspension and repeat this process for 10 -3 , 10 -4 , 10 -5 and 10 -6 Dilution. Pipette 0.1ml of each concentration gradient soil dilution solution into Gao's synthetic agar medium No. 1, and evenly apply it on the surface of the medium with a spreader. Each concentration is replicated three times, and cultured at a constant temperature of 30℃.

[0097] (2) Strain purification

[0098] After 5-7 days of culture on the plate, single colonies were picked with an inoculating loop and purified and cultured on Gao's medium No. 1 by streak isolation. Culture was then continued at 30°C for 5-15 days. Purification was repeated twice using the same method to obtain pure strain F9.

[0099] Example 2 Identification of Streptomyces

[0100] The obtained pure strains were identified by morphology, physiology, biochemistry and molecular biology. The results are as follows:

[0101] (1) Morphological characteristics:

[0102] The colonies are dry and opaque, with irregular white protrusions on the surface and blue spores. They adhere tightly to the culture medium and are difficult to pick up, and are not easily broken when picked up. The front and back of the colonies are inconsistent in color, and the surface of the culture medium at the edge of the colonies is deformed. The outer wall is smooth and uniform, and the bacterial cells are filamentous and multi-branched, as shown in Figure 1.

[0103] (2) Physiological and biochemical characteristics:

[0104] The fatty acid composition of the strain was tested by the Microbial Fatty Acid Rapid Identification System (MIDI), and the results showed that the main fatty acids of the strain were C14:0iso, C16:0, C16:0iso, C15:0anteiso, and C15:0iso, with contents of 16.21%, 15.35%, 14.94%, 11.12%, and 6.11%, respectively. The specific fatty acid composition results are shown in Table 1, and the results indicate that the purified strain obtained by the present disclosure screened meets the main cellular fatty acid characteristics of Streptomyces.

[0105] Table 1 Streptomyces fatty acid data

[0106] The results of Biolog GENIII test are shown in Table 2, which show that the purified strain meets the biochemical metabolic characteristics of Streptomyces.

[0107] Table 2 Streptomyces Biolog GENIII data

[0108] Note: + indicates positive; - indicates negative; w indicates weakly positive.

[0109] (3) Molecular biological characteristics:

[0110] 1) The basic characteristics of the genome of the purified strain are analyzed as follows:

[0111] The genome of strain F9 was 11,175,236 bp in size, with a G+C content of 70.21%, and its genomic characteristics were basically consistent with those of other species of the genus Streptomyces.

[0112] Table 3 Analysis of basic characteristics of the purified strain genome

[0113] 2) The 16S rDNA gene sequence (1416 bp) and phylogenetic tree analysis results of the purified strain are as follows:

[0114] The 16S rDNA gene sequence of the purified strain was determined using bacterial 16S rDNA gene universal primers 27F and 1492R (27F 5'-GTTTGATCMTGGCTCAG-3', SEQ ID NO: 2; 1492R 5'-TACGGYTACCTTGTTACGACTT-3', SEQ ID NO: 3) to obtain a 1416 bp gene fragment:

[0115] The gene sequence was compared with that of known type bacteria, and the results are shown in Table 4. The purified strain had the highest similarity with the known Streptomyces geranii type species A301T, which was 99.34%.

[0116] Table 4 Comparative analysis of 16S rRNA gene sequences of purified strains

[0117] The phylogenetic tree constructed based on its 16S rDNA gene sequence and related taxa is shown in Figure 2. The results showed that the strain was closely related to Streptomyces geranii A301 T Gathered into the same branch.

[0118] 3) The ANI value of the purified strain was analyzed using the JSpeciesWS website. The results are shown in Table 5. It can be concluded that the strain is similar to the type strain A301 of the known species Streptomyces geranii. T The ANI of the strain was 87.29%. Furthermore, the ANI values ​​of the strain and its related species were all below 95%. Generally speaking, the ANI values ​​between strains of the same species are ≥ 95%. Therefore, it can be concluded that the purified strain is a new species and, according to international nomenclature rules, is named Streptomyces linyiensis.

[0119] Table 5 Analysis of Streptomyces ANI values

[0120] Example 3 Effect of Linyi Streptomyces fermentation broth on tomato yield and rhizosphere microbial community structure

[0121] First, prepare the Linyi Streptomyces fermentation broth according to the following method:

[0122] Streptomyces was fermented in Gao's No. 1 liquid culture medium at a temperature of 30° C. and a rotation speed of 180 rpm for 4 days to obtain a Streptomyces fermentation broth.

[0123] A field experiment with tomatoes was conducted, divided into two groups: a control group and a treatment group. The control group was irrigated with water, while the treatment group was irrigated with a Streptomyces fermentation broth. Ninety days after planting, the tomatoes were harvested, and rhizosphere soil samples were collected for soil metagenomic analysis and rhizosphere microbial community structure analysis. The results showed that compared with the control group, the tomato yield in the treatment group increased by 16.7%, nitrogenase activity in the rhizosphere increased by 20%, and nitrogen uptake by the plants increased by 13%. Analysis of the rhizosphere microbial community structure, as shown in Table 6, showed that compared with the control group, the abundance of Actinomycetes in the rhizosphere soil of the treatment group increased by 8.6%, Streptomyces by 9.1%, Rhizobia by 12.8%, Mesorhizobium by 6.7%, and Micromonas by 14.1%.

[0124] Table 6 Effects of Streptomyces fermentation broth on tomato yield and rhizosphere microbial community structure

[0125] Example 4 Effect of a Streptomyces-containing inoculant on corn yield and rhizosphere microbial community structure

[0126] First, prepare the inoculum according to the following method:

[0127] Gao's No. 1 liquid culture medium was added to the bran with a solid-liquid mass ratio of 4:1 to make a semi-solid culture medium. After inoculation with Streptomyces, the medium was allowed to ferment at 30°C for 5 days, dried at 40°C, and crushed to obtain a Streptomyces inoculum. The number of viable bacteria of Streptomyces Linyi in the inoculum was 2×10 9 CFU.

[0128] A field experiment with corn was conducted, divided into two groups: a control group and a treatment group. The control group was flushed with water, while the treatment group was flushed with a microbial inoculant containing Streptomyces. After 90 days of planting, yield was measured, rhizosphere soil was collected, soil metagenomes were analyzed, and the rhizosphere microbial community structure was analyzed. The results showed that compared with the control group, the corn yield in the treatment group increased by 5.3%, rhizosphere nitrogenase activity increased by 24.3%, and plant nitrogen uptake increased by 8.0%. Analysis of the rhizosphere microbial community structure, as shown in Table 7, showed that compared with the control group, the abundance of Actinomycetes in the rhizosphere soil of the treatment group increased by 4.3%, Streptomyces by 9.9%, Rhizobia by 16%, Mesorhizobium by 7.9%, and Micromonas by 10.3%.

[0129] Table 7 Effects of Streptomyces fermentation broth on rhizosphere microbial community structure of corn yield

[0130] Example 5 Effect of liquid fertilizer containing Streptomyces on cabbage yield and rhizosphere microbial community structure

[0131] A liquid microbial fertilizer was prepared by mixing Streptomyces fermentation broth with nutrients such as potassium dihydrogen phosphate, urea, monoammonium phosphate, potassium sulfate, potassium humate, and trace elements. The liquid microbial fertilizer was applied to cabbage plants to verify its effectiveness. A field experiment with cabbage was arranged and divided into two groups: a control group and a treatment group. The control group was applied with isonutrient fertilizer, while the treatment group was applied with isonutrient microbial fertilizer containing Streptomyces fermentation broth. After 60 days of growth, the yield was measured, rhizosphere soil was collected, soil metagenome was measured, and the rhizosphere microbial community structure was analyzed. The results showed that compared with the control group, the cabbage yield in the treatment group increased by 19.7%, the rhizosphere nitrogenase activity increased by 23.1%, and the nitrogen absorption of the plants increased by 15.5%. The structure of the rhizospheric microbial community was analyzed, and the results are shown in Table 8. Compared with the control group, the abundance of Actinomycetes in the rhizospheric soil of the treatment group increased by 10.5%, the abundance of Streptomyces increased by 16.7%, the abundance of Rhizobium increased by 11.9%, the abundance of Mesorhizobium increased by 12.6%, and the abundance of Xanthomonas increased by 9.6%.

[0132] Table 8 Effects of liquid microbial fertilizer containing Streptomyces on cabbage yield and rhizosphere microbial community structure

[0133] Example 6 Effect of solid bacterial fertilizer containing Streptomyces on the structure of cabbage rhizosphere microbial community

[0134] A solid microbial fertilizer made from Streptomyces solid inoculum was mixed with nutrients such as potassium dihydrogen phosphate, urea, monoammonium phosphate, potassium sulfate, potassium humate, and trace elements to prepare the solid microbial fertilizer. The fertilizer was applied to cabbage plants to verify its effectiveness. A field experiment with cabbage was conducted and divided into two groups: a control group and a treatment group. The control group was treated with an isonutrient fertilizer, while the treatment group was treated with an isonutrient microbial fertilizer containing a fermentation broth of Streptomyces. After 60 days of growth, the yield was measured, rhizosphere soil was collected, soil metagenomes were measured, and the rhizosphere microbial community structure was analyzed. The results showed that compared with the control group, the cabbage yield in the treatment group increased by 19.8%, the nitrogenase activity in the rhizosphere increased by 24.1%, and the nitrogen absorption of the plants increased by 16.4%. The structure of the rhizospheric microbial community was analyzed, and the results are shown in Table 9. Compared with the control group, the abundance of Actinomycetes in the rhizospheric soil of the treatment group increased by 9.6%, the abundance of Streptomyces increased by 9.6%, the abundance of Rhizobium increased by 10.4%, the abundance of Mesorhizobium increased by 13.2%, and the abundance of Xanthomonas increased by 10.5%.

[0135] Table 9 Effects of solid microbial fertilizer containing Streptomyces on cabbage yield and rhizosphere microbial community structure

[0136] Example 7 Effect of a composite agent containing Streptomyces on cucumber yield and rhizosphere microbial community structure

[0137] First, prepare the inoculum according to the following method:

[0138] Gao's No. 1 liquid medium was added to the bran with a solid-liquid mass ratio of 4:1 to make a semi-solid medium. After inoculation with Streptomyces, the medium was allowed to ferment at 30°C for 5 days, dried at 40°C, and crushed to obtain Streptomyces powder. The Streptomyces powder was compounded with Streptomyces leucoderma and Bacillus jelly powder to obtain composite agent 1. The Streptomyces powder was compounded with Streptomyces leucoderma powder, Bacillus jelly powder, Bacillus subtilis powder, Bacillus amyloliquefaciens powder, and Streptomyces freundii powder to obtain composite agent 2. The number of viable cells of Streptomyces linyi in composite agents 1 and 2 was 2×10 9 CFU.

[0139] A field experiment with cucumbers was conducted, with two groups divided into a control group and a treatment group. The control group was flushed with water, while the treatment group was flushed with a composite inoculant containing Streptomyces. After 90 days of planting, yield was measured, rhizosphere soil samples were collected, soil metagenomes were analyzed, and the rhizosphere microbial community structure was analyzed. Results showed that compared with the control group, the cucumbers treated with composite inoculant 1 increased yield by 18.3%, rhizosphere nitrogenase activity by 16.4%, and plant nitrogen uptake by 14.8%. Analysis of the rhizosphere microbial community structure, as shown in Table 10, showed that compared with the control group, the abundance of Actinomycetes in the rhizosphere soil of the treatment group increased by 6.4%, with Streptomyces increasing by 13.9%, Rhizobium by 10.3%, Mesorhizobium by 8.8%, and Micromonas by 10%. The cucumbers treated with composite inoculant 2 increased yield by 20.9%, rhizosphere nitrogenase activity by 18.1%, and plant nitrogen uptake by 15.9%. The structure of the rhizospheric microbial community was analyzed, and the results are shown in Table 11. Compared with the control group, the abundance of Actinomycetes in the rhizospheric soil of the treatment group increased by 5.6%, the abundance of Streptomyces increased by 15.7%, the abundance of Rhizobium increased by 11.4%, the abundance of Mesorhizobium increased by 9.7%, and the abundance of Micromonas increased by 11.2%.

[0140] Table 10 Effects of composite agents containing Streptomyces on cucumber yield and rhizospheric microbial community structure

[0141] Example 8 Effects of Biopesticides Containing Streptomyces on Corn Yield and Rhizosphere Microbial Community Structure

[0142] A biopesticide containing Streptomyces linyiensis was prepared by mixing a fermentation broth of Streptomyces linyiensis with insecticides such as thiamethoxam and avermectin. The results were then validated by applying it to corn plants. A field experiment was conducted on corn plants, divided into two groups: a control group and a treatment group. The control group received an isonutrient fertilizer, while the treatment group received a biopesticide containing Streptomyces linyiensis. After 120 days of growth, yield was measured, rhizosphere soil was collected, soil metagenomes were analyzed, and the rhizosphere microbial community structure was analyzed. The results showed that compared to the control group, corn yield in the treatment group increased by 25.6%, rhizosphere nitrogenase activity increased by 25.7%, and plant nitrogen uptake increased by 14.7%. Analysis of the rhizosphere microbial community structure, as shown in Table 11, showed that compared to the control group, the abundance of Actinomycetes in the rhizosphere soil of the treatment group increased by 4.3%, Streptomyces by 7.6%, Rhizobia by 10.3%, Mesorhizobia by 12.1%, and Xanthomonas by 6.5%.

[0143] Table 11 Effects of biopesticides containing Streptomyces linyiensis on corn yield and rhizosphere microbial community structure

[0144] Example 9 Effects of Biopesticides Containing Streptomyces on Soybean Yield and Rhizosphere Microbial Community Structure

[0145] A biopesticide containing Streptomyces linyiensis was prepared by mixing Streptomyces powder with insecticides such as thiamethoxam and avermectin. The results were verified by applying it to soybean plants. A field experiment was conducted on soybeans, divided into two groups: a control group and a treatment group. The control group received an isonutrient fertilizer, while the treatment group received a biopesticide containing Streptomyces linyiensis. After 160 days of growth, yield was measured, rhizosphere soil was collected, soil metagenomes were analyzed, and the rhizosphere microbial community structure was analyzed. The results showed that compared with the control group, soybean yield in the treatment group increased by 5.9%, rhizosphere nitrogenase activity increased by 14.0%, and plant nitrogen uptake increased by 8.2%. Analysis of the rhizosphere microbial community structure, as shown in Table 12, showed that compared with the control group, the abundance of Actinomycetes in the rhizosphere soil of the treatment group increased by 4.4%, Streptomyces by 8.3%, Rhizobia by 13.8%, Mesorhizobia by 10.4%, and Xanthomonas by 12.9%.

[0146] Table 12 Effects of biological pesticides containing Streptomyces linyiensis on soybean yield and rhizospheric microbial community structure

[0147] In summary, the Streptomyces provided herein can effectively increase the abundance of nitrogen-fixing microbial strains in crop rhizosphere soil, for example, increasing the abundance of Actinomycetes, Streptomyces, Rhizobium, Mesorhizobium, Micromonas, and Luteomonas. The provided Streptomyces can be prepared into a microbial agent and used as a microbial fertilizer or added to a microbial fertilizer to increase the abundance of nitrogen-fixing microorganisms in crop rhizosphere soil, increase nitrogenase activity in crop rhizosphere soil, improve the soil microecological environment, promote crop growth, and increase crop yield.

[0148] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and features of different embodiments or examples, unless they are mutually inconsistent.

[0149] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A microorganism, characterized in that The microorganism is Streptomyces linyiensis, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 28, 2022, with the deposit number CGMCC No.25834.

2. The microorganism according to claim 1, characterized in that The microorganism has a 16S rDNA sequence as shown in SEQ ID NO:

1.

3. A fermentation broth, characterized in that: The fermentation broth is fermented by the microorganism according to claim 1 or 2.

4. A microbial agent, characterized in that: The microbial agent comprises the microorganism according to claim 1 or 2, or the fermentation broth according to claim 3.

5. The microbial agent according to claim 4, characterized in that: The microbial agent is in dry powder form, and the effective viable bacteria count of Streptomyces linyi in each gram of the microbial agent is at least 2×10 9 CFU.

6. The microbial agent according to claim 4, characterized in that: The microbial agent is obtained by the following steps: Fermenting and culturing the Linyi Streptomyces to obtain a fermentation product; The fermentation product is spray-dried and pulverized to obtain the microbial agent.

7. A composite bacterial agent, characterized in that: The composite bacterial agent comprises the microbial agent according to any one of claims 4 to 6.

8. The composite bacterial agent according to claim 7, characterized in that: The composite microbial agent comprises a first microbial agent and a second microbial agent, wherein the first microbial agent is the microbial agent according to any one of claims 4 to 6, and the second microbial agent comprises at least one selected from Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus gelatinosa, Streptomyces freundii, and Streptomyces tenuifolius.

9. The composite bacterial agent according to claim 8, characterized in that: The composite microbial agent comprises 10-50 parts by weight of the first microbial agent and 5-20 parts by weight of the second microbial agent; The second microbial agent comprises 0-5 parts by weight of Streptomyces tenuiflavos, 0-10 parts by weight of Bacillus gelatinosa, 0-5 parts by weight of Bacillus subtilis, 0-5 parts by weight of Bacillus amyloliquefaciens, and 0-5 parts by weight of Streptomyces freundii.

10. The composite bacterial agent according to claim 8 or 9, characterized in that: The effective viable bacteria count of Streptomyces linyi in each gram of the composite bacterial agent is at least 2×10 9 CFU, and at least one selected from the following: The effective viable count of Streptomyces tenuifolius is at least 5×10 8 CFU; The effective viable count of Bacillus jelly is at least 5×10 8 CFU; The effective viable count of Bacillus subtilis is at least 5×10 8 CFU; The effective viable count of Bacillus amyloliquefaciens is at least 5×10 8 CFU; The effective viable count of Streptomyces freundii is at least 5×10 8 CFU.

11. Use of the microorganism according to claim 1 or 2, the fermentation liquid according to claim 3, the microbial agent according to any one of claims 4 to 6, or the composite agent according to any one of claims 7 to 10 in the preparation of microbial fertilizers or biological pesticides.

12. A microbial fertilizer, characterized in that: The microbial fertilizer comprises the microorganism according to claim 1 or 2, the fermentation liquid according to claim 3, the microbial agent according to any one of claims 4 to 6, or the composite agent according to any one of claims 7 to 10.

13. The microbial fertilizer according to claim 12, characterized in that: The microbial fertilizer is solid fertilizer or liquid fertilizer.

14. The microbial fertilizer according to claim 13, characterized in that: The microbial fertilizer is a solid fertilizer, and the number of effective live microorganisms contained in each gram of the microbial fertilizer is at least 2×10 9 CFU.

15. The microbial fertilizer according to claim 13, characterized in that: The microbial fertilizer is a liquid fertilizer, and the number of effective live microorganisms contained in each liter of the microbial fertilizer is at least 2×10 9 CFU.

16. The microbial fertilizer according to any one of claims 12 to 15, characterized in that: The microbial fertilizer further includes a basic fertilizer, and the basic fertilizer includes at least one selected from compound fertilizers, water-soluble fertilizers, organic fertilizers, and foliar fertilizers.

17. A biological pesticide, characterized in that: It comprises the microorganism according to claim 1 or 2, the fermentation broth according to claim 3, the microbial agent according to any one of claims 4 to 6, or the composite agent according to any one of claims 7 to 10.

18. The biopesticide according to claim 17, characterized in that: The number of effective live microorganisms contained in each liter of the biological pesticide is at least 2×10 9 CFU.

19. Use of the microorganism according to claim 1 or 2, the fermentation liquid according to claim 3, the microbial agent according to any one of claims 4 to 6, the composite agent according to any one of claims 7 to 10, the microbial fertilizer according to any one of claims 12 to 16, or the biological pesticide according to claim 17 or 18 in increasing the abundance of nitrogen-fixing microorganisms in the rhizosphere soil of crops, or increasing the nitrogenase activity in the rhizosphere soil of crops.

20. The use according to claim 19, wherein the nitrogen-fixing microorganism comprises at least one of the genera Actinomycetes, Streptomyces, Rhizobium, Mesorhizobium, Micromonas, and Xanthomonas.

21. A method for increasing the abundance of nitrogen-fixing microorganisms in crop rhizosphere soil, characterized in that: include: Applying the microorganism according to claim 1 or 2, the fermentation liquid according to claim 3, the microbial agent according to any one of claims 4 to 6, the composite agent according to any one of claims 7 to 10, the microbial fertilizer according to any one of claims 12 to 16, or the biological pesticide according to claim 17 or 18 to crops.

22. The method according to claim 21, characterized in that The crops include at least one selected from corn, wheat, soybean, rice, potato, sorghum, cucumber, pepper, tomato, peanut, eggplant and cotton.

23. A method for increasing nitrogenase activity in crop rhizosphere soil, characterized in that: include: Applying the microorganism according to claim 1 or 2, the fermentation liquid according to claim 3, the microbial agent according to any one of claims 4 to 6, the composite agent according to any one of claims 7 to 10, the microbial fertilizer according to any one of claims 12 to 16, or the biological pesticide according to claim 17 or 18 to crops.

24. The method according to claim 23, characterized in that The crops include at least one selected from corn, wheat, soybean, rice, potato, sorghum, cucumber, pepper, tomato, peanut, eggplant and cotton.

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