Streptomyces YCB062 and application thereof

Abstract

The invention belongs to the technical field of agricultural microorganisms, and discloses a streptomyces strain YCB062 and application thereof. The Streptomyces sp.YCB062 has the preservation number of GDMCC NO: 66856, the Streptomyces sp.YCB062 is a new species of Streptomyces, has a good bacteriostatic effect on different plant pathogenic fungi and pathogenic bacteria, and shows broad-spectrum bacteriostatic activity; in addition, the strain also has relatively strong secondary metabolite synthesis capability, has the activity of promoting growth such as potassium decomposition, siderophore production, amylase and casein production and the like, and is a disease-resistant growth-promoting strain with a good application prospect.

Description

Technical Field

[0001] This application relates to the field of agricultural microbiology technology, and in particular to a Streptomyces strain YCB062 and its applications. Background Technology

[0002] Plant diseases caused by pathogenic fungi such as *Anthracnose fungus of Camellia oleifera*, *Geotrichum candida* of citrus, and *Ralstonia solanacearum* of tomato have seriously harmed the agricultural industry. The extensive use of chemical pesticides easily leads to environmental pollution, pesticide residues, and other problems, endangering human health. Disease-resistant and growth-promoting microorganisms possess both the function of resisting plant diseases and promoting plant growth. Microbial agents or biofertilizers prepared from these microorganisms can not only reduce dependence on chemical pesticides but also improve crop yield and quality, playing a vital role in maintaining sustainable agricultural development.

[0003] Streptomyces are a group of Gram-positive bacteria that are widely distributed and produce abundant secondary metabolites. Due to differences in their growth habitats and nutritional conditions, Streptomyces from different sources also exhibit diversity in the synthesis of natural products. The development of Streptomyces resources with excellent characteristics can effectively delay the harm to the ecological environment and human health caused by the overuse of chemical pesticides. Summary of the Invention

[0004] The purpose of this application is to provide a Streptomyces strain YCB062 and its applications. Strain YCB062 has broad-spectrum antibacterial activity and various life-promoting properties, and has a strong inhibitory effect on common plant pathogenic fungi and bacteria.

[0005] To achieve the purpose of this application, in the first aspect, this application provides a new species of Streptomyces sp. YCB062 with biocontrol activity, which is classified as Streptomycess p. This strain was isolated from soil samples collected from the Camellia oleifera planting base of Xingning Xiangwang Agricultural and Forestry Technology Co., Ltd. in Meizhou, Guangdong (24°24'38”N; 115°42'23”E; 436.0m). It was deposited on August 6, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC; address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; postcode: 510070), with accession number GDMCCNO: 66856.

[0006] Secondly, this application provides inoculants, biopesticides, or biofertilizers prepared from Streptomyces YCB062 or its fermentation broth.

[0007] Thirdly, this application provides a biocontrol agent or antibacterial agent prepared from Streptomyces YCB062.

[0008] Fourthly, this application provides any of the following applications of Streptomyces YCB062 or its inoculants: 1) for antagonizing plant pathogenic fungi; 2) for antagonizing plant pathogenic bacteria; 3) for preparing bio-fertilizers; 4) for promoting plant growth; 5) for controlling tomato gray mold; 6) for controlling camellia anthracnose; 7) for controlling citrus white mold.

[0009] Fifthly, this application provides the application of Streptomyces YCB062 in resistance to pathogens and plant diseases caused by pathogens, including pathogenic fungi and pathogenic bacteria.

[0010] The pathogens described in this application include, but are not limited to, pathogenic fungi and pathogenic bacteria; wherein, the pathogenic fungi include Botrytis cinerea (Tomato gray mold), Citrus white mold (Citrustrichoderma), Geotrichum candidum (Tobacco aspergillus), and Colletotrichum camelliae (Camellia anthracnose).

[0011] The pathogenic bacteria described in this application include Ralstorinia solanacearum (RSH) and / or Xanthomanos oryza epv. Oryza.

[0012] Furthermore, the plant diseases mentioned include, but are not limited to, tomato gray mold, camellia anthracnose, and citrus white mold.

[0013] Sixthly, this application provides any of the following applications of Streptomyces YCB062: i) for potassium solubilization; ii) for iron carrier production; iii) for starch hydrolysis; and iiii) for casein hydrolysis.

[0014] This application conducted a multiphase classification and identification of Streptomyces YCB062, an antagonistic strain with broad-spectrum antibacterial activity isolated from the soil environment of a camellia oleifera planting base in Meizhou, Guangdong. This bacterium has potential biocontrol application value and good development and application prospects.

[0015] After identification, it was found to be similar to Sterptomyces roseifaciens MBT76. T , Streptomyceshiroshimensis NBRC 3839 T , Streptomyces abikoensis NBRC 13860 T and Streptomyces blastmyceticus NRRL B-5480 TThe highest homology was observed, with 98.7-99.0% similarity in the 16S rRNA gene sequence. Furthermore, YCB062 and S. roseifaciens MBT76... T S. hiroshimensis NBRC 3839 T S. abikoensis NBRC 13860 T and S. blastmyceticus NRRL B-5480 T The average nucleotide similarity (ANI) value of the genome was 81.3-81.9%, and the digital DNA hybridization value (dDDH) value was 25.0-25.3%. These values ​​were all less than the species threshold values ​​of 95-96% and 70%, respectively, indicating that strain YCB062 is a new species of Streptomyces.

[0016] The Streptomyces YCB062 provided in this application has a strong growth inhibitory effect on four tested fungi and two tested bacteria; in the experiment on the antagonistic activity against plant pathogens, the antibacterial efficiency of YCB062 against plant pathogens reached 66%-75%, which shows that Streptomyces YCB062 has a significant inhibitory effect on the tested plant pathogens.

[0017] The Streptomyces YCB062 provided in this application has a strong ability to synthesize secondary metabolites. Experimental tests have shown that it has the ability to promote growth, such as producing siderophores, solubilizing potassium, hydrolyzing starch, and hydrolyzing casein. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a description of the morphological characteristics of strain YCB062 in one embodiment of this application; (wherein A: cell morphology of strain YCB062 after 2 days of growth on nutrient agar NA medium; B: cell morphology of strain YCB062 after 7 days of growth on nutrient agar NA medium; C: individual morphological characteristics of YCB062).

[0020] Figure 2 This is a maximum likelihood (ML) phylogenetic tree of YCB062 and its similar strains constructed based on the 16S rRNA gene, which is one embodiment of this application.

[0021] Figure 3This application describes the antagonistic effect of strain YCB062, one of the embodiments of this application, against plant pathogenic fungi; (the figures shown are, in order, *Gyromyces lutea* TSF05, *Gyromyces albopictus* TSF19, *Aspergillus fumigatus* TSF4, and *Anthracnose fungus of Camellia oleifera* HU1397).

[0022] Figure 4 This application describes the quantitative antifungal effect of strain YCB062, one of the embodiments of this application, against four plant pathogenic fungi; ** indicates p<0.01;

[0023] Figure 5 This application demonstrates the antagonistic effect of strain YCB062, one of the embodiments of this application, against plant pathogenic bacteria; (the figures show Ralstonia solanacearum RSH and Xanthomonas oryzae Xoo, in that order).

[0024] Figure 6 The physiological and biochemical characteristics of strain YCB062, one embodiment of this application, are as follows: (where A: potassium solubilization ability test; B: siderophore production ability test; C: starch hydrolysis ability test; D: casein hydrolysis ability test). Detailed Implementation

[0025] To make the objectives, advantages, and features of the present invention more readily understood, specific embodiments of the present invention are described in detail below with reference to examples. Several embodiments of the present invention are given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of the present invention will be more thorough and complete:

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Unless otherwise specified, all methods described in the following examples are conventional. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] The novel Streptomyces species provided by this invention is Streptomyces sp. YCB062, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC; address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province; postcode: 510070), with a deposit date of August 6, 2025, and accession number GDMCCNO: 66856. This novel Streptomyces species YCB062 was isolated from soil samples collected from the Camellia oleifera planting base of Xingning Xiangwang Agricultural and Forestry Technology Co., Ltd. in Meizhou, Guangdong Province (24°24'38”N; 115°42'23”E; 436.0m).

[0029] The reagents and culture media used in the following examples are as follows:

[0030] Potassium-solubilizing medium: 10.0g sucrose, 0.5g yeast extract, 1.0g ammonium sulfate, 2.0g K2HPO4, 0.5g MgSO4·7H2O, 1.0g CaCO3, 1.0g potassium feldspar powder, 1L distilled water, pH 7.2, sterilized at 121℃ for 20min.

[0031] CAS detection culture medium:

[0032] (1) CAS medium (g / L): glucose 2.0, casein 3.0, CaCl2 0.1, MgSO4 4.9, agar 17.0, bring to a final volume of 1L, sterilize at 121℃ for 20min;

[0033] (2) CAS staining solution (g / L): Resazurin 0.3, FeCl3 0.1, hexadecyltrimethylammonium bromide 1.5, bring to a final volume of 1L, sterilize at 115℃ for 20min;

[0034] (3) When the temperature is cooled to 60°C, slowly add 5 mL each of 0.1 mol / L phosphate buffer and 5% CAS staining solution to the CAS medium to make the CAS detection medium.

[0035] Example 1: Isolation and purification of strain YCB062

[0036] 1.1 Sample Collection and Pretreatment

[0037] Approximately 200g of soil samples from the 10-20cm soil layer were collected from the camellia oleifera planting base of Xingning Xiangwang Agricultural and Forestry Technology Co., Ltd. in Meizhou, Guangdong (24°24'38”N; 115°42'23”E; 436.0m). After removing visible roots and other debris, the soil was passed through a 2mm sieve, mixed thoroughly, and then allowed to air dry naturally at room temperature.

[0038] 1.2 Isolation and Purification of Soil Bacteria

[0039] Take 1g of air-dried soil and place it into a 50mL centrifuge tube containing 9mL of sterile physiological saline. Place the centrifuge tube in a shaker at 160rpm for 2 hours. On a clean bench, use a pipette to transfer 0.1mL of the mixed soil suspension into a centrifuge tube containing 0.9mL of sterile physiological saline. Mix well to obtain 10... -1 Diluent. Prepare 10 solutions at a 1:10 ratio. -2 10 -3 10 -4 10 -5 10 -6Serial dilutions. Take 0.2 mL of each serial dilution and spread it evenly on the surface of 0.2 TSA solid medium. Incubate at 30°C upside down for different times. After colonies grow, pick single colonies with different morphological characteristics and streak them onto 0.2 TSA medium for purification. Store the purified strains in 30% glycerol at -80°C.

[0040] Example 2: Identification of strain YCB062

[0041] 2.1 Morphological identification of strain YCB062

[0042] Figure 1 The morphological characteristics of YCB062 are shown, among which... Figure 1 A represents the colony morphology of this strain after 2 days of cultivation on NA solid medium. Figure 1 B represents the colony morphology of YCB062 after 7 days of growth on NA medium. Early colonies are yellow, later developing a brown precipitate; colonies are spherical, raised, dry, and difficult to pick up. Figure 1 C represents the individual morphological characteristics observed under 100x oil immersion, and the results show that YCB062 is a Gram-positive, filamentous bacterium.

[0043] 2.2 Physiological and biochemical characteristics of strain YCB062

[0044] The physiological and biochemical characteristics of strain YCB062 are shown in Table 1. YCB062 showed positive oxidase and reductase activities, grew in a temperature range of 15-36℃, tolerated a pH range of 5.0-8.0, and had a maximum tolerance of 5.5% NaCl. It could grow on NA, R2A, TSA, LB, and Gao's No. 1 media. Its methyl red, methyl ester, citrate, and VIP tests were all negative.

[0045] Table 1

[0046]

[0047]

[0048] 2.3 Molecular identification of strain YCB062

[0049] Genomic DNA was extracted from YCB062 using the Magen Bacterial DNA Extraction Kit and used as a template for 16S rRNA gene amplification using universal primers (27F / 1492R). The PCR reaction mixture (20 μL) consisted of: 10 μL 2×phanta Max Mix, 0.5 μL 27F, 0.5 μL 1492R, 0.5 μL DNA template, and 8.5 μL ddH2O. The reaction program was: 95℃ for 5 min, 95℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min, 30 cycles, followed by 72℃ for 10 min. 5 μL of the PCR product was subjected to agarose gel electrophoresis. After confirmation, the remaining sample was sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rRNA gene sequencing.

[0050] The 16S rRNA gene sequence of strain YCB062 was uploaded to the EzBiocloud (https: / / www.ezbiocloud.net / ) database and compared with known 16S rRNA genes for homology sequence. Standard strains with high homology were selected to construct a maximum likelihood (ML) phylogenetic tree.

[0051] The results are as follows Figure 2 Strain YCB062 was identified as belonging to the genus *Streptomyces*, which is related to *Sterptomyces roseifaciens* MBT76. T , Streptomyces hiroshimensis NBRC 3839 T , Streptomyces abikoensis NBRC 13860 T and Streptomyces blastmyceticus NRRL B-5480 T The two rRNA gene sequences showed the greatest homology, with a 16S rRNA sequence similarity of 98.7-99.0%. ML phylogenetic tree results showed that YCB062 is related to Streptomyces meridianus MTZ3.1. T , Streptomyces gamaensis NEAU-Ggll T They clustered on a large branch.

[0052] 2.4 Whole-genome sequencing and comparative analysis of genome ANI and dDDH of strain YCB062

[0053] After culturing YCB062 on NA medium for 3 days, the bacterial cells were collected and sent to Shanghai Meiji Biotechnology Co., Ltd. for whole-genome sequencing on the Illumina Hiseq platform. After reading filtering of the raw genome data, high-quality paired reads were assembled into contigs and scaffolds data. The average nucleotide identity (ANI) of strain YCB062 with its most similar strain was calculated using the online ANI calculator (https: / / www.ezbiocloud.net / tools / ani) on the EzBiocloud platform; the digital DNA-DNA hybridization (dDDH) was calculated using the online Genome-to-Genome Calculator GGDCC (http: / / ggdc.dsmz.de / home.php) developed by DSMZ.

[0054] The results showed that YCB062 and S. roseifaciens MBT76 T S. hiroshimensis NBRC 3839 T S. abikoensis NBRC 13860 T and S. blastmyceticus NRRL B-5480 T The genome ANI is 81.3-81.9% and the dDDH value is 25.0-25.3%, both of which are less than the species threshold values ​​of 95-96% and 70%, respectively, further proving that YCB062 is a new species of the genus Streptomyces.

[0055] Based on the morphological characteristics, physiological and biochemical characteristics, 16S rRNA gene sequence similarity, ML phylogenetic tree, and comparison results of genome ANI and dDDH values, strain YCB062 is a new species of Streptomyces, and its 16S rRNA gene sequence is shown in SEQ ID NO.1 (Streptomyces sp. YCB062 16S rRNA gene sequence).

[0056] Example 3: Study on the antagonistic activity of strain YCB062 against plant pathogens

[0057] 3.1 Study on the antagonistic activity of strain YCB062 against plant pathogenic fungi

[0058] Mycelial blocks of *Botrytis cinerea* (Tomato gray mold), *Citrus trichoderma* (Citrus white mold), *Geotrichum candidum* (Tobacco aspergillus), and / or *Colletotrichum camelliae* (Camellia anthracnose) HU1397 were inoculated in the center of NA solid agar medium. The test strain YCB062 was inoculated at a cross-shaped point 1 cm from the center agar block. Plates inoculated only with the aforementioned fungal pathogens served as controls. The plates were incubated upside down at 30°C for 5 days. The inhibitory effect of strain YCB062 on plant pathogenic fungi was observed, and the diameter of the inhibition zone was measured.

[0059] The inhibition rate is calculated using the following formula: Inhibition rate (%) = (Coronavirus colony diameter in control group - Coronavirus colony diameter in treatment group) / Coronavirus colony diameter in control group × 100%.

[0060] The results are as follows Figure 3 and Figure 4 As shown, YCB062 exhibits significant antifungal effects against four plant pathogenic fungi, with an inhibition rate of 66%–75%.

[0061] 3.2 Study on the antagonistic activity of strain YCB062 against plant pathogenic bacteria

[0062] Plant pathogenic bacteria *Ralstorinia solanacearum* RSH and *Xanthomanos oryzae pv. Oryza* Xoo were inoculated into NA solid medium and cultured overnight at 30°C. The bacterial cells were then prepared into a bacterial suspension and the OD was adjusted. 600 The value was approximately 1.0. 100 μL of bacterial suspension was evenly spread onto NA solid medium and allowed to air dry. YCB062 cells were spotted onto a plate containing plant pathogenic bacteria using a toothpick. The plate was incubated upside down at 30°C for 3 days to observe the antibacterial effect of YCB062 against plant pathogenic bacteria.

[0063] The results are as follows Figure 5 As shown, YCB062 exhibits significant antibacterial activity against both plant pathogenic bacteria.

[0064] Example 4: Study on the life-promoting properties and hydrolytic enzyme activity of strain YCB062

[0065] YCB062 was inoculated onto potassium-solubilizing medium and CAS detection medium, respectively, and incubated at 30℃ for 2-5 days. The presence of a clear zone on the potassium-solubilizing medium and the change in color around the bacterial cells from blue-purple to red on the CAS detection medium were observed to determine whether the strain had the ability to solubilize potassium and produce siderophores.

[0066] After YCB062 was inoculated onto starch hydrolysis medium and cultured for 3 days, iodine solution was added to the medium. The presence of a clear zone was observed, indicating that the strain had amylase hydrolysis activity. In addition, after YCB062 was inoculated onto casein medium and cultured for 3 days, the presence of a clear zone around the cells was observed to determine whether the strain had casein hydrolysis activity.

[0067] The results are as follows Figure 6 As shown, YCB062 has potassium solubilization ( Figure 6 A) Siderotrophic activity (6B), hydrolyzed starch (6C), and casein (6D).

[0068] The above description is merely a preferred embodiment of this application and is not intended to limit this application in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of this application shall fall within the scope of the technical solution of this application.

Claims

1. Streptomyces sp. YCB062, characterized by: The Streptomyces strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 66856.

2. The microbial agent, biopesticide, or biofertilizer prepared from Streptomyces or its fermentation broth as described in claim 1.

3. The biocontrol agent or antibacterial agent prepared from Streptomyces as described in claim 1.

4. Any of the following applications of the Streptomyces or its inoculants as described in claim 1: 1) for antagonizing plant pathogenic fungi; 2) for antagonizing plant pathogenic bacteria; 3) for preparing bio-fertilizers; 4) for promoting plant growth; 5) for controlling anthracnose in camellia oleifera; 6) for controlling gray mold in tomatoes; 7) for controlling white mold in citrus.

5. The application according to claim 4, characterized in that: The plant pathogenic fungi include Botrytis cinerea (tomato gray mold), Citrus trichoderma (citrus white mold), Geotrichum candidum (tobacco aspergillus), and Colletotrichum camelliae (tea anthracnose).

6. The application according to claim 4, characterized in that: The plant pathogenic bacteria include Ralstorinia solanacearum and / or Xanthomanos oryzaepv. Oryza.

7. The application of Streptomyces according to claim 1 in resistance to pathogens and plant diseases caused by pathogens, wherein the pathogens include pathogenic fungi and pathogenic bacteria; wherein, The pathogenic fungi include *Botrytiscinerea*, *Citrus trichoderma*, *Geotrichum candidum*, and *Colletotrichum camelliae*; the pathogenic bacteria include *Ralstorinia solanacearum* and / or *Xanthomanos oryzaepv. Oryza*; the plant diseases include tomato gray mold, citrus white mold, and *Alternaria oleifera* anthracnose.

8. Any of the following applications of the Streptomyces of claim 1: i) for potassium solubilization; ii) for iron carrier production; iii) for starch hydrolysis; iiii) for casein hydrolysis.

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