Kitasporium Ak67 and its application

Abstract

This invention belongs to the field of biological control, specifically relating to a new strain of *Kitasatospora* and its application in plant disease control. The first aspect of this invention is to provide a strain of *Kitasatospora* sp. Ak67, which was deposited on November 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29076. The strain provided by this invention exhibits broad-spectrum antibacterial activity.

Description

Technical Field

[0001] This invention belongs to the field of biological control, specifically relating to a new species of *Synthia spp.* and its application in the control of plant diseases. Background Technology

[0002] Plant diseases severely impact plant growth, leading to decreased yield and quality, and are one of the major threats to agricultural production. Fungal diseases comprise over 30,000 species, accounting for more than 70% of all plant diseases and representing one of the most significant biological factors in their occurrence. Several or even dozens of fungal diseases can be found in a single crop. Furthermore, the strong resistance of fungal spores makes control particularly challenging. While chemical control is the most common method for managing plant diseases and offers significant short-term effects, long-term use can lead to drug resistance in pathogens, potentially causing environmental pollution and food security issues. Biological control technology, with its advantages of low environmental pollution, low risk of drug resistance, and safety for humans, livestock, and natural enemies, has become a key technological support for the green control of plant fungal diseases.

[0003] *Streptomyces kimosporum*, belonging to the phylum Actinobacteria in the family Streptomycetes, is a microorganism with diverse biological activities. It can inhibit the growth of some plant pathogenic fungi, produce indoleacetic acid, and promote phosphate dissolution and release to boost plant growth. *Streptomyces kimosporum* is also an important source of natural products. Comparative analysis of large-scale bacterial secondary metabolite gene synthesis clusters (BGCs) revealed that the average number of BGCs in the *Streptomyces kimosporum* genome is greater than that in *Streptomyces*, indicating that *Streptomyces kimosporum* is a valuable source for future discovery of novel bioactive substances. However, new antagonistic *Streptomyces kimosporum* species and new bioactive substances are rarely discovered. The results of this study will provide new strain resources for the biological control of various plant fungal diseases, and the discovery of new bioactive substances will also provide theoretical and technical support for the development of biocontrol agents. Summary of the Invention

[0004] The present invention aims to provide a new species of *Cyperus nitida* with broad-spectrum antibacterial activity, a fungal agent, and its application.

[0005] The first aspect of the present invention is to provide a strain of Kitasatospora sp. Ak67, which was deposited on November 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29076.

[0006] As a second aspect of the present invention, the present invention provides a microbial inoculant prepared from the Ak67 strain of *Cyperus niger*.

[0007] Furthermore, the method for preparing the *Spora nigra* Ak67 inoculum is as follows: Ak67 strain is activated on NA solid plates and cultured at 28°C for 2 days. Single colonies are picked and inoculated onto NB seed culture medium, and cultured at 28°C with shaking at 200 rpm / min for 1 day. Then, it is inoculated at a ratio of 5% onto Gao's No. 1 liquid culture medium and cultured at 28°C with shaking at 200 rpm / min for 5 days to obtain the fermentation broth of strain Ak67, with a viable count reaching 1 × 10⁻⁶. 9 ~1×10 10 CFU / mL.

[0008] As a third aspect of the present invention, the present invention also provides the application of the above-mentioned new species of *Nyctaginus niger* in the prevention and control of plant pathogens.

[0009] Furthermore, the plant pathogen is a plant pathogenic fungus.

[0010] Preferably, the plant pathogenic fungi are Fusarium graminearum, Fusarium oxysporum, Fusarium proliferatum, Sporisorium scitamineum, and Alternaria solani. Attached Figure Description

[0011] Figure 1 The colony morphology of strain Ak67 on the culture medium;

[0012] Figure 2 A phylogenetic tree of strain Ak67 constructed based on its whole genome;

[0013] Figure 3 This is a diagram showing the antifungal effect of strain Ak67 against plant pathogenic fungi.

[0014] Figure 4 The inhibition of Fusarium graminearum mycelial growth by strain Ak67 is shown in Figure 1. A: Blank control, B: Ak67.

[0015] Figure 5 This describes the growth-promoting effect of strain Ak67 on maize. Detailed Implementation

[0016] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0017] The culture media used in the following examples are as follows:

[0018] NB medium: beef extract 3g / L, peptone 10g / L, sodium chloride 5g / L;

[0019] Gao's No. 1 medium: KNO3 1g / L, soluble starch 20g / L, NaCl 0.5g / L, K2HPO4·3H2O 0.5g / L, MgSO4·7H2O 0.5g / L, FeSO4·7H2O 0.01g / L, agar powder 15g / L;

[0020] PDA medium: 200 g / L peeled potato, 20 g / L glucose, 15 g / L agar powder;

[0021] MYX medium: sodium glutamate 5 g / L, yeast extract 1 g / L, MgSO4·7H2O 1 g / L, glucose 2 g / L, agar powder 15 g / L;

[0022] PDB medium: 200 g / L peeled potato, 20 g / L glucose.

[0023] Example 1

[0024] This example demonstrates the isolation and identification of Kitasporium Ak67:

[0025] I. Isolation and Preservation of Strains

[0026] Soil samples were collected from the rhizosphere of the disease-suppressing plant in Sanming City, Fujian Province on May 10, 2021, using a 5-point sampling method. The Ak67 strain was obtained on MYX medium using the dilution-spread method and preserved in a 25% glycerol solution at -80°C. The colony morphology of strain Ak67 is shown in the attached figure. Figure 1 As shown.

[0027] II. Identification of Ak67 strain

[0028] Following the method of Liu et al. (2021), crude DNA was extracted. A small amount of bacterial cells was scraped, and 50 μL of 5% Chelex solution was added. The mixture was heated at 95℃ for 20 min on a PCR instrument to obtain a PCR template. PCR amplification was performed using universal primers 27F and 1492R for bacterial 16S rDNA. The PCR products were sent to a company for sequencing. The sequencing results of the 16S rDNA are shown in SEQ ID NO.1. The sequence was uploaded to EzBioCloud for sequence alignment. The results showed that Kitasatospora xanthocidica NBRC 13469 had the highest similarity (99.16%). To further determine the taxonomic position of strain Ak67, we performed whole-genome sequencing on strain Ak67. The whole-genome sequence of strain Ak67 was uploaded to the Type (Strain) Genome Server (TYGS) to calculate the digital DNA-DNA hybridization (dDDH) value between Ak67 and other strains. The average nucleotide identity (ANI) among strains was automatically calculated using PHANTASM v1.0.4. First, the LPSN taxonomic data was manually updated to the latest version. Then, the phylogenetic marker for strain Ak67 was calculated, selecting the DNA-directed RNA polymerase subunit beta protein as the phylogenetic marker. The software automatically downloaded the complete genomes of closely related species, and ANI values ​​were calculated using Pyani v0.2.11. A phylogenetic tree was constructed using FastTree v2.1.1 based on the tandem alignment results of 426 single-copy core genes (see attached). Figure 2 The results showed that the maximum dDDH value between Ak67 and K. aureofaciens ATCC 10762 was 30.3%, which was much lower than the threshold of 70% for new species classification. Ak67 clustered together with K. aureofaciens ATCC 10762, K. xanthocidica JCM 4862, and K. purpeofusca NRRL B-1817 to form an independent branch, with ANI values ​​of 88.4%, 88.4%, and 87.3%, respectively, which were lower than the threshold of 95% for new species classification. Therefore, Ak67 was identified as a new species of the genus *Katariopsis*.

[0029] The aforementioned strain Ak67 was deposited on November 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.29076.

[0030] Example 2

[0031] Determination of the broad-spectrum antibacterial activity of strain Kitasatospora sp. Ak67:

[0032] Example 1 shows that Ak67 isolated from the fungus inhibits the growth of various crop fungal pathogens.

[0033] The following information is provided regarding plant pathogens: 1) Fusarium graminearum is the main pathogen causing stem rot in crops; 2) Fusarium oxysporum is a globally distributed soil-borne pathogen that can cause wilt in more than 100 kinds of plants, including cucurbits, solanaceae, bananas, cotton, legumes, and flowers; 3) Fusarium moniliforme is an opportunistic pathogen that mainly causes root rot, wilt, and other diseases; 4) Sugarcane smut is an important pathogen causing sugarcane smut. The pathogen forms dikaryotic hyphae through sexual mating of haploid basidiospores of different mating types, which then infect the host and cause disease; 5) Alternaria solanacea is the pathogen causing early blight in tomatoes.

[0034] For *Fusarium oxysporum*, *Fusarium solani*, and *Alternaria solanacea*, the fungi to be tested were inoculated into PDA medium for activation and later use. *Alternaria nigra* Ak67 cells (described in this invention) were picked and inoculated into 10 mL of NB medium, incubated at 200 rpm and 28°C for 2 days. Using a blue pipette tip, mycelial cakes were created at the edge of the fungal plate containing hyphae and inoculated into the center of a new PDA plate. Ak67 cells were streaked 2 cm from the center. NB medium was used as a control group, and the plates were incubated upside down at 28°C for 3–5 days. Mycelial growth was observed, and the inhibition effect was statistically analyzed. Each treatment was repeated three times.

[0035] For *Fusarium graminearum*, the fungus to be tested was inoculated into PDA medium for activation and later use. Ak67 cells of *Rhizopus nigra*, as described in this invention, were picked and inoculated into 10 mL of NB medium, incubated at 200 rpm and 28°C for 2 days. Using a blue pipette tip, mycelial cakes were created at the edge of the fungal plate containing hyphae and transferred to the edge of a new PDA plate. Ak67 cells were streaked in the center of the plate. NB medium was used as a control group, and the plates were incubated upside down at 28°C for 3–5 days. Mycelial growth was observed, and the inhibition effect was statistically analyzed. Each treatment was repeated three times.

[0036] Inhibition rate (%) = (diameter of mycelium in control group - diameter of inhibited mycelium in treatment group) / diameter of mycelium in control group.

[0037] For *Ustilago maydis*, basidiospores of the "+" and "-" types were inoculated into PDA medium for activation. An appropriate amount of colonies was scraped into a sterile centrifuge tube, fully resuspended in sterile water, and the OD600 was adjusted to 1.5. *Ustilago maydis* Ak67 cells described in this invention were inoculated into 10 mL of NB medium and cultured at 200 rpm and 28°C for 5 days. Equal amounts of positive and negative basidiospores were mixed thoroughly, and 100 μL was spread onto PDA medium. Using a blue pipette tip, 50 μL of Ak67 culture solution was inoculated into the medium. The medium was then placed upright in a 28°C incubator and cultured for two days. Mycelial growth was observed, and the inhibitory effect was statistically analyzed. Each treatment was repeated three times.

[0038] The results of the antifungal spectrum determination of strain Ak67 against plant pathogenic fungi are as follows: Figure 3 As shown, Ak67 has a strong inhibitory effect on Fusarium graminearum, Ustilago maydis, and Alternaria solanaceae, and also has a relatively strong inhibitory effect on Fusarium oxysporum and Fusarium moniliforme.

[0039] Table 1. Inhibition rate of strain Ak67 against plant pathogenic fungi

[0040] Pathogens Antibacterial rate (%) Fusarium graminearum 51.5±1.36 Fusarium oxysporum 40.0±1.88 Fusarium moniliforme 39.7±1.1 Alternaria solanacearum 61.5±2.34

[0041] Example 3

[0042] Growth inhibition test of Ak67 strain on Fusarium graminearum mycelium:

[0043] This invention employs the plate confrontation method, culturing *Fusarium moniliforme* Ak67 and *Fusarium graminearum* in a 28°C incubator. A plate without inoculation of the antagonistic strain serves as a blank control group. After 5 days, mycelial samples from the fungal ends are observed under a microscope to assess the mycelial growth of *Fusarium graminearum* in both the experimental and control groups. (See attached image) Figure 4 Figure A shows the blank control group without Ak67 inoculation, where the hyphae grow normally; Figure B shows the treatment group inoculated with Ak67, where the hyphae tips become shorter, swollen and deformed, and have more branches.

[0044] Example 4

[0045] Preparation of Ak67 strain inoculant:

[0046] The Ak67 strain, stored at -80℃, was streaked onto NA solid medium and incubated upside down at 28℃ for 24 hours. Single colonies were picked and inoculated onto NB liquid medium, and cultured with shaking at 28℃ for 2 days to obtain the seed culture. Ak67 was then inoculated at a ratio of 5% onto Gao's No. 1 medium and cultured at 28℃ and 200 rpm for 5 days to obtain the Ak67 bacterial agent, with a viable count of 1×10⁻⁶. 9 ~1×10 10 CFU / mL.

[0047] Example 5

[0048] The growth-promoting effect of Ak67 on greenhouse potted corn:

[0049] Select plump, uniformly sized seeds and sow them in seedling trays. When the corn reaches the 3-leaf stage, rinse the soil around the roots with clean water and soak the roots.

[0050] Ak67 was cultured in NB medium for 2 days, centrifuged at 4000 rpm for 10 min at 4℃, and diluted with sterile water to an OD600 of 1.0. Corn roots were immersed in the bacterial solution for 1 minute, then removed and planted in new seedling trays. The control group received sterile water. One month after planting, traits such as corn plant height were recorded. (See attached image) Figure 5 As shown in Table 2, the maize seedling height in the Ak67 treatment group was 89.04 cm, which was 20.47% higher than that in the control group (73.91 cm), showing a significant difference (p<0.001). The stem diameter of the maize seedlings in the Ak67 treatment group was 20.77 mm, which was 21.53% higher than that in the control group (17.09 mm), showing a significant difference (p<0.001). The results indicate that Ak67 has a good growth-promoting effect on maize.

[0051] Table 2 Effects of strain Ak67 on maize growth

[0052]

[0053] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Kitasori ( Kitasatospora sp. Ak67, with accession number CGMCC No. 29076.

2. Contains *Tetrasporium kiwifruit* as described in claim 1 (… Thesatospora (spe.)Ak67 microbial inoculant.

3. The microbial agent as described in claim 2, characterized in that, The viable count of *Symplocos kiwi* Ak67 in the microbial agent is 1 × 10⁻⁶. 9 ~1×10 10 CFU / mL.

4. The Kitasa polymorpha according to claim 1 ( Thesatospora The application of sp.) Ak67 in the preparation of plant disease control products is characterized by, The plant disease is Fusarium graminearum (Fusarium graminearum). Fusarium graminearum Caused by ).

5. The *Tetrasporium kiwifruit* according to claim 1 (… Thesatospora The application of sp.) Ak67 in the preparation of plant disease control products is characterized by, The plant disease is *Fusarium solani* (…). Fusarium proliferatum Caused by ).

6. The *Tetrasporium kiwifruit* according to claim 1 (… Thesatospora The application of sp.) Ak67 in the preparation of plant disease control products is characterized by, The plant disease is *Smutella canis* ( ). Sporisorium scitamineum Caused by ).

7. The *Tetrasporium kiwifruit* according to claim 1 (… Thesatospora The application of sp.) Ak67 in the preparation of plant disease control products is characterized by, The plant disease is Alternaria alternata (Solanum lycopersicum). Alternaria solani Caused by ).

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