Leptospora sp. Ak67 and application thereof

Abstract

The invention belongs to the field of biological prevention and control, and particularly relates to a new Leptospora sp. Strain and application thereof in plant disease prevention and control. The first aspect of the invention is to provide a strain of Kitasatospora sp. Ak67, the strain Ak67 has been preserved in China General Microbiological Culture Collection Center on November 21, 2023, called CGMCC for short, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 29076. The strain provided by the invention has a broad-spectrum bacteriostatic effect.

Description

Technical Field

[0001] The invention belongs to the field of biological control, and specifically relates to a new species of Kitasatosporium and application thereof in plant disease control. Background Art

[0002] Plant diseases seriously affect plant growth, leading to a decline in yield and quality, and are one of the main threats to agricultural production. There are more than 30,000 types of fungal diseases, accounting for more than 70% of plant diseases. They are one of the most important biological factors for the occurrence of plant diseases. Several or even dozens of fungal diseases may be found on one crop. At the same time, due to the strong resistance of fungal spores, prevention and control are also quite challenging. Chemical control is the most commonly used method for controlling plant diseases. Although it is effective in the short term, long-term use can easily lead to drug resistance in pathogens, which in turn leads to potential problems such as environmental pollution and food security. Biological control technology has become a key technical support for the green prevention and control of plant fungal diseases due to its advantages such as low environmental pollution, low risk of drug resistance, and safety for humans, animals and natural enemies.

[0003] Kitasatospora belongs to the Actinomycetes of the family Streptomycetes and is a microorganism with multiple biological activities. Kitasatospora can inhibit the growth of some plant pathogenic fungi, produce indoleacetic acid, and promote the dissolution and release of phosphates to promote plant growth. Kitasatospora is also an important source of natural products. A large-scale comparative analysis of bacterial secondary metabolite gene synthesis clusters (BGCs) found that the average number of BGCs in the Kitasatospora genome is greater than that of Streptomyces. These indicate that Kitasatospora is a valuable source for the discovery of new active substances in the future. However, new antagonistic Kitasatospora species and new active substances are rarely discovered. The results of this study will provide new strain resources for the biological control of various plant fungal diseases. At the same time, the discovery of new active substances also provides theoretical and technical support for the development of biocontrol agents. Summary of the invention

[0004] The present invention aims to provide a new strain of Kitasatosporium with broad-spectrum antibacterial effect, a bacterial agent and application thereof.

[0005] The first aspect of the present invention is to provide a strain of Kitasatospora sp. Ak67. The strain Ak67 was deposited in the General Microbiology Center of the China Culture Collection Administration on November 21, 2023, referred to as CGMCC. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 29076.

[0006] As a second aspect of the present invention, the present invention provides a microbial agent prepared from the Kitasatosporium Ak67 strain.

[0007] Furthermore, the bacterial preparation method of Kitasatosporium Ak67 is as follows: the strain Ak67 is activated on a NA solid plate, cultured at 28°C for 2 days, a single colony is picked and inoculated into a NB seed medium, cultured at 28°C and 200rpm / min for 1 day, inoculated into Gao's No. 1 liquid medium at a ratio of 5%, and cultured at 28°C and 200rpm / min for 5 days to obtain a fermentation liquid of the strain Ak67, and the number of viable bacteria reaches 1×10 9 ~1×10 10 CFU / mL.

[0008] As a third aspect of the present invention, the present invention also provides the use of the above-mentioned new species of Kitasatosporium in preventing and controlling 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. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0012] Figure 2 This is the phylogenetic tree of strain Ak67 based on the whole genome;

[0013] Figure 3 This is a graph showing the antibacterial effect of strain Ak67 on plant pathogenic fungi;

[0014] Figure 4 The inhibition of strain Ak67 on the mycelial growth of Fusarium graminearum, A: blank control, B: Ak67;

[0015] Figure 5 This shows the growth promotion effect of strain Ak67 on corn. DETAILED DESCRIPTION

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

[0017] The culture medium used in the following examples is as follows:

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

[0019] Gao's medium No. 1: KNO3 1g / L, soluble starch 20g / L, NaCl 0.5g / L, K2HPO4·3H2O0.5g / L, MgSO4·7H2O 0.5g / L, FeSO4·7H2O 0.01g / L, agar powder 15g / L;

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

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

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

[0023] Example 1

[0024] This example is the separation and identification of Kitasatosporium Ak67:

[0025] 1. Strain Isolation and Preservation

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

[0027] 2. Identification of Ak67 strain

[0028] Refer to the method of Liu Qing et al. (2021) for crude DNA extraction, scrape a small amount of bacteria, add 50 μL of 5% Chelex solution, heat it at 95°C for 20 minutes on a PCR instrument, and use it as a PCR template. Use bacterial 16S rDNA universal primers 27F and 1492R for PCR amplification, and send the PCR product to the company for sequencing. The sequencing results of 16s rDNA are shown in SEQ ID NO.1. The sequence was uploaded to EzBioCloud for sequence alignment, and the results showed that Kitasatospora xanthocidica NBRC 13469 had the highest similarity of 99.16%. In order to further determine the taxonomic status of strain Ak67, we sequenced the whole genome of strain Ak67. The whole genome sequence of strain Ak67 was uploaded to Type (Strain) Genome Server (TYGS) to calculate the digital DNA-DNA hybridization (dDDH) value between Ak67 and other strains. The average nucleotide identity (ANI) between strains was automatically calculated using PHANTASM v1.0.4. The classification data of LPSN was manually updated to the latest version, and then the phylogenetic markers of strain Ak67 were calculated. DNA-directed RNA polymerase subunit beta protein was selected as the phylogenetic marker. The software automatically downloaded the whole genome of related species and the ANI value was calculated using Pyani v0.2.11. A phylogenetic tree was constructed based on the tandem alignment results of 426 single-copy core genes by FastTree v2.1.1 (see Appendix). 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, K.aureofaciens ATCC 10762, K.xanthocidica JCM 4862, and K.purpeofusca NRRL B-1817 clustered together to form an independent branch, and their ANI values ​​were 88.4%, 88.4%, and 87.3%, respectively, which were lower than the threshold of 95% for new species classification, thus confirming that Ak67 was a new species of the genus Kitasatosporium.

[0029] The above-mentioned strain Ak67 was deposited on November 21, 2023 in the General Microbiology Center of the China Culture Collection Administration, referred to as CGMCC. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 29076.

[0030] Example 2

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

[0032] The Ak67 isolated in Example 1 was tested for growth inhibition of various crop fungal pathogens.

[0033] Information on plant pathogens is as follows: 1) Fusarium graminearum, the main pathogen of crop stem rot; 2) Fusarium oxysporum is a soil-borne pathogenic fungus distributed worldwide, which can cause wilt of more than 100 species of plants such as melons, Solanaceae, bananas, cotton, legumes and flowers; 3) Fusarium solani is a conditional pathogen that mainly causes root rot, wilt and other diseases; 4) Ustilago officinalis is an important pathogen that causes sugarcane smut. The pathogen forms binucleate hyphae through sexual mating of haploid basidiospores of different mating types to infect the host and cause disease; 5) Alternaria solani is the pathogen that causes early blight of tomatoes.

[0034] For Fusarium oxysporum, Fusarium laminarum, and Alternaria solani, the fungi to be tested were inoculated into PDA medium for activation and standby use. Pick the Ak67 bacteria of Kitasatosporium described in the present invention and inoculate them into 10mL NB medium, 200rpm, and culture at 28°C for 2 days. Use a blue gun tip to beat the mycelium at the edge of the fungus plate, inoculate it in the center of a new PDA plate, and streak Ak67 at 2cm from the center. Use NB medium as the control group, invert and culture at 28°C for 3 to 5 days, observe the growth of mycelium, and statistically analyze the inhibitory effect. Each treatment is repeated three times.

[0035] For Fusarium graminearum, the fungus to be tested was inoculated into PDA medium for activation and standby. Pick the Ak67 bacteria of Kitasatosporium described in the present invention and inoculate it into 10mL NB medium, 200rpm, 28°C for 2 days. Use a blue gun tip to beat the fungus cake at the edge of the fungus plate containing hyphae, inoculate it to the edge of a new PDA plate, and streak Ak67 inoculated in the center of the plate. Use NB medium as the control group, invert and culture at 28°C for 3 to 5 days, observe the growth of hyphae, and statistically analyze the inhibitory effect. Each treatment is repeated three times.

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

[0037] For sugarcane whip smut fungus, "+" and "-" type basidiospores were inoculated into PDA medium for activation and standby use. Scrape an appropriate amount of colonies into a sterile centrifuge tube, fully suspend with sterile water, and adjust OD600 to 1.5. Pick the Ak67 bacteria of Kitasatosporium described in the present invention and inoculate them into 10mL NB medium, 200rpm, 28°C for 5 days. Mix equal amounts of positive and negative basidiospores, take 100μL and apply it to PDA medium, use a blue gun tip to punch holes, inoculate 50μL of Ak67 culture solution, place it upright at 28°C incubator, culture for two days, observe the growth of mycelium, and statistically analyze the inhibitory effect. Each treatment was repeated three times.

[0038] The results of the antibacterial 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 officinalis, and Alternaria solani, and also has a relatively strong inhibitory effect on Fusarium oxysporum and Fusarium solani.

[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 39.7±1.1 Alternaria solani 61.5±2.34

[0041] Example 3

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

[0043] The present invention adopts the plate confrontation method, and the Kitasatosporium Ak67 and the Fusarium graminearum are cultured in a 28°C incubator, and a plate without antagonistic strains is used as a blank control group. After 5 days, the hyphae at the end of the fungus are observed under a microscope to observe the growth of the Fusarium graminearum hyphae in the experimental group and the control group (see Appendix). Figure 4 Figure A is the blank control group without Ak67 inoculation, with normal mycelium growth; Figure B is the treatment group inoculated with Ak67, with mycelium tips shortened, swollen and deformed, and more branches.

[0044] Example 4

[0045] Preparation of Ak67 strain inoculum:

[0046] The strain Ak67 was taken out from the -80℃ refrigerator, streaked on NA solid medium and inverted at 28℃ for 24h, and a single colony was picked and inoculated into NB liquid medium, and incubated in a shaker at 28℃ for 2 days as seed liquid. At a ratio of 5%, Ak67 was inoculated into Gao's No. 1 medium and cultured at 28℃ and 200rpm / min for 5 days to obtain Ak67 bacterial agent, and the number of viable bacteria was 1×10 9 ~1×10 10 CFU / mL.

[0047] Example 5

[0048] Ak67 promotes the growth of greenhouse potted corn:

[0049] Select seeds with full grains and uniform size and sow them in pots. When the corn just 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°C, and diluted with sterile water to an OD600 of 1.0. Corn roots were immersed in the bacterial solution and taken out after 1 minute and planted in new pots. The control group was sterile water. After 1 month of planting, the height and other traits of corn were counted. Figure 5 As shown in Table 2, the height of corn seedlings in the Ak67 treatment group was 89.04 cm, which was 20.47% higher than that in the blank group (73.91 cm), with a significant difference (p<0.001); the stem diameter of corn seedlings in the Ak67 treatment group was 20.77 mm, which was 21.53% higher than that in the blank group (17.09 mm), with a significant difference (p<0.001). The results showed that Ak67 had a good growth-promoting effect on corn.

[0051] Table 2 Effect of strain Ak67 on corn growth

[0052]

[0053] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Kitasatospora sp. Ak67, whose accession number is CGMCC No.29076.

2. A microbial agent containing Kitasatospora sp. Ak67 as claimed in claim 1.

3. The microbial agent according to claim 2, characterized in that The microbial agent comprises live bacteria of Kitasatosporium Ak67 and fermentation liquid containing active substances.

4. The microbial agent according to claim 3, characterized in that The viable count of Kitasatosporium Ak67 was 1×10 9 ~1×10 10 CFU / mL.

5. Use of Kitasatospora sp. Ak67 according to claim 1 in the preparation of a microbial agent for preventing and controlling plant diseases and promoting plant growth.

6. The use according to claim 5, characterized in that: The plant disease is caused by Fusarium graminearum, Fusarium oxysporum, Fusarium proliferatum, Sporisorium scitamineum or Alternaria solani.

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