Streptomycete YCB063 capable of preventing and controlling camellia oleifera diseases and application of streptomycete YCB063

Abstract

The invention belongs to the technical field of agricultural microorganisms, and particularly relates to streptomyces YCB063 capable of preventing and controlling camellia oleifera diseases and application of the streptomyces YCB063. The streptomyces sp. YCB063 disclosed by the invention is a new species, and the preservation number of the streptomyces sp. YCB063 is GDMCC No: 65663. The strain has a good antagonistic effect on five camellia oleifera pathogenic fungi, and the bacteriostasis rate reaches 60%-83%; in addition, the streptomyces is extremely easy to culture and easy for industrial production, active substances in fermentation liquor of the streptomyces have a remarkable antibacterial effect, mycelial growth of camellia oleifera pathogenic bacteria can be inhibited, and the streptomyces can be used for preparing biological agents for preventing and treating camellia oleifera diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microorganisms, and particularly relates to a Streptomyces YCB063 capable of preventing and controlling oil tea diseases and its application. Background Art

[0002] Oil tea (Camellia oleifera Abel) belongs to the genus Camellia of the family Theaceae and is a unique woody oil crop in China, widely planted in provinces such as Guangdong, Hunan, and Shanxi. Oil tea not only has a broad market prospect in the field of edible oil, but its by-products also show extremely high economic value in multiple related fields such as medicine, cosmetics, industry, and agriculture.

[0003] There are more than 50 kinds of oil tea diseases in China, among which the most common diseases are Camellia anthracnose (Camelliagloeosporioides) and soft rot of oil tea. Camellia anthracnose is caused by fungi of the genus Colletotrichum spp., mainly damaging the leaves and fruits of oil tea, resulting in fruit cracking and dropping, seriously affecting the yield and quality of oil tea. Since oil tea is an important economic tree species for edible oil, the use of chemical pesticides should be minimized during the prevention and control process to avoid food source pollution. Therefore, the development of highly efficient, low-toxic, and broad-spectrum biological pesticides has become an important means for preventing and controlling oil tea diseases.

[0004] Streptomyces is a type of Gram-positive bacteria widely distributed in nature. Currently, 1236 species have been listed on the LPSN website (https: / / www.bacterio.net / ). Streptomyces can produce a variety of metabolites with novel structures and good activities and is an important source of agricultural antibiotics. According to statistics, about 75% of the antibiotics globally are produced by Streptomyces. In addition to antibiotics, Streptomyces can also produce hormone-like compounds, glucanase, chitinase, etc. that promote plant growth and have important application potential in the biological prevention and control of plant diseases and pests. In addition, a large number of secondary metabolite synthesis gene clusters are hidden in the Streptomyces genome, and the metabolites encoded by the vast majority of these gene clusters have not been isolated and identified. Therefore, Streptomyces has great research potential and application prospects in the development of biological pesticides and the prevention and control of plant diseases. Summary of the Invention

[0005] The object of the present invention is to provide a new species of Streptomyces YCB063 capable of preventing and controlling Camellia oleifera diseases, namely Streptomyces sp. YCB063, which is deposited in the Guangdong Microbial Culture Collection Center (abbreviated as GDMCC; address: 5th Floor, Building 59, 100th Yard, Middle Xianlie Road, Guangzhou; postal code: 510070), with the deposit date being December 23, 2024, and the deposit number being GDMCC No: 65663.

[0006] The second object of the present invention is to provide a preparation containing the Streptomyces YCB063 of claim 1 or its culture.

[0007] The third object of the present invention is to provide the application of the Streptomyces YCB063 or the preparation in preventing and controlling Camellia oleifera diseases.

[0008] The fourth object of the present invention is to provide the application of the Streptomyces YCB063 or the preparation of claim 2 in preventing and controlling Camellia oleifera pathogenic fungal diseases.

[0009] Preferably, the Camellia oleifera pathogenic fungus is one or more of Colletotrichum aeschynomenes HU1397, Colletotrichum tomentosae HU1424, Lasiodiplodia pseudotheobromae HU1186 of the genus Botrytis, Botryosphaeria qinlingensis HU1401 of the genus Botryosphaeria, and Diaporthe searlei HU1392 of the genus Diaporthe.

[0010] The fifth object of the present invention is to provide a method for preventing and controlling Camellia oleifera diseases, which is to use the Streptomyces YCB063 or the preparation to prevent and control Camellia oleifera diseases.

[0011] Preferably, the Camellia oleifera disease is a Camellia oleifera disease caused by one or more of Colletotrichum aeschynomenes HU1397, Colletotrichum tomentosae HU1424, Lasiodiplodia pseudotheobromae HU1186 of the genus Botrytis, Botryosphaeria qinlingensis HU1401 of the genus Botryosphaeria, and Diaporthe searlei HU1392 of the genus Diaporthe.

[0012] A new species of Streptomyces, YCB063, was isolated from the rhizosphere soil of Camellia oleifera in Xingning, Guangdong. This strain has good antagonistic effects against five pathogenic fungi of Camellia oleifera, namely Colletotrichum aeschynomenes HU1397, Colletotrichum tomentosae HU1424, Lasiodiplodia pseudotheobromae HU1186, Botryosphaeria qinlingensis HU1401, and Diaporthe searlei HU1392, showing significant antibacterial effects with an inhibition rate of 60% - 83%. In addition, this Streptomyces is extremely easy to culture and suitable for industrial production. The active substances in its fermentation broth have significant antibacterial effects and can inhibit the mycelial growth of Camellia oleifera pathogenic bacteria, and can be used to prepare biological agents for preventing and controlling Camellia oleifera diseases. Description of the Drawings

[0013] Figure 1 : A: Mycelial morphology of YCB063; B: Individual morphological characteristics of YCB063.

[0014] Figure 2 : Maximum likelihood (ML) phylogenetic tree constructed based on the 16S rRNA gene.

[0015] Figure 3 : Antagonistic effects of strain YCB063 against 5 pathogenic fungi of Camellia oleifera; A: Colletotrichum aeschynomenes HU1397; B: Colletotrichum tomentosae HU1424; C: Lasiodiplodia pseudotheobromae HU1186; D: Botryosphaeria qinlingensis HU1401; E: Diaporthe searlei HU1392; F - J: Antagonistic effect diagrams of YCB063 against the above 5 pathogenic fungi of Camellia oleifera; K: Quantitative detection diagram of YCB063 against the above 5 pathogenic fungi of Camellia oleifera.

[0016] Figure 4 : Antagonistic effects of the fermentation supernatant of strain YCB063 against 2 Colletotrichum gloeosporioides of Camellia oleifera; A: Colletotrichum aeschynomenes HU1397; B: Colletotrichum tomentosae HU1424. Detailed Embodiments

[0017] In order to make the technical problems, technical solutions and technical advantages to be solved by the present invention clearer, the following will be described in detail with reference to specific examples, but the protection scope of the present invention is not limited to the following specific embodiments, and the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and are not limitations of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0018] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0019] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0020] The new species of Streptomyces provided by the present invention is Streptomyces sp. YCB063, which is deposited in Guangdong Microbiological Culture Collection Center (GDMCC for short; address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou; Postal Code: 510070), with a deposit date of December 23, 2024, and a deposit number of GDMCC No: 65663. The new species of Streptomyces of the present invention was isolated from a soil sample of Longao Camellia oleifera planting base (24°24'10"N; 115°40'45"E; 257.5m) in Xingning City, Meizhou, Guangdong.

[0021] Example 1: Isolation and purification of strains

[0022] (1) Sample collection and processing:

[0023] The rhizosphere soil samples of Camellia oleifera were collected from the Long’ao Camellia oleifera planting base in Xingning, Guangdong (24°24'10”N; 115°40'45”E; 257.5m). The rhizosphere surface soil was removed with a shovel, and the soil within 10-20 cm was put into a sterile bag and brought back to the laboratory. After removing the dead branches and leaves and the roots, the soil samples were crushed and passed through a 2 mm pore sieve. The filtered soil was stored in a 4°C refrigerator for later use.

[0024] (2) Isolation and purification of soil bacteria:

[0025] Weigh 5 g of soil sample and add it to a flask containing 45 mL of sterile deionized water. Place the flask in a shaker at 30°C and 160 rpm and incubate for 2 h. On a clean bench, use a pipette to pipette 0.1 mL of the mixed soil suspension into a centrifuge tube containing 0.9 mL of sterile saline. Mix well to obtain 10-1 Diluent. According to the ratio of 1:10, 10 -1 , 10 -2 , 10 -3 and 10 -4 gradient diluents were prepared. Using a pipette, 100 μL of different diluents were respectively and evenly spread on the surface of 0.1 N A medium, and then placed upside down in a constant temperature incubator at 30 °C for cultivation and observation at different times. Single colonies were picked and purified by the four-zone streaking method until the single colonies grown in the fourth zone were completely consistent in size, shape, and color. The bacteria were mixed into 25% glycerol and stored in a -80 °C refrigerator.

[0026] Example 2: Identification of Strain YCB063

[0027] (1) Morphological identification:

[0028] The strain YCB063 was cultured on N A medium for 2 - 3 days, and the morphology of the colony, such as size, shape, color, and edge regularity, was observed. YCB063 was subjected to Gram staining using a Gram staining kit (HKM, Huankai Microbial).

[0029] Figure 1 A shows the morphological characteristics of the isolated strain YCB063. The colonies on the N A plate are small, dry, and milky white; Figure 1 B shows the individual morphological characteristics of the strain. The results show that YCB063 is a Gram-positive bacterium, rod-shaped and linear.

[0030] (2) Molecular identification:

[0031] The DNA of strain YCB063 was extracted using a Magen bacterial DNA extraction kit. Using it as a template, PCR amplification of the 16S rDNA gene was carried out using universal primers (27F / 1492R). Among them, the PCR reaction system (20 μL): 2×phanta MaxMix: 10 μL, 27F: 0.5 μL, 1492R: 0.5 μL, DNA template 0.5 μL, ddH 2 O 8.5 μL. PCR reaction conditions: pre-denaturation temperature and time 95 °C, 5 min; denaturation temperature and time 95 °C, 30 s; annealing temperature and time 56 °C, 30 s; extension temperature and time 72 °C, 1 min, 32 cycles; final extension temperature and time 72 °C, 5 min. After the reaction, 5 μL of the PCR product was subjected to agarose gel electrophoresis. After confirmation, the PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for gene sequencing.

[0032] The obtained sequencing results were uploaded to the EzBiocloud website (EzBioCloud.net|Search about Bacteria or Archaea), and homology sequence alignment was performed with the known 16S rRNA gene. Standard strains with higher homology were selected to construct a maximum likelihood (ML) phylogenetic tree.

[0033] The results were as Figure 2 , and strain YCB063 was identified as the genus Streptomyces, which had the highest similarity of 98.47% with the 16S rRNA gene of the standard strain Streptomyces ferralitis SFOp68 T . Secondly, the similarity with Streptomyces rubrisoli FXJ1.725 T was 98.32%. These values were all less than the species boundary value of 98.65%. In addition, the results of the ML phylogenetic tree showed that YCB063 formed an independent branch and clustered with S. ferralitis SFOp68 T and S. rubrisoli FXJ1.725 T on a large branch, indicating that YCB063 is a new species of the genus Streptomyces.

[0034] Example 3: Antagonistic effect of strain YCB063 against 5 Camellia oleifera pathogenic fungi

[0035] Five pathogenic fungi of Camellia oleifera, Colletotrichum acutatum HU1397, Colletotrichum tomentosae HU1424, Lasiodiplodia pseudotheobromae HU1186, Botryosphaeria qinlingensis HU1401, and Diaporthe searlei HU1392, on PDA plates were punched into blocks using a sterile borer, and the blocks were transferred to the center of NA plates. At a distance of 1 cm from the blocks, the cells of YCB063 were picked with a sterilized toothpick for spot inoculation. Four spots were inoculated on each plate and cultured at 30 °C for 3 d. Whether an inhibition zone was formed on the plate was observed, and the diameter of the inhibition zone was measured.

[0036] The inhibition rate was calculated according to the following formula: Inhibition rate (%) = (colony diameter of the control pathogenic fungus - colony diameter of the treated pathogenic fungus) / colony diameter of the control pathogenic fungus × 100%.

[0037] The results were as Figure 3, compared with the control, YCB063 showed significant antibacterial effects against 5 pathogenic fungi of Camellia oleifera, with the antibacterial rate reaching 60% - 83%.

[0038] Example 4: Detection of the antagonistic effect of the fermentation supernatant of YCB063 against 2 kinds of Camellia oleifera anthracnose by the poisoned plate method

[0039] (1) Preparation of the fermentation supernatant of YCB063:

[0040] Inoculate a single colony of the newly activated strain YCB063 into a test tube containing 3 mL of NB liquid medium, and culture it with shaking at 200 rpm and 30 °C for 1 day. Then, inoculate it into a triangular flask containing 100 mL of NB liquid medium at an inoculation amount of 1%, and culture it with shaking at 200 rpm and 30 °C for 2 days. After that, centrifuge at 12000 rpm for 10 min to collect the fermentation supernatant of YCB063.

[0041] (2) Preparation of poisoned plates and control plates:

[0042] Filter and sterilize the fermentation supernatant of YCB063 with a 0.22 μm filter; heat and melt the solid NA medium. After the medium cools to about 50 °C, add the filtered and sterilized fermentation supernatant of YCB063 at a ratio of 20% (v / v) to prepare poisoned plates. Add 20% of NB liquid medium to the NA medium to make control plates.

[0043] (3) Detection of the antibacterial effect of the fermentation supernatant of YCB063 against 2 kinds of Camellia oleifera anthracnose on poisoned plates

[0044] Transfer the Camellia oleifera anthracnose pathogens Colletotrichum aeschynomenes HU1397 and Colletotrichum tomentosae HU1424 grown on PDA medium for 3 days to the poisoned plates and control plates, and after culturing at 30 °C for 5 days, measure the colony diameters on the poisoned plates and control plates.

[0045] The antibacterial rate is calculated according to the following formula: Antibacterial rate (%) = (colony diameter of the pathogen on the control plate - colony diameter of the pathogen on the poisoned plate) / colony diameter of the pathogen on the control plate × 100%.

[0046] The results are as Figure 4 shown. The fermentation supernatant containing 20% of YCB063 could significantly inhibit the growth of Camellia oleifera anthracnose pathogens, and the antibacterial rate against Colletotrichum aeschynomenes HU1397 reached 78.59%, and the antibacterial rate against Colletotrichum tomentosae HU1424 reached 67.68%.

[0047] It is understood that the above specific embodiments are further descriptions of the present invention and are not used to limit the protection scope of the present invention. For those skilled in the art, all other retouches and modifications obtained without creative labor belong to the protection scope of the present invention.

[0048]

[0049]

Claims

1. A strain of Streptomyces sp. YCB063, whose deposit number is GDMCC No: 65663.

2. A preparation, characterized in that Containing the Streptomyces YCB063 of claim 1 or a culture thereof.

3. Use of the Streptomyces YCB063 described in claim 1 or the preparation described in claim 2 in preventing and controlling oil-tea camellia diseases.

4. Use of the Streptomyces YCB063 according to claim 1 or the preparation according to claim 2 in preventing and controlling pathogenic fungal diseases of tea oil.

5. The use according to claim 4, characterized in that: The oil-tea camellia pathogenic fungi are one or more of the oil-tea camellia anthracnose fungi Colletotrichum aeschynomenes HU1397, Colletotrichum tomentosae HU1424, Lasiodiplodia pseudotheobromae HU1186, Botryosphaeria qinlingensis HU1401, and Diaporthe searlei HU1392.

6. A method for preventing and controlling oil tea diseases, characterized in that: The method comprises using the Streptomyces YCB063 described in claim 1 or the preparation described in claim 2 to prevent and control tea oil diseases.

7. The control method according to claim 6, characterized in that: The oil-tea camellia disease is caused by one or more of the oil-tea camellia anthracnose fungi Colletotrichum aeschynomenes HU1397, Colletotrichum tomentosae HU1424, Lasiodiplodia pseudotheobromae HU1186, Botryosphaeria qinlingensis HU1401, and Diaporthe searlei HU1392.

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