A strain of Streptomyces gaoyangensis, a bacterial agent, its application and its preparation method

Abstract

The present invention discloses a strain of Streptomyces koyangensis, a microbial agent, its application, and a preparation method. This strain has excellent control effects against anthrax and rice blast, providing a new solution for the prevention and control of these diseases. Furthermore, this Streptomyces koyangensis is salt-alkali and heavy metal-tolerant, making it a potential strain for controlling plant diseases in saline-alkali land and heavy metal-contaminated soil environments. It has promising market application prospects for its application in cultivation in saline-alkali land, secondary salinization, and heavy metal-contaminated soil.

Description

Technical Field

[0001] The present invention relates to the field of biological control of plant diseases, and in particular to a strain of Streptomyces galangatus, a bacterial agent, its application and a preparation method thereof. Background Art

[0002] Staphylococcus aureus, a member of the genus Staphylococcus, is a representative Gram-positive bacterium and a common foodborne pathogen. It commonly inhabits the skin, nasal cavity, throat, stomach, intestines, carbuncles, and suppurative sores of humans and animals, and is also ubiquitous in air, sewage, and other environmental environments. Under appropriate conditions, S. aureus can produce enterotoxins, causing food poisoning. Food poisoning caused by S. aureus accounts for approximately 25% of foodborne microbial food poisoning incidents, making it the third most common microbial pathogen after Salmonella and Bacillus parahaemolyticus. This strain exhibits significant inhibitory activity against S. aureus, and further research is needed into its active metabolites.

[0003] Cucumber anthracnose is a disease of cucumbers caused by species of the Cucurbitaceae family, such as Colletotrichum spinulosum. Cucumber anthracnose can occur from the seedling stage to the adult stage. In seedlings, semi-elliptical light brown lesions often appear on the edges of the cotyledons, with orange-yellow dot-like colloids growing on them. In severe cases, the base of the stem near the ground turns yellow-brown and gradually shrinks, causing the seedling to fall over. The cucumber anthracnose pathogen overwinters in the soil as mycelium and pseudosclerotia along with diseased remains, and seeds can also carry the bacteria. The pathogen can also survive saprophytic damage on wooden frames in greenhouses and sheds. After overwintering, it produces a large number of conidia, which become the source of initial infection. In the field, conidia are spread by wind, rain, insects and agricultural operations.

[0004] Rice blast is a major rice disease, causing significant yield losses, ranging from 40% to 50% in severe cases to total crop failure. The disease occurs in various locations, most commonly affecting leaves and nodes, resulting in varying degrees of yield loss. Early and severe panicle or node blast, in particular, can cause white panicles and ultimately total crop failure. The pathogen overwinters primarily as conidia and mycelium on rice straw and grains. The following year, conidia are produced and spread to rice plants by wind and rain. They germinate, invade the host, and spread to neighboring cells, forming a centrally infected plant. Conidia formed in the diseased area are then transported by wind and rain for reinfection.

[0005] Chemical control options for cucumber anthracnose and rice blast are limited. Commonly used chemical agents include carbendazim, iprodione, and peracetic acid for anthracnose, and tricyclazole, chlorpyrifos, and chlorpyrifos for rice blast. Long-term overuse of chemical agents increases the risk of pathogen resistance, leading to reduced control effectiveness. Furthermore, pesticide residues can cause environmental pollution and pose a high risk to humans and animals. The ban on highly toxic pesticides also complicates the selection of chemical control options. In recent years, with growing environmental awareness in agricultural production, the discovery and utilization of more environmentally compatible biological control resources has attracted widespread attention from researchers, as they are beneficial to sustainable agricultural development and better meet the needs of green control and ecological security. Therefore, the development of biocontrol agents that are effective against both anthracnose and rice blast is imperative.

[0006] Soil salinization, a major type of soil degradation, is a significant constraint on agricultural development and crop growth. The development of biocontrol agents based on salt-tolerant microorganisms is crucial for improving land productivity and maintaining the red line for cultivated land. Furthermore, beneficial microorganisms can enhance plant salt tolerance and promote plant growth.

[0007] Arable land slightly contaminated by heavy metals is still an important agricultural production resource in my country. In such plots, the excessive presence of heavy metals causes a certain degree of immune damage to plants, making them more susceptible to infection by pathogens and diseases. However, chemical pesticides are also one of the sources of heavy metal pollution in farmland, so the best choice for plant disease prevention and control in such plots is environmentally friendly microbial pesticides. The ability of biocontrol bacteria to colonize and reproduce well in the application microhabitat is the primary condition for microbial pesticides to play a preventive and control role. Therefore, in plots slightly contaminated by heavy metals, microbial pesticides containing biocontrol bacteria with good heavy metal tolerance (resistance) can better play a role in preventing and controlling plant diseases. Summary of the Invention

[0008] In order to obtain a strain that is resistant to salt, alkali and heavy metals and can prevent and control anthrax and rice blast, the present invention provides a strain of Streptomyces gaoyangensis. The Streptomyces gaoyangensis of the present invention can prevent and control cucumber anthrax and rice blast, and its antibacterial effect is more than 40%. At the same time, the strain has strong salt, alkali and heavy metal resistance, indicating that the strain or biocontrol agent has good application prospects in the field of preventing and controlling cucumber anthrax and rice blast and in saline-alkali and heavy metal polluted environments.

[0009] The present invention provides a strain of Streptomyces koyangensis, wherein the Streptomyces koyangensis is WY23, ​​which was deposited in the China Center for Type Culture Collection on June 14, 2024, with a deposit number of CCTCC NO: M20241230, and the address of the collection center is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0010] The second object of the present invention is to provide the use of the Streptomyces gaoyangensis in antibacterial activities.

[0011] The antibacterial agent includes: inhibiting Colletotrichum orbiculare, Pyricularia oryzae and Staphylococcus aureus.

[0012] Furthermore, the pathogen of anthrax is Colletotrichumorbiculare of the Cucurbitaceae family.

[0013] Furthermore, the pathogen of rice blast is Pyricularia oryzae.

[0014] The third object of the present invention is to provide the application of the Streptomyces gaoyangensis in preventing and controlling plant diseases in saline-alkali and heavy metal-contaminated soil environments.

[0015] The plant diseases include: inhibiting diseases caused by Colletotrichum orbiculare or Pyricularia oryzae.

[0016] The fourth object of the present invention is to provide a biocontrol agent prepared by the Streptomyces gaoyangensis.

[0017] The fifth object of the present invention is to provide a method for preparing the biocontrol agent, wherein the biocontrol agent is prepared by activating and fermenting the Streptomyces gaoyangensis.

[0018] The fermentation culture was carried out in ISP2 liquid medium at 28° C. with shaking for 3 days.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention isolates a strain of Streptomyces koyangensis, and the biocontrol agent prepared from the strain is cultured in opposition to Colletotrichum spp. The test results show that the biocontrol agent has a significant inhibitory effect on the growth of Colletotrichum spp., with an inhibition rate of 42.2%; the agent can prevent and control anthracnose. The biocontrol agent prepared by the present invention is co-cultured with Pyricularia oryzae, and the test results show that the biocontrol agent has a significant inhibitory effect on Pyricularia oryzae, with an inhibition rate of 40.4%; the agent can prevent and control rice blast. The biocontrol agent prepared from the biocontrol bacterium WY23 disclosed in the present invention has a good control effect on anthracnose and rice blast, provides a new solution for the control of anthracnose and rice blast, and has good market application prospects.

[0021] 2. The Streptomyces koyangensis provided by the present invention is tolerant to salt, alkali, and heavy metals, and can be used as a potential strain for preventing and controlling plant diseases in saline-alkali land and heavy metal-contaminated soil environments. It can be used in planting in saline-alkali land and in secondary salinization and heavy metal-contaminated soils.

[0022] Information on the deposit of biological materials

[0023] WY23, ​​referred to as Streptomyces koyangensis in this application, was deposited in the China Center for Type Culture Collection on June 14, 2024, with the deposit number CCTCC NO: M 20241230. The address of the collection center is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the classification name is Streptomyces koyangensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0025] Figure 1 This is a colony morphology diagram of Streptomyces gaoyangensis WY23 in the present invention.

[0026] Figure 2 This is a mycelial morphology diagram of Streptomyces gaoyangensis WY23 in the present invention.

[0027] Figure 3 This is the phylogenetic analysis of Streptomyces gaoyangensis WY23 in the present invention.

[0028] Figure 4 This is a safety test of Streptomyces gaoyangensis WY23 in the present invention. DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. The experimental methods described in the examples of the present invention are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0030] Example 1: A strain of Streptomyces gaoyangensis and its application.

[0031] 1. Isolation and identification of strains

[0032] The bacteria were isolated and purified from rice rhizosphere soil. The specific method was as follows: 10 g of rhizosphere soil was added to 90 mL of sterile distilled water with glass beads under sterile conditions, shaken and centrifuged, and diluted 10-fold gradiently. 100 μL of 10 -2dilution and 10 -3 The 100-fold dilution was evenly spread on ISP2 solid culture medium, and nalidixic acid and cycloheximide were added to the ISP2 culture medium at final concentrations of 20 mg / L and 25 mg / L, respectively, to inhibit the growth of bacteria and fungi.

[0033] After culturing at 28°C for 5-7 days, single colonies were inoculated onto ISP2 medium plates. The strain was purified by streaking, and single colonies of the purified strain were inoculated onto the same slant as the ISP2 medium for initial preservation. Finally, the pure spore suspension was stored in 25% glycerol tubes and frozen at -80°C.

[0034] ISP2 medium formula (g / L): yeast powder 4, malt extract powder 10, glucose 4, agar 15-20, distilled water 1L, natural pH.

[0035] Based on the morphological, physiological and biochemical characteristics, 16s rRNA sequence, and specific primer analysis of Streptomyces, it was ultimately identified as Streptomyces koyangensis. The identification results are as follows:

[0036] 1. Morphological characteristics of bacteria:

[0037] Gram staining was positive. The aerial hyphae of strain WY23 on ISP2 medium were luxuriant and grayish white, the spores were white and powdery, and the hyphae in the base were light brown ( Figure 1 ). Observing the fungus under a microscope, the mycelium is linear, the spore threads are straight or curved, the spores are nearly round and smooth, and the spores can grow in strings. ( Figure 2 ).

[0038] 2. Physiological and biochemical characteristics

[0039] The physiological and biochemical characteristics of Streptomyces gaoyangii WY23 are shown in Table 1. WY23 hydrolyzes starch, liquefies gelatin, peptizes milk, and reduces nitrates and H2S. However, it does not degrade fat or cellulose, and does not produce melanin. It grows on α-D-glucose, L-arabinose, D-fructose, D-mannitol, and D-xylose, but not on triglycerides, raffinose, rhamnose, sucrose, or L-inositol.

[0040] Table 1 Analysis of physiological and biochemical characteristics of Streptomyces gaoyangensis WY23

[0041]

[0042] Note: +: positive, -: negative.

[0043] 3. 16S rDNA sequence analysis

[0044] DNA was extracted, and a 16s rDNA fragment was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR system (50 μL) was as follows: 10 pmol of primer, 0.2 mM dNTP, 1.5 mM MgCl₂, 2.5 U Taq DNA polymerase (TaKaRa), 1× PCR buffer (TaKaRa), and 25 ng of genomic DNA template. PCR reaction conditions were: 94°C denaturation for 5 min, 95°C denaturation for 30 s, 58°C annealing for 45 s, and 72°C extension for 90 s. After 30 cycles, a full extension at 72°C for 10 min was performed. PCR products were separated and purified by 1% agarose gel electrophoresis and analyzed on an ABI 3730 DNA sequencer (Sangon Biotech Co., Ltd.). The phylogenetic tree was constructed using the Neighbor-Joining method (NJ) using the software Culstal W2 and Mega 7.0. Figure 3 Based on colony and bacterial morphological observations, physiological and biochemical assays, combined with 16S rDNA sequence alignment and phylogenetic analysis, strain WY23 was preliminarily identified as Streptomyces koyangensis. This strain was deposited with the China Center for Type Culture Collection on June 14, 2024, with the accession number CCTCC NO: M 20241230. The collection center address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0045] 2. Antibacterial Analysis

[0046] 1. Pathogen selection

[0047] Colletotrichum orbiculare and Pyricularia oryzae were used as test pathogens.

[0048] 2. Antibacterial experiment

[0049] Spread the spores of Streptomyces WY23 on ISP2 medium plates and incubate at 28°C for 3 days. Use a borer (8 mm diameter) to punch holes in the plate where the bacterial lawn grows evenly to prepare agar blocks for later use.

[0050] After activating the test pathogenic fungi, take a bacterial cake with a diameter of 8 mm and place it at the center of the PDA culture medium. Streptomyces agar blocks are placed at three sites around the pathogens. Each pathogen is cultured alone as a control. Three parallel cultures of each strain are cultured in an inverted manner at 28°C for 5-7 days to observe whether an inhibition zone is produced and measure the width of the inhibition zone to calculate its average value.

[0051] Prepare a suspension of Staphylococcus aureus in sterile water and spread evenly onto a NB agar plate. Subsequently, place a Streptomyces agar block on the NB agar plate coated with S. aureus in triplicate. After incubation at 30°C for 2-3 days, observe for the formation of inhibition zones and record the size of these zones using the cross-hatch method, calculating the average value.

[0052] The average width of the inhibition zone or the average diameter of the inhibition circle in each treatment group is shown in Table 2.

[0053] The formula of PDA culture medium (g / L): potato 200, glucose 20, agar 15-20, distilled water 1L, natural pH.

[0054] The formula of NB culture medium (g / L): beef extract 3, peptone 10, sodium chloride 5, agar 15-20, distilled water 1L, pH 7.0-7.2.

[0055] Table 2 Antibacterial activity of Streptomyces gaoyangensis WY23

[0056]

[0057] 3. Preparation of biocontrol agents

[0058] The biocontrol bacteria WY23 was cultured in ISP2 liquid medium at 28°C with shaking for 3 days, and the obtained bacterial solution was the biocontrol agent.

[0059] 4. Salt and alkali resistance test

[0060] Biocontrol strain WY23 was cultured in liquid ISP2 medium with shaking for 48 hours. The culture was then diluted 100-fold and inoculated into ISP2 liquid medium (pH values ​​of 5, 6, 7, 8, 9, 10, 11, and 12, and NaCl concentrations of 0, 1%, 3%, 5%, 7%, 10%, and 12%, respectively). Culture was continued for 2-5 days. Growth was determined by observing turbidity, biofilm formation, and precipitation at the bottom of the shake flask. The results (Tables 3 and 4) indicate that biocontrol strain WY23 is salt- and alkali-tolerant and has potential as a biocontrol resource in saline- and alkaline environments. The bacterial concentration initially increased and then decreased with increasing pH. The highest concentration of the biocontrol strain was achieved at pH 9, indicating the fastest growth and optimal growth. In addition, as the NaCl concentration increased, the bacterial concentration first increased and then decreased. When the NaCl concentration was 5%, the concentration of biocontrol bacteria was the highest, indicating that the biocontrol bacteria reproduced the fastest and was most suitable for growth.

[0061] Table 3 Acid and alkali resistance test of Streptomyces gaoyangensis WY23

[0062]

[0063] Note: +: positive, -: negative.

[0064] Table 4 Salt stress tolerance test of Streptomyces gaoyangensis WY23

[0065]

[0066] Note: +: positive, -: negative.

[0067] 5. Heavy metal resistance testing

[0068] The biocontrol strain WY23 was cultured in liquid ISP2 medium with shaking for 48 hours. The activated WY23 strain was then streaked onto ISP2 agar plates supplemented with varying concentrations of K₂Cr₂Oₐ, CdCl₂, ZnSO₄·7H₂O, MnSO₄·H₂O, CuSO₄·5H₂O, Pb(NO₃)₂, and CoCl₃. WY23 culture medium without any heavy metals served as a control. Three replicates were used for each treatment, and the strains were incubated at 28°C for 5-7 days. The minimum inhibitory concentration (MIC) for each metal was determined by the presence or absence of WY23 growth on the plates. Table 5 shows the minimum inhibitory concentrations (MICs) for various heavy metals against WY23, ​​indicating that the strain exhibits a moderate tolerance to common heavy metals.

[0069] Table 5 Minimum inhibitory concentrations of different heavy metals on WY23

[0070]

[0071] VI. Safety Testing of Biocontrol Bacteria

[0072] The biocontrol bacteria WY23 was inoculated on blood agar medium and cultured at 28℃ for 3 days. It was found that no transparent circle appeared around the colony of the biocontrol bacteria WY23 ( Figure 4 ), indicating that the biocontrol bacteria has no hemolytic effect, indicating that the biocontrol bacteria has good biocontrol bacteria safety.

Claims

1. A strain of Streptomyces gaoyangensis ( Streptomyces koyangensis ), characterized in that The Streptomyces gaoyangensis strain is WY23, ​​and its deposit number is CCTCC M 20241230.

2. The use of Streptomyces gaoyangensis according to claim 1 in antibacterial treatment; the antibacterial treatment is: inhibiting anthracnose of melons ( Colletotrichum orbiculare ), Rice blast fungus ( Pyricularia oryzae ), Staphylococcus aureus ( Staphylococcus aureus ).

3. The use of the Streptomyces gaoyangensis according to claim 1 in preventing and controlling plant diseases in saline-alkali and heavy metal-contaminated soil environments; the plant diseases are: anthracnose of cucurbits ( Colletotrichum orbiculare ) or rice blast fungus ( Pyricularia oryzae ) caused by diseases.

4. A biocontrol agent prepared from the Streptomyces gaoyangensis according to claim 1.

5. A method for preparing the biocontrol agent according to claim 4, characterized in that: The biocontrol agent is prepared by activating and fermenting the Streptomyces gaoyangensis.

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