Streptomycete for preventing and treating oak tree ailanthic guerin bacteria as well as application and fermentation method of streptomycete
By optimizing the fermentation conditions, the sterile fermentation broth was prepared using Streptocytica HZW strain of Streptocytica, which solved the problem that the inability to effectively inhibit the genus Cytosaccharide in the prior art, and achieved effective inhibition of the bacteria and prevention and treatment of plant diseases.
Patent Information
- Application Number
- CN202510067201.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-16
- Publication Date
- 2025-05-27
AI Technical Summary
The prior art lacks the bio-defensive bacteria that can effectively inhibit the genus Cyperus, resulting in the inability to effectively prevent and treat plant diseases caused by the bacteria.
The Streptocytica HZW strain was used for fermentation, and a sterile fermentation broth with strong antibacterial effect was prepared by optimizing fermentation conditions such as culture medium, temperature and pH.
This method can significantly inhibit saccharin, provide an effective microbial preparation for preventing and treating plant diseases, and has good development and application prospects.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical fields of microbial fermentation and biological control. More specifically, it relates to a Streptomyces strain against Phytophthora cinnamomi of oak trees, its application and fermentation method. Background Art
[0002] China is one of the origin and modern distribution centers of Quercus plants. The area and stock volume of Quercus tree species rank first in the national forest resources, playing an important role in maintaining the stability of the ecosystem structure such as water conservation and wind prevention and sand fixation. Oak leaves are rich in nutritional value, containing proteins, mineral elements and trace elements, and are often used as feed for tussah silkworms, livestock and wild animals. However, due to the long-term growth of Quercus plants in the natural environment, they are susceptible to various pests and diseases.
[0003] As an important pathogen of Quercus plants, the genus Phytophthora has been found in many countries and regions. The fungi of this genus can infect one or more Fagaceae plants, mainly including Quercus, Castanea and Fagus, causing leaf spot and leaf blight symptoms in plants. In addition, there are reports of other host plants, such as plants in the families Altingiaceae, Anacardiaceae and Davidiaceae. At present, this disease is still in the stage of pathogen isolation and identification, and there is no report on the control using chemical agents or biocontrol agents.
[0004] Therefore, it is of great significance to study biocontrol bacteria that can effectively control Phytophthora cinnamomi and thus prevent and control the plant diseases caused by Phytophthora cinnamomi. Summary of the Invention
[0005] The present invention aims to solve the problem of the lack of biocontrol bacteria that can inhibit Phytophthora cinnamomi in the prior art, provides a Streptomyces asoensis HZW strain that can inhibit Phytophthora cinnamomi, and provides a fermentation culture method with the best antibacterial effect for the HZW strain.
[0006] The first object of the present invention is to provide the application of Streptomyces asoensis HZW strain in inhibiting Phytophthora cinnamomi, or the application in preparing a product for inhibiting Phytophthora cinnamomi.
[0007] The second object of the present invention is to provide a fermentation method of Streptomyces for inhibiting Phytophthora cinnamomi.
[0008] The third object of the present invention is to provide the application of the fermentation product obtained by the above fermentation method.
[0009] The above objects of the present invention are achieved by the following technical solutions:
[0010] The application of Streptomyces asoensis HZW strain in inhibiting Phytophthora cinnamomi.
[0011] The application of Streptomyces asoensis HZW strain in preparing a product for inhibiting Phytophthora cinnamomi.
[0012] Specifically, the Streptomyces asoensis HZW strain was deposited at the Guangdong Microbial Culture Collection Center on January 17, 2024, with the deposit number GDMCC NO: 64293.
[0013] Use of the above Streptomyces asoensis HZW strain in controlling plant diseases caused by Keishibacterium tsukii.
[0014] Use of the above Streptomyces asoensis HZW strain in preparing a product for controlling plant diseases caused by Keishibacterium tsukii.
[0015] The present invention provides a Streptomyces fermentation method for inhibiting Keishibacterium tsukii. The Streptomyces asoensis is inoculated into a fermentation medium and fermented at 25 - 35°C for 4 - 10 days.
[0016] Preferably, the Streptomyces asoensis is the Streptomyces asoensis HZW strain described in claim 1.
[0017] Preferably, the fermentation is carried out at 25 - 30°C.
[0018] More preferably, the fermentation is carried out at 25 - 28°C.
[0019] Preferably, the fermentation is carried out for 6 - 10 days.
[0020] More preferably, the fermentation is carried out for 6 days or 8 - 10 days.
[0021] Preferably, the fermentation medium is any one of TSB fermentation medium, YPG fermentation medium, PDB fermentation medium, and ISP2 fermentation medium.
[0022] More preferably, the fermentation medium is YPG fermentation medium.
[0023] Preferably, the pH value of the fermentation medium is 6 - 9.
[0024] More preferably, the pH value of the fermentation medium is 6 - 7.
[0025] As a preferred embodiment, the Streptomyces fermentation method is: inoculating the Streptomyces asoensis HZW strain into a YPG fermentation medium with a pH value of 6 and fermenting at 25°C for 9 days.
[0026] Use of the Streptomyces fermentation product obtained by the above Streptomyces fermentation method in inhibiting Keishibacterium tsukii.
[0027] Use of the Streptomyces fermentation product obtained by the above Streptomyces fermentation method in preparing a product for inhibiting Keishibacterium tsukii.
[0028] Use of the streptomyces fermentation product prepared by the above streptomyces fermentation method in preventing and treating plant diseases caused by *Trichococcus tsubakiensis*.
[0029] Use of the streptomyces fermentation product prepared by the above streptomyces fermentation method in preparing a product for preventing and treating plant diseases caused by *Trichococcus tsubakiensis*.
[0030] Specifically, the fermentation product is the fermentation broth after removing the thalli.
[0031] The present invention has the following beneficial effects:
[0032] 1. The sterile fermentation broth of the *Streptomyces asoensis* HZW strain of the present invention has a good effect of inhibiting *Trichococcus tsubakiensis*, providing a basis for the production of a *Trichococcus tsubakiensis* control agent.
[0033] 2. According to the medium optimization principle, the present invention optimizes the conditions of three factors, namely, the culture days, temperature, and pH of the screened fermentation medium, provides a nutrient environment more suitable for the growth and reproduction of *Streptomyces* HZW to produce active substances, and further verifies the optimal fermentation conditions by using the response surface optimization experimental design to improve the antibacterial ability of the fermentation broth.
[0034] 3. *Streptomyces* HZW of the present invention can be used to prevent and treat the diseases of *Trichococcus tsubakiensis* on oak trees. The sterile fermentation broth of *Streptomyces* HZW can be used to prepare a microbial agent for preventing and treating *Trichococcus tsubakiensis*, and has good development and application prospects in the related field of microbial pesticides. Description of the Drawings
[0035] Figure 1 Shows the antibacterial effects of different types of fermentation media.
[0036] Figure 2 Shows the measurement results of the antibacterial diameters of different types of fermentation media.
[0037] Figure 3 Shows the stability test results of the sterile fermentation broth of YPG medium.
[0038] Figure 4 Shows the comparison results of the antibacterial effects of the fermentation broth at different culture days.
[0039] Figure 5 Shows the comparison results of the antibacterial effects of the fermentation broth at different culture temperatures.
[0040] Figure 6 Shows the comparison results of the antibacterial effects of the fermentation broth under different pH conditions.
[0041] Figure 7 Shows the hyphal morphology diagram of *Trichococcus tsubakiensis* in the control group of Example 6.
[0042] Figure 8 Shows the hyphal morphology diagram of *Trichococcus tsubakiensis* in the experimental group of Example 6.
[0043] Figure 9 It is the response surface plot of the interactive effects of culture days, temperature, and initial pH on the antibacterial activity.
[0044] Figure 10 It is the antibacterial effects of propineb solutions with different concentrations on *Unaspis yanonensis* (Figure a shows the antibacterial effect of 1 g / L propineb on *Unaspis yanonensis*; Figure b shows the antibacterial effect of 0.5 g / L propineb on *Unaspis yanonensis*; Figure c shows the antibacterial effect of 0.25 g / L propineb on *Unaspis yanonensis*; Figure d shows the antibacterial effect of 0.125 g / L propineb on *Unaspis yanonensis*; Figure e shows the antibacterial effect of 0.0625 g / L propineb on *Unaspis yanonensis*).
[0045] Figure 11 It is the antibacterial effects of different biocontrol bacteria fermentation broths on *Unaspis yanonensis* (Figure a shows the antibacterial effect of K3-11 fermentation broth on *Unaspis yanonensis*; Figure b shows the antibacterial effect of RSTC-DS32 fermentation broth on *Unaspis yanonensis*; Figure c shows the antibacterial effect of LJ-6 fermentation broth on *Unaspis yanonensis*; Figure d shows the antibacterial effect of the CK group on *Unaspis yanonensis*; Figure e shows the antibacterial effect of HZW fermentation broth on *Unaspis yanonensis*).
[0046] Figure 12 It is the antibacterial effects of carbendazim solutions with different concentrations on *Unaspis yanonensis* (Figure a shows the antibacterial effect of 1 g / L carbendazim on *Unaspis yanonensis*; Figure b shows the antibacterial effect of 0.5 g / L carbendazim on *Unaspis yanonensis*; Figure c shows the antibacterial effect of 0.25 g / L carbendazim on *Unaspis yanonensis*; Figure d shows the antibacterial effect of 0.125 g / L carbendazim on *Unaspis yanonensis*; Figure e shows the antibacterial effect of 0.0625 g / L carbendazim on *Unaspis yanonensis*).
[0047] Figure 13 It is the determination results of the antibacterial diameters of *Unaspis yanonensis* under different treatments (BSX represents propineb, DJL represents carbendazim, and the suffix number represents the concentration). Detailed implementation manners
[0048] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.
[0049] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.
[0050] Propineb (active ingredient 70%) was purchased from SinoAgro United (Tianjin) Agrochemical Co., Ltd.
[0051] Carbendazim (active ingredient 50%) was purchased from Yingkou Leike Pesticide Co., Ltd., Liaoning Province.
[0052] Gao's No. 1 liquid medium, purchased from Coolaber Science & Technology, product number mm34131732;
[0053] Yeast extract, purchased from Guangdong Huankai Microbial Sci & Tech Co., Ltd., product number 3211079;
[0054] PDB medium, purchased from Guangdong Huankai Microbial Sci & Tech Co., Ltd., product number 240319A20;
[0055] TSA medium, purchased from Guangdong Huankai Microbial Sci & Tech Co., Ltd., product number 240822A21;
[0056] Bacteriological peptone, purchased from Beijing Aoboxing Biotechnology Co., Ltd., product number 01 - 001;
[0057] The formula of PDA medium is: potato (peeled) 200 g / L, glucose 20 g / L, agar 14 g / L, chloramphenicol 0.1 g / L.
[0058] Glucose, purchased from Biosharp, product number 24096561.
[0059] In the antagonistic tests in the following examples, three replicate tests were set up, the results were averaged, the data were analyzed by one-way ANOVA, the significance level was set at 0.05, and the analysis and plotting software was GraphPad Prism 10.
[0060] The strain used in the examples is Streptomyces asoensis HZW, preserved in the Guangdong Microbial Culture Collection Center, preservation number GDMCC NO.64293, and the pathogen indicator bacterium is Tubakiaseoraksanensis, Latin name, preserved in the Asia-Pacific Sericulture Training Center. The Streptomyces asoensis HZW strain has been disclosed in the prior patent application (application number 2024101735066) of the inventors of the present application.
[0061] The biocontrol bacterium used is Bacillus tequilensis K3 - 11, preserved in the Guangdong Microbial Culture Collection Center, preservation number GDMCC No: 63276, and disclosed in the patent "A Bacillus tequilensis K3 - 11 and Its Application".
[0062] The biocontrol bacterium RSTC-DS32 is deposited in the Guangdong Provincial Culture Collection of Microorganisms with the deposit number GDMCC No: 63051 and is disclosed in the paper "Identification of Gonatophragmium mori Causing Mulberry Zonate Leaf Spot Disease and Characterization of Their Biological Enemies in Guangxi, China".
[0063] The biocontrol bacterium LJ-6 is deposited in the Guangdong Provincial Culture Collection of Microorganisms with the deposit number GDMCC No: 64766 and is disclosed in the patent "An extreme orientalized Pseudomonas strain LJ-6 and its application in controlling mulberry diseases".
[0064] Example 1 Screening of liquid medium
[0065] I. Test materials
[0066] Gause's No. 1 liquid medium;
[0067] ISP2 fermentation medium: yeast extract 4 g / L, maltose 10 g / L, glucose 4 g / L;
[0068] PDB fermentation medium: potato extract 300 g / L, glucose 20 g / L;
[0069] TSA fermentation medium: casein peptone digest 15 g / L, soybean flour papain digest 5 g / L, sodium chloride 5 g / L;
[0070] Glycerol fermentation medium: glycerol 10 mL / L, bacteriological peptone 10 g / L, yeast extract 2 g / L, glucose 5 g / L;
[0071] YPG fermentation medium: yeast extract 10 g / L, bacteriological peptone 20 g / L, glucose 20 g / L.
[0072] II. Test methods
[0073] (1) Using a 50 mL conical flask as the container, inoculate the Streptomyces sp. HZW strain into 30 mL of Gause's No. 1 liquid medium for culture to obtain a seed solution.
[0074] (2) Add 100 μL of the seed solution to 30 mL of different fermentation media (ISP2 fermentation medium, PDB fermentation medium, TSA fermentation medium, glycerol fermentation medium, YPG fermentation medium) respectively, and carry out fermentation culture at 28 °C and 160 rpm for 6 days to obtain fermentation culture solutions.
[0075] (3) Filter the fermentation culture broth using a sterile filter membrane to obtain sterile fermentation broth of different fermentation media, i.e., the biocontrol agent.
[0076] (4) Adopt the plate antagonism test. Punch a 6-mm hole in the center of the PDA medium, add 50 μL of sterile fermentation broth of different fermentation media, and use the addition of 50 μL of sterile water as the control group (CK).
[0077] Additionally, set up a bacterial cell treatment group: Add 100 μL of the seed liquid to the Gao's No. 1 broth and culture for 2 d (160 rpm / min, 28 °C), then aspirate 50 μL of the bacterial liquid and add it to the hole in the PDA medium.
[0078] Use *Phytophthora cactorum* as the pathogen indicator bacterium to spot inoculate on the PDA medium. Conduct three replicates for each group, and verify whether the fermentation broth has the ability to antagonize *Phytophthora cactorum* based on the diameter of the inhibition zone.
[0079] III. Experimental Results
[0080] The results are as Figure 1 、 Figure 2 shown. The results indicate that the fermentation broth obtained by culturing and filtering with the YPG fermentation medium has the best antibacterial effect against *Phytophthora cactorum*. The diameter of the inhibition zone is higher than that of the sterile fermentation broth of the glycerol medium, ISP2 medium, PDB medium, and TSB medium. The antibacterial effect of the sterile fermentation broth of the YPG fermentation medium is significantly higher than that of these 4 groups.
[0081] Example 2 Stability of the Fermentation Broth
[0082] I. Test Method
[0083] Take the sterile fermentation broth of the YPG fermentation medium in Example 1 and set up different treatment groups;
[0084] Stock solution group: Do not treat the sterile fermentation broth;
[0085] UV treatment group: Irradiate the sterile fermentation broth with UV light at a wavelength of 254 nm for 30 min;
[0086] Proteinase K treatment group: Add 20 μL of a 2 μg / mL proteinase K solution to the sterile fermentation broth, and then incubate in a constant temperature water bath at 56 °C for 30 min;
[0087] High-temperature treatment group; Conduct high-temperature treatment of the sterile fermentation broth at 100 °C.
[0088] Additionally, set up sterile water as the control group (CK). Use the treated sterile fermentation broth of different treatment groups and sterile water to conduct the plate antagonism test against *Phytophthora cactorum*. The method refers to Example 1.
[0089] II. Test Results
[0090] The test results are as Figure 3 shown. The results indicate that there is no statistically significant difference (P>0.05) in the diameter of the inhibition zone of the fermentation filtrate of the HZW strain between the ultraviolet treatment group and the proteinase K treatment group compared with the untreated original solution group, indicating that the antibacterial active substance produced by the HZW strain has good ultraviolet irradiation stability and proteinase thermal stability.
[0091] The antibacterial activity of the fermentation filtrate of the HZW strain in the high-temperature treatment group decreased significantly, and there was no significant difference compared with the control group (CK group). This shows that in an extremely high-temperature environment, its antibacterial effect will be significantly weakened, and the antibacterial active substance has limited tolerance to high temperatures.
[0092] Antibacterial Ability of Sterile Fermentation Broth at Different Culture Days in Example 3
[0093] I. Test Method
[0094] 1. Using a 50 mL conical flask as the container, inoculate the Streptomyces HZW strain into 30 mL of Gause's No. 1 liquid medium for cultivation to obtain a seed solution.
[0095] 2. Using a 50 mL conical flask as the container, add 100 μL of the seed solution to 30 mL of YPG fermentation medium (initial pH value is 6.8 - 7.2), and carry out fermentation culture under the culture conditions of 28 °C and 160 rpm / min for 4, 5, 6, 7, 8, 9, and 10 days respectively; use a sterile filter membrane to filter the bacteria to obtain sterile fermentation broths with different culture days.
[0096] 3. Adopt the plate antagonism test. Punch a 6 mm hole in the center of the PDA medium, add 50 μL of sterile fermentation broths with different culture days, use 50 μL of sterile water as the control group, and use Aspergillus luchuensis as the pathogen indicator bacterium to spot inoculate on the PDA medium, and use the inhibition diameter as the evaluation index of antibacterial ability.
[0097] II. Test Results
[0098] The test results are as Figure 4As shown in the figure, the results indicate that among the fermentation broths with a culture period ranging from 8d to 10d, their antibacterial activities are significantly superior to those of the fermentation broths with culture periods of 4, 5, and 7d. It is speculated that in the initial stage of fermentation, the metabolic regulation system of Streptomyces promotes the gene expression and enzyme activities related to the synthesis of metabolites, causing the activity of metabolites to gradually increase until it reaches the peak. When the metabolites accumulate to a certain extent, Streptomyces will feedback-inhibit the gene expression and enzyme activities, resulting in a decrease in the activity of metabolites. As fermentation continues, Streptomyces adapts to environmental changes and metabolite accumulation through its own regulatory mechanisms, such as inducing the production of new enzyme systems and activating silent genes, thereby upregulating the metabolite activity again. Therefore, the antibacterial effect of the fermentation broth of HZW strain on the 7th day of culture is inferior to that on the 6th day and the 8th - 10th days. Within the culture period of 8 - 10d, there are no obvious differences in the antibacterial properties of the fermentation broths on different days.
[0099] Example 4 Antibacterial Ability of Sterile Fermentation Broths at Different Culture Temperatures
[0100] I. Test Method
[0101] 1. Using a 50 mL conical flask as the container, inoculate the Streptomyces HZW strain into 30 mL of Czapek - Dox liquid medium for culture to obtain the seed liquid.
[0102] 2. Using a 50 mL conical flask as the container, add 100 μL of the seed liquid to 30 mL of YPG fermentation medium (initial pH value is 6.8 - 7.2), and perform fermentation culture at 160 rpm / min for 8d. Set different temperature gradients for culture, and the culture temperatures are 25°C, 28°C, 30°C, and 35°C respectively; use a sterile filter membrane to filter the bacteria to obtain sterile fermentation broths under different temperature cultures.
[0103] 3. Adopt the plate antagonism test. Punch a 6 mm hole in the center of the PDA medium, add 50 μL of sterile fermentation broths under different temperature cultures, use the addition of 50 μL of sterile water as the control group, and use the fungus of Togashi Keisuke as the pathogen indicator bacteria to spot - inoculate on the PDA medium. Use the inhibition diameter as the evaluation index for antibacterial ability.
[0104] II. Test Results
[0105] The test results are as Figure 5 shown. The results indicate that when using the YPG fermentation medium to ferment the HZW strain, after culturing for 8d at four different temperature gradients from 25 to 35°C, the obtained fermentation broths can effectively inhibit the growth of the hyphae of the fungus of Togashi Keisuke, and there are significant differences compared with the control group. In particular, under the conditions of 25°C, 28°C, and 30°C, the fermentation effect is more significant.
[0106] Example 5 Antibacterial Ability of Sterile Fermentation Broths Obtained from Media with Different pH Values
[0107] I. Test method
[0108] 1. Using a 50 mL conical flask as a container, inoculate Streptomyces sp. HZW strain into 30 mL of Czapek No. 1 liquid medium for cultivation to obtain a seed solution.
[0109] 2. Using a 50 mL conical flask as a container, add 100 μL of the seed solution to 30 mL of YPG fermentation medium, adjust the initial pH values of the YPG fermentation medium to 3, 4, 5, 6, 7, 8, and 9 respectively, and then carry out fermentation cultivation for 8 days under the cultivation conditions of 28 °C and 160 rpm / min; use a sterile filter membrane to filter the bacteria to obtain sterile fermentation broths under different initial pH value cultivations.
[0110] 3. Adopt the plate antagonism test. Punch a 6 mm hole in the center of the PDA medium, add 50 μL of sterile fermentation broths under different initial pH value cultivations, use the addition of 50 μL of sterile water as the control group, use Phytophthora cinnamomi Rands as the pathogen indicator bacterium to spot inoculate on the PDA medium, and use the inhibition diameter as the evaluation index of antibacterial ability.
[0111] II. Test results
[0112] The test results are as Figure 6 shown. The results show that under the cultivation conditions of 28 °C and 160 rpm / min, Streptomyces sp. HZW strain is cultivated in the YPG fermentation medium with a pH value ranging from 3 to 9 for a total of 8 days. The antibacterial effects of the fermentation broths within the pH value range of 6 - 9 are significantly better than those of the fermentation broths within the pH value range of 3 - 5 and the control group (CK). Especially, the antibacterial effect of the fermentation broth cultivated under the condition of pH value 6 is the most significant.
[0113] Effect of the fermentation broth treatment in Example 6 on the mycelial morphology of the pathogen
[0114] I. Test method
[0115] Under the cultivation conditions of 28 °C and 160 rpm / min, use the YPG medium (initial pH value is 6) to cultivate Streptomyces sp. HZW strain for 8 days, and use a sterile filter membrane to filter the bacteria to obtain a sterile fermentation broth.
[0116] Set up a control group and an experimental group:
[0117] Control group: 1 mL of YPG liquid medium + a 6 mm Phytophthora cinnamomi Rands bacterial cake;
[0118] Experimental group: 1 mL of the sterile fermentation broth of Streptomyces sp. HZW strain + a 6 mm Phytophthora cinnamomi Rands bacterial cake.
[0119] Each group had 3 replicates. The control group and the experimental group were cultured at 100 rpm / min and 28 °C for 48 h. After picking the bacterial cakes to prepare slides and performing Gram staining, the microscopic morphology of the hyphae was observed using a microscope (magnification: 100 times).
[0120] The hyphal morphology of the control group is shown in Figure 7 as follows, and the hyphal morphology of the experimental group is shown in Figure 8 as follows. The experimental results showed that the morphological characteristics of the hyphae in the control group were more slender and plump, no fracture phenomenon was observed, and it had a significant branching structure; for the pathogen treated with the sterile fermentation broth of the HZW strain, obvious swelling occurred in its hyphae, substances inside some hyphae began to overflow, the hyphal structure was damaged, and even ruptured. This indicates that the sterile fermentation broth of the HZW strain has an obvious destructive and growth-inhibiting effect on *Cercospora kikuchii*.
[0121] Example 7 Optimization of liquid culture by response surface
[0122] On the basis of the previous single-factor experiments, the Plackett-Burman design was used to optimize three factors in the culture process, namely the culture days (A), culture temperature (B), and initial pH of the medium (C). Each factor was set at three levels, and a total of 15 experiments were carried out to explore their effects on the culture effect. The design software used was Design-Expert 13.
[0123] The coding and level selection of each factor and the test results are shown in Table 1 and Table 2; the response surface optimization result diagram is shown in Figure 9 as follows. The results showed that it was significantly different to use the quadratic model recommended by the software for processing and analysis, with R 2 = 0.9685, Adjusted R 2 = 0.9370, Predicted R 2 = 0.7661, and Adeq Precision was 17.470. The value greater than 4 indicates that it is acceptable and the signal is sufficient.
[0124] Under this model, regression analysis was carried out to obtain the quadratic polynomial regression equation:
[0125] Y = 38.26 - 0.5363A - 0.6825B - 1.21C - 0.5675AB + 1.05BC - 0.4233B 2 - 0.9158C 2 (Y
[0126] is the antibacterial diameter). According to the experimental results in Table 2, the sterile fermentation broth of the HZW strain had the strongest antibacterial effect when cultured at 25 °C and the initial pH value of the medium was 6 for 9 days.
[0127] Table 1 Test factors and levels
[0128]
[0129] Table 2 Test results
[0130]
[0131] Example 8
[0132] The antibacterial effects of commercially available fungicides (propineb and carbendazim), 3 biocontrol bacteria (biocontrol bacterium Bacillus tequilensis K3-11, biocontrol bacterium RSTC-DS32, and biocontrol bacterium LJ-6), and the fermentation broth of HZW strain against Cercospora kikuchii were determined. The following treatment groups were specifically set up;
[0133] Propineb treatment group (BSX): Using sterile water as a solvent, propineb solutions with concentrations of 1 g / L, 0.5 g / L, 0.25 g / L, 0.125 g / L, and 0.0625 g / L were respectively prepared as treatment solutions;
[0134] Biocontrol bacterium treatment groups (K3-11, RSTC-DS32, LJ-6): After Bacillus tequilensis K3-11, biocontrol bacterium RSTC-DS32, and biocontrol bacterium LJ-6 were cultured at 28 °C and 160 rpm / min for 6 days, the cells were filtered through a 0.22 μm sterile filter membrane to obtain a sterile fermentation broth, which was used as the treatment solution;
[0135] HZW fermentation broth treatment group (HZW): After the HZW strain was cultured in YPG medium (pH value = 8) at 25 °C and 160 rpm / min for 9 days, the cells were filtered through a 0.22 μm sterile filter membrane to obtain the sterile fermentation broth of the HZW strain;
[0136] Carbendazim treatment group (DJL): Using sterile water as a solvent, carbendazim solutions with concentrations of 1 g / L, 0.5 g / L, 0.25 g / L, 0.125 g / L, and 0.0625 g / L were respectively prepared as treatment solutions;
[0137] CK group: YPG medium.
[0138] The plate confrontation method was adopted. A 6 mm punch was used to make holes in the center of the medium, and 4 mm indicator bacteria blocks (Cercospora kikuchii) were inoculated equidistantly around the holes. 50 μL of the treatment solution of each treatment group was added to the holes. After culturing for 14 days, the cross-cross method was used to measure the diameter of the antibacterial circle of each group.
[0139] The antibacterial effects of propineb solutions with different concentrations, fermentation broths of different biocontrol bacteria, and carbendazim solutions with different concentrations against Cercospora kikuchii are respectively as Figure 10 、 Figure 11 and Figure 12As shown, the determination results of the antibacterial diameter of *Trichoderma harzianum* under different treatments are as follows Figure 13 As shown, the antibacterial test results indicate that the antibacterial effect of the HZW fermentation broth is significantly better than that of the propineb solutions at five different concentrations and the fermentation broths of the biocontrol bacteria K3-11, RSTC-DS32, and LJ-6; the antibacterial effect is the same as that of the carbendazim solution at 0.0625 g / L, and it can be used to control the fungal diseases caused by *Trichoderma harzianum*.
[0140] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.
Claims
1. Use of Streptomyces asunciaria HZW strain in inhibiting Acanthocephala, or in preparing a product for inhibiting Acanthocephala, characterized in that: The HZW strain of Streptomyces asuncifolius was deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 17, 2024, and its deposit number is GDMCC NO: 64293.
2. Use of the Streptomyces asunciaria HZW strain according to claim 1 in preventing and controlling plant diseases caused by A. tsunemotoi, or in preparing products for preventing and controlling plant diseases caused by A. tsunemotoi.
3. A method for inhibiting the fermentation of Streptomyces by using Streptomyces, characterized in that: The Streptomyces australis was inoculated into the fermentation medium for fermentation culture for 4-10 days.
4. The Streptomyces fermentation method according to claim 3, characterized in that: The Streptomyces asuncifolius is the Streptomyces asuncifolius HZW strain described in claim 1.
5. The Streptomyces fermentation method according to claim 3, characterized in that: The pH value of the fermentation medium is 6-9.
6. The Streptomyces fermentation method according to claim 3, characterized in that: The fermentation culture is carried out at 25-35°C.
7. The Streptomyces fermentation method according to claim 3, characterized in that: The fermentation medium is any one of TSB fermentation medium, YPG fermentation medium, PDB fermentation medium and ISP2 fermentation medium.
8. The Streptomyces fermentation method according to claim 7, characterized in that: The fermentation medium is YPG fermentation medium.
9. Use of a Streptomyces fermentation product obtained by the Streptomyces fermentation method according to any one of claims 3 to 7 in inhibiting Aconitifolia, or in preparing a product for inhibiting Aconitifolia.
10. Use of a Streptomyces fermentation product obtained by the Streptomyces fermentation method according to any one of claims 3 to 7 in preventing and controlling plant diseases caused by A. tsunemotoi, or in preparing a product for preventing and controlling plant diseases caused by A. tsunemotoi.