Microbial agent containing pseudomonas proteinans and ochrobactrum intermedium and application of microbial agent

Abstract

The invention relates to a microbial agent containing pseudomonas deformation and ochrobactrum intermedium and application of the microbial agent. The microbial agent comprises the pseudomonas deformation and the ochrobactrum intermedium. The pseudomonas mutans is classified and named as pseudomonas mutans, and the preservation number of the pseudomonas mutans is CGMCC (China General Microbiological Culture Collection Center) No.14475; the classification name of the ochrobactrum intermedium is Ochrobactrum intermedium, and the preservation number of the ochrobactrum intermedium is CGMCC (China General Microbiological Culture Collection Center) No.33645. The invention further discloses a preparation method of the ochrobactrum intermedium. The microbial agent disclosed by the invention can be used for efficiently, stably and environmentally-friendly biological control of plant diseases.

Description

Technical Field

[0001] This application belongs to the field of microbial technology. Specifically, it relates to a microbial inoculant containing Pseudomonas plecoglossicida and Ochrobactrum intermedium and its application. Background Art

[0002] Plant diseases are important factors affecting global agricultural production, leading to a significant decrease in crop yields and quality every year, and causing huge losses to the agricultural economy. Traditional plant disease control mainly relies on chemical pesticides.

[0003] In recent years, biological control, as a green and sustainable plant disease control strategy, has received extensive attention. Biological control mainly uses beneficial microorganisms or their metabolites to inhibit the growth and reproduction of pathogenic bacteria, thereby achieving the purpose of disease control. Compared with chemical control, biological control has many advantages, such as being environmentally friendly, not easily producing pesticide residues, reducing the impact on non-target organisms, and to a certain extent avoiding the generation of pathogen resistance. However, biological control still faces some challenges in practical applications. On the one hand, the control effects of existing biological control strains or preparations are often not stable enough, being greatly affected by environmental factors (such as temperature, humidity, soil pH, etc.), resulting in obvious differences in control effects in different regions or seasons. On the other hand, the relatively scarce high-efficiency biological control microbial resources available for large-scale production and application limit the popularization and application of biological control technologies. In addition, the research on the action mechanism of biological control is not deep enough, which also hinders the research and development and optimization of new biological control products.

[0004] In view of the above background, there is an urgent practical need to develop an efficient, stable and environmentally friendly plant disease biological control method and related products. Summary of the Invention

[0005] The purpose of the present disclosure is to provide a microbial inoculant containing Pseudomonas plecoglossicida and Ochrobactrum intermedium and its application. The microbial inoculant of the present disclosure can carry out biological control of plant diseases efficiently, stably and environmentally friendly.

[0006] To achieve the above purpose, in the first aspect of the present disclosure, there is provided a microbial inoculant for biological control of plant diseases, and the microbial inoculant includes Pseudomonas plecoglossicida and Ochrobactrum intermedium; The classification and naming of the Pseudomonas plecoglossicida is Pseudomonas plecoglossicida Pseudomonas plecoglossicida , and its deposit number is CGMCC No. 14475; the classification and naming of the Ochrobactrum intermedium is Ochrobactrum intermedium Ochrobactrum intermedium , and its deposit number is CGMCC No. 33645.

[0007] Optionally, the dosage form of the microbial inoculant includes a liquid agent and / or a solid agent.

[0008] Optionally, the microbial inoculant is a liquid agent. In the microbial inoculant, the viable count of Pseudomonas plecoglossicida is 10 7 ~10 9 CFU / mL.

[0009] Optionally, the microbial inoculant is a solid agent. In the microbial inoculant, the viable count of Pseudomonas plecoglossicida is 10 8 ~10 10 CFU / g.

[0010] Optionally, in the microbial inoculant, the ratio of the viable count of Pseudomonas plecoglossicida to the viable count of Ochrobactrum intermedium is (1~3):1.

[0011] Optionally, the method for preparing the microbial inoculant includes: preparing a bacterial suspension of Pseudomonas plecoglossicida and Ochrobactrum intermedium.

[0012] The second aspect of the present disclosure provides the application of the microbial inoculant described in the first aspect of the present disclosure in the biological control of plant diseases.

[0013] Optionally, the plant diseases include one or more of cucumber fusarium wilt, rice blast, cucumber root rot, and ginger blast.

[0014] Optionally, the application method of the microbial inoculant includes the spraying method, and the application amount of the microbial inoculant is 1.0×10 7 to 1.0×10 9 CFU / m 2 .

[0015] Optionally, the application method of the microbial inoculant includes the root irrigation method, and the application amount of the microbial inoculant is 2.0×10 6 to 2.0×10 8 CFU / plant.

[0016] Through the above technical solutions, the present disclosure selects specific Ochrobactrum intermedium and Pseudomonas plecoglossicida to prepare a microbial inoculant. The Ochrobactrum intermedium and Pseudomonas plecoglossicida therein have a synergistic inhibitory effect on plant pathogens and have a significant control effect on plant diseases. The inoculant of the present disclosure is less affected by environmental factors, the disease control effect is stable, and it can maintain a good control effect in different seasons and regions, meeting the control requirements; and it is environmentally friendly, without chemical residues, meeting the development requirements of green agriculture.

[0017] Other features and advantages of the present disclosure will be described in detail in the subsequent specific implementation part.

[0018] Biological material preservation information Ochrobactrum intermedium of the present disclosure Ochrobactrum intermediumn It is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CGMCC). The preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is February 24, 2025, and the strain preservation number is: CGMCC No. 33645. Brief description of the drawings

[0019] Figure 1 It is the inhibitory effect diagram of the microbial agent in Example 1 of the present disclosure on the pathogen of rice blast. Detailed implementation manners

[0020] The following will describe in detail the specific implementation manners of the present disclosure with reference to the accompanying drawings. It should be understood that the specific implementation manners described herein are only for explaining and understanding the present disclosure, and are not used to limit the present disclosure.

[0021] The first aspect of the present disclosure provides a microbial agent for biological control of plant diseases. The microbial agent includes Pseudomonas plecoglossicida and Ochrobactrum intermedium; The classification and naming of the Pseudomonas plecoglossicida is Pseudomonas plecoglossicida Pseudomonas plecoglossicida , and the preservation number is CGMCC No. 14475; the classification and naming of the Ochrobactrum intermedium is Ochrobactrum intermedium Ochrobactrum intermedium , and the preservation number is CGMCC No. 33645.

[0022] The inventors of the present disclosure isolated Ochrobactrum intermedium and Pseudomonas plecoglossicida, and compounded the two into a microbial agent. Compared with a single-agent, the compounded agent has a synergistic inhibitory effect, has a significant control effect on plant diseases, has low environmental sensitivity, stable control effect, can be used in different seasons and regions, has a simple preparation method, and can meet the control requirements; compared with chemical agents, the microbial agent of the present disclosure is environmentally friendly, has no chemical residues, can reduce the impact on non-target organisms, and meets the development needs of green agriculture.

[0023] According to an embodiment of the present disclosure, the microbial agent is composed of Pseudomonas plecoglossicida, Ochrobactrum intermedium, optional sterile water, and optional buffer solution.

[0024] According to an embodiment of the present disclosure, the dosage form of the microbial agent includes a liquid agent and / or a solid agent. When the microbial agent is a solid agent, its usage method can be solid application, or it can be configured into an aqueous agent for application; the liquid agent can be directly applied after being cultured in a liquid medium, or can be configured into a liquid microbial agent for application after being cultured.

[0025] According to an embodiment of the present disclosure, the microbial inoculant is a liquid agent. In the microbial inoculant, the viable count of Pseudomonas plecoglossicida is 10 7 ~10 9 CFU / mL, including but not limited to 10 7 CFU / mL, 0.2×10 8 CFU / mL, 0.33×10 8 CFU / mL, 0.5×10 8 CFU / mL, 0.67×10 8 CFU / mL, 0.8×10 8 CFU / mL, 0.83×10 8 CFU / mL, 0.99×10 8 CFU / mL, 10 8 CFU / mL, 0.5×10 9 CFU / mL, 0.75×10 9 CFU / mL, 10 9 CFU / mL, or the range composed of any two of them; the microbial inoculant with the above viable count has a good plant disease control effect.

[0026] According to an embodiment of the present disclosure, the microbial inoculant is a solid agent. In the microbial inoculant, the viable count of Pseudomonas plecoglossicida is 10 8 ~10 10 CFU / g, including but not limited to 10 8 CFU / g, 0.2×10 9 CFU / g, 0.5×10 9 CFU / g, 0.8×10 9 CFU / g, 10 9 CFU / g, 0.2×10 10 CFU / g, 0.5×10 10 CFU / g, 0.8×10 10 CFU / g, 10 10 CFU / g, or the range composed of any two of them; the microbial inoculant with the above viable count has a good plant disease control effect.

[0027] In order to enable the microbial inoculant to have a good effect on preventing and controlling plant diseases, according to an embodiment of the present disclosure, in the microbial inoculant, the ratio of the viable count of Pseudomonas plecoglossicida to the viable count of Ochrobactrum intermedium is (1-3):1, preferably (2-2.5):1, including but not limited to 1:1, 1.2:1, 1.5:1, 1.8:1, 2:1, 2.2:1, 2.5:1, 2.75:1, 2.9:1, 3:1, or the range composed of any two of them.

[0028] According to an embodiment of the present disclosure, the microbial inoculant is a liquid agent. The method for preparing the microbial inoculant includes: configuring Pseudomonas plecoglossicida and Ochrobactrum intermedium into a suspension; specifically, mixing the bacterial suspension of Pseudomonas plecoglossicida with the bacterial suspension of Ochrobactrum intermedium; centrifuging or filtering the cultured Pseudomonas plecoglossicida and Ochrobactrum intermedium respectively, collecting the bacterial cells, and resuspending them with sterile water or buffer solution respectively to obtain the bacterial suspension of Pseudomonas plecoglossicida and the bacterial suspension of Ochrobactrum intermedium, and then mixing the two; or directly resuspending the cultured Pseudomonas plecoglossicida cells and Ochrobactrum intermedium cells with sterile water or buffer solution to configure a suspension; the types of buffer solutions are conventional in the art.

[0029] According to an embodiment of the present disclosure, the microbial inoculant is a solid agent. The preparation method includes: mixing the cultured Pseudomonas plecoglossicida and Ochrobactrum intermedium cells; or loading them on a solid carrier, and the types of solid carriers are not particularly limited.

[0030] According to an embodiment of the present disclosure, Pseudomonas plecoglossicida can be cultured in a medium. The types of media and culture methods are not particularly limited. For example, it can be tryptone soy broth liquid medium (TSB), and cultured with shaking at 28-30 °C and 150-200 rpm for 24 hours until the OD600 of the bacterial liquid reaches 1.0-1.2. The specific formula of the medium is not particularly limited.

[0031] According to an embodiment of the present disclosure, Ochrobactrum intermedium can be cultured in a medium. The types of media and culture methods are not particularly limited. For example, it can be nutrient broth medium (NB), and cultured at 32 °C and 180 rpm for 48-60 h to make the viable count reach 10 8 to 10 9 CFU / mL, and the specific formula of the medium is not particularly limited.

[0032] The second aspect of the present disclosure provides the application of the microbial inoculant described in the first aspect of the present disclosure in the biological control of plant diseases.

[0033] According to an embodiment of the present disclosure, the plant diseases include one or more of cucumber fusarium wilt, rice blast, cucumber root rot, and ginger blast.

[0034] According to an embodiment of the present disclosure, the application method of the microbial inoculant includes the spraying method, that is, spraying the microbial inoculant on the surface of plant leaves; the application amount of the microbial inoculant is 1.0×10 7 to 1.0×10 9 CFU / m 2 , including but not limited to 1.0×10 7 CFU / m 2 , 0.2×10 8 CFU / m 2 , 0.8×10 8 CFU / m 2 , 1.0×10 8 CFU / m 2 , 0.2×10 9 CFU / m 2 , 0.5×10 9 CFU / m 2 , 0.8×10 9 CFU / m 2 , 1.0×10 9 CFU / m 2 , or the range composed of any two of them; the dosages of Pseudomonas plecoglossicida and Ochrobactrum intermedium are calculated relative to the land area; adopting the above application amounts can improve the control effect while reducing the application amount of the inoculant and reducing the treatment cost.

[0035] According to an embodiment of the present disclosure, the application method of the microbial inoculant includes the root irrigation method, that is, irrigating the microbial inoculant into the soil around the roots of plants; the application amount of the microbial inoculant is 2.0×10 6 to 2.0×10 8 CFU / plant, including but not limited to 2.0×10 6 CFU / plant, 0.3×10 7 CFU / plant, 0.5×10 7 CFU / plant, 0.8×10 7 CFU / plant, 1.0×10 8 CFU / plant, 1.2×10 8 CFU / plant, 1.5×10 8 CFU / plant, 1.8×10 8 CFU / plant, 2.0×10 8 CFU / plant, or the range composed of any two of them; adopting the above application amounts can improve the control effect while reducing the application amount of the inoculant and reducing the treatment cost.

[0036] The present invention will be further described in detail below in conjunction with embodiments, but the scope of the present invention is not limited to the following embodiments.

[0037] Unless otherwise specified, the culture media used are all commercially available products.

[0038] Example 1 1. Strain culture (1) Pseudomonas plecoglossicida CGMCC No. 14475 was inoculated into TSB liquid medium and cultured at 30 °C and 200 rpm for 24 hours until the OD600 of the bacterial liquid reached 1.0.

[0039] TSB liquid medium formula: Tryptone (17 g / L), Soy peptone (3 g / L), Sodium chloride (5 g / L), Dipotassium hydrogen phosphate (2.5 g / L), Glucose (2.5 g / L).

[0040] (2) Ochrobactrum intermedium CGMCC No. 33645 was inoculated into NB liquid medium and cultured at 32 °C and 180 rpm for 48 hours until the viable cell count reached 10 8 to 10 9 CFU / mL.

[0041] NB liquid medium formula: Trypticase peptone (10 g / L), Beef extract powder (3 g / L), NaCl (5 g / L).

[0042] 2. Bacterial liquid preparation (1) The cultured Pseudomonas plecoglossicida CGMCC No. 14475 and Ochrobactrum intermedium CGMCC No. 33645 were centrifuged at 4 °C and 8000 rpm for 10 minutes respectively to collect the bacterial cells.

[0043] (2) The bacterial cells were resuspended with sterile water and prepared into bacterial suspensions with a concentration of 10 8 CFU / mL respectively.

[0044] 3. Preparation of microbial inoculant The bacterial suspension of Pseudomonas plecoglossicida CGMCC No. 14475 was mixed with the bacterial suspension of Ochrobactrum intermedium CGMCC No. 33645 in a certain proportion to obtain microbial inoculant A1, and the parameters are listed in Table 1.

[0045] Figure 1 is the inhibitory effect diagram of microbial inoculant A1 on the pathogen of rice blast. According to Figure 1 it can be seen that the microbial inoculant of the present disclosure has a good inhibitory effect on the pathogen of rice blast.

[0046] Examples 2 - 4 The microbial agents A2 - A4 were prepared by the method of Example 1, except that the ratio of the cell suspension of Pseudomonas plecoglossicida CGMCC No. 14475 to the cell suspension of Ochrobactrum intermedium CGMCC No. 33645 was adjusted, such that the concentration and / or ratio of Ochrobactrum intermedium and Pseudomonas plecoglossicida in the agent was different from that in Example 1. The parameters are listed in Table 1.

[0047] Comparative Example 1 The microbial agent D1 was prepared by the method of Example 1, except that the cell suspension of Ochrobactrum intermedium was not added. The parameters are listed in Table 1.

[0048] Comparative Example 2 The microbial agent D2 was prepared by the method of Example 1, except that the cell suspension of Pseudomonas plecoglossicida was not added. The parameters are listed in Table 1.

[0049] Comparative Example 3 The microbial agent D3 was prepared by the method of Example 1, except that the cell suspension of Ochrobactrum intermedium CGMCC No. 33645 was replaced with a solution of Ochrobactrum intermedium GDMCC 60409 (Guangdong Provincial Culture Collection Center of Microorganisms) at the same concentration. The parameters are listed in Table 1.

[0050] Comparative Example 4 The microbial agent D4 was prepared by the method of Example 1, except that the cell suspension of Pseudomonas plecoglossicida CGMCC No. 14475 was replaced with a cell suspension of Pseudomonas plecoglossicida ACCC 03304 (purchased from China Center for Type Culture Collection of Agricultural Microorganisms) at the same concentration. The parameters are listed in Table 1.

[0051] Table 1

[0052] Test Example 1 Control of Rice Blast Each time, treatment plots with an area of 0.2 mu were randomly selected for the experiment, and the experiment was repeated 3 times. The microbial agent and chemical pesticide were applied by the spraying method. The application rates of the microbial agent are listed in Table 2, and the application rate of the control agent (40% isoprothiolane EC) was 100 mL per mu. Spraying was carried out once at the booting stage and once at the full heading stage, for a total of 2 times. After application, the incidence of rice blast in the field plots was investigated, and the yield was measured at harvest. The final results are shown in Table 2 below.

[0053] According to the "Rules for Forecast and Investigation of Rice Blast" (GB / T 15790-2009), the rice blast disease situation is divided into 6 levels: level 0, disease-free; level 1, with few and small lesions, and the lesion area accounts for less than 1% of the leaf area; level 2, with small and numerous lesions, or large and few lesions, and the lesion area accounts for 1% - 5% of the leaf area; level 3, with large and relatively numerous lesions, and the lesion area accounts for 5% - 10% of the leaf area; level 4, with large and numerous lesions, and the lesion area accounts for 10% - 50% of the leaf area; level 5, with the lesion area accounting for more than 50% of the leaf area, and the whole leaf will wither and die.

[0054] Among them, the calculation method for the control effect of rice blast: Control effect = (Disease index - Disease index of the blank group) / Disease index of the blank group × 100%; The calculation method for the disease index: Disease index = 100 × ∑(Number of diseased leaves at each level × Representative value at each level) / (Total number of leaves investigated × Representative value of the highest level); The calculation method for the yield increase rate: Yield increase rate = (Actual yield - Actual yield of the blank group) / Actual yield of the blank group × 100%.

[0055] Table 2

[0056] According to the above data, it can be seen that under the condition of using the same dosage of the microbial inoculant, the microbial inoculant of the present disclosure has an obvious control effect on rice blast, can increase the rice yield, and can avoid chemical residues caused by chemical agents; further, according to the comparison between microbial inoculant A4 and other inoculants, it can be known that under the condition that other conditions are the same, when the ratio of the viable count of Pseudomonas plecoglossicida to the viable count of Ochrobactrum intermedium in the inoculant is in the range of (1 - 3):1, a better control effect can be obtained; according to the comparison between microbial inoculant A1 and inoculants A2 and A3, it can be known that when the ratio is in the preferred range of (2 - 2.5):1, the control effect can be further improved.

[0057] Test Example 2 Control of Cucumber Fusarium Wilt Each time, 20 cucumber seedlings were randomly selected for the experiment, and the experiment was repeated 3 times. When the cucumber seedlings grew to 2 true leaves, a spore suspension of cucumber Fusarium oxysporum was poured along the edge of the pot for pathogen inoculation (15 mL / plant), and at the same time, the microbial inoculant was applied by the root irrigation method, and the dosage of the inoculant is listed in Table 3. After 12 days of cultivation in the greenhouse, the incidence degree of Fusarium wilt was investigated, and the disease index and control effect were calculated. The final results are shown in Table 3 below.

[0058] According to the agricultural industry standard of the People's Republic of China "Technical Regulations for Identification of Disease Resistance of Main Diseases of Cucumber - Part 3: Technical Regulations for Identification of Cucumber Resistance to Fusarium Wilt" (NY / T 1857.3 - 2010), the indoor disease levels of cucumber Fusarium wilt are divided into 5 levels: level 0, asymptomatic; level 1, cotyledons turn yellow, but do not wilt; level 2, cotyledons wilt; level 3, cotyledons and true leaves wilt or the plant is dwarfed; level 4, wither and die.

[0059] Among them, the calculation method of the control effect: Control effect = (disease index - disease index of the blank group) / disease index of the blank group × 100%; The calculation method of the disease index: Disease index = 100 × ∑(number of diseased leaves at each level × representative value at each level) / (total number of leaves surveyed × representative value of the highest level).

[0060] Table 3

[0061] According to the above data, it can be seen that under the condition of the same application amount of the bacterial agent, the microbial bacterial agent of the present disclosure has an obvious control effect on cucumber fusarium wilt, meeting the control requirements while avoiding chemical residues caused by chemical agents; Further, according to the comparison between the microbial bacterial agent A4 and other bacterial agents, it can be known that under the condition that other conditions are the same, when the ratio of the viable count of Pseudomonas plecoglossicida to the viable count of Ochrobactrum intermedium in the bacterial agent is in the range of (1 - 3):1, a better control effect can be obtained; According to the comparison between the microbial bacterial agent A1 and the bacterial agents A2 and A3, it can be known that when the ratio is in the preferred range of (2 - 2.5):1, the control effect can be further improved.

[0062] The preferred embodiments of the present disclosure have been described in detail above. However, the present disclosure is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

[0063] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable way. To avoid unnecessary repetition, the present disclosure will not separately describe various possible combination methods.

[0064] In addition, any combination can be made between various different embodiments of the present disclosure as long as it does not violate the idea of the present disclosure, and it should also be regarded as the content disclosed by the present disclosure.

Claims

1. A microbial inoculum for biological control of plant diseases, characterized in that, The microbial inoculum includes Pseudomonas plecoglossicida and Ochrobactrum intermedium; The classification and naming of the said Pseudomonas pleomorphica is Pseudomonas pleomorphica Pseudomonas plecoglossicida , and its deposit number is CGMCC No. 14475; the classification and naming of the said Ochrobactrum intermedium is Ochrobactrum intermedium Ochrobactrum intermedium , and its deposit number is CGMCC No. 33645.

2. The microbial inoculant according to claim 1, wherein, The dosage form of the microbial inoculum includes liquid agent and / or solid agent.

3. The microbial inoculant according to claim 2, wherein, The microbial inoculant is a liquid agent. In the microbial inoculant, the viable count of Pseudomonas plecoglossicida is 10 7 ~10 9 CFU / mL.

4. The microbial inoculant according to claim 2, wherein The microbial inoculant is a solid agent, and in the microbial inoculant, the viable count of Pseudomonas plecoglossicida is 10 8 ~10 10 CFU / g.

5. The microbial inoculant according to claim 3 or 4, wherein, In the microbial inoculum, the ratio of the viable count of Pseudomonas plecoglossicida to the viable count of Ochrobactrum intermedium is (1-3):

1.

6. The microbial inoculum according to claim 1, wherein, The method for preparing the microbial inoculum includes: preparing a bacterial suspension of Pseudomonas plecoglossicida and Ochrobactrum intermedium.

7. Use of the microbial inoculum according to any one of claims 1-6 in the biological control of plant diseases.

8. The application according to claim 7, wherein, The plant diseases include one or more of cucumber fusarium wilt, rice blast, cucumber root rot and ginger blast.

9. The application according to claim 7, wherein The application method of the microbial inoculant includes the spraying method, and the application amount of the microbial inoculant is 1.0×10 7 to 1.0×10 9 CFU / m 2 .

10. The application according to claim 7, wherein, The application method of the microbial inoculum includes the root irrigation method, and the application amount of the microbial inoculum is 2.0×10 6 to 2.0×10 8 CFU / strain.

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