Acinetobacter, bacterial agent, plant growth promoter, biological fertilizer and application thereof

Abstract

The present application relates to the field of microbial technology and provides a kind of Acinetobacter, a bacterial agent, a plant growth promoter, a biological fertilizer and an application. The Acinetobacter is named as Acinetobacter sp.11.12.7.6. The strain has been deposited in the General Microbiological Center of China Microorganism Culture Collection Committee on December 8, 2023, with the deposit number CGMCC No.29270. The Acinetobacter has the functions of degrading ursolic acid produced by asparagus, preventing and treating asparagus stem blight, promoting plant growth and nitrogen fixation.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and more specifically, to a kind of Acinetobacter, a bacterial agent, a plant growth promoter, a biological fertilizer and applications. Background Art

[0002] Asparagus, also known as Asparagus officinalis, is a dioecious perennial herb of the genus Asparagus in the family Liliaceae. Asparagus is rich in a variety of amino acids, proteins and vitamins, and its content is higher than that of ordinary vegetables and fruits. In particular, asparagus contains trace elements such as aspartamide, selenium, molybdenum, and manganese, which have the effect of regulating body metabolism and improving body immunity. It is known as the "king of vegetables" internationally.

[0003] Stem blight is the number one disease in asparagus cultivation, often causing devastating losses and seriously affecting the yield and quality of Chinese asparagus. The reduction in asparagus production is not only due to asparagus stem blight. During the 15-20 years of continuous cropping of asparagus, ursolic acid, as one of the allelopathic autotoxic substances of asparagus, seriously affects the growth of asparagus, resulting in a reduction in asparagus production. In addition, the chlorophyll content during the growth of asparagus also seriously affects the yield of asparagus. At present, the application of chemical fertilizers is a common method to increase asparagus production, but the residues of chemical fertilizers seriously damage the agricultural ecosystem and cause environmental pollution. Summary of the invention

[0004] The present application is provided to solve the above defects in the prior art. An acinetobacter, a bacterial agent, a plant growth promoter, a biological fertilizer and an application are needed. Experiments have shown that the acinetobacter can degrade ursolic acid produced by asparagus, prevent and treat asparagus stem blight, promote plant growth and fix nitrogen.

[0005] The present application provides an Acinetobacter, which is named Acinetobacter sp.11.12.7.6. The strain has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Committee on December 8, 2023, with a deposit number of CGMCC No. 29270. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, Chaoyang District, Beijing.

[0006] The present application also provides a bacterial agent, the active ingredients of which include the Acinetobacter described in the examples of the present application.

[0007] The present application also provides a plant growth promoter, which contains the Acinetobacter described in the examples of the present application.

[0008] The present application also provides a biofertilizer, which contains the Acinetobacter described in the examples of the present application.

[0009] The Acinetobacter in the present application has been proved through experiments that the Acinetobacter can degrade ursolic acid produced by asparagus, prevent and treat asparagus stem blight, promote plant growth and fix nitrogen.

[0010] In some embodiments, the above-mentioned bacterial agents, plant growth promoters and biological fertilizers can be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0011] In some embodiments, the above-mentioned bacterial agent, in addition to the Acinetobacter, further contains a carrier, and the carrier may be a carrier commonly used in the field of bacterial agents and biologically inert. The carrier may be a solid carrier or a liquid carrier; the solid carrier may be a mineral material, a plant material or a polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material may be at least one of corn flour, soybean flour and starch; the polymer compound may be polyvinyl alcohol and / or polyglycol.

[0012] In some embodiments, the liquid carrier may be an organic solvent, vegetable oil, mineral oil or water; the organic solvent may be decane and / or dodecane.

[0013] As required, a binder, a stabilizer (such as an antioxidant), etc. may be added to the bacterial agent, which is not specifically limited here.

[0014] The present application also provides an application of the above-mentioned Acinetobacter, bacterial agent, plant growth promoter or biofertilizer in degrading ursolic acid produced by asparagus. Acinetobacter has a good degradation rate for ursolic acid produced by asparagus in the range of 0.1-100 mg / L, and still has a degradation effect of 30% even under the condition of high concentration of ursolic acid of 500 mg / L.

[0015] The present application also provides an application of the above-mentioned Acinetobacter, bacterial agent, plant growth promoter or biological fertilizer in the prevention and treatment of asparagus diseases, preferably, in the prevention and treatment of asparagus stem blight. Experiments have shown that after the bacterial liquid of Acinetobacter is fermented and cultured with asparagus, the effect of asparagus in preventing and treating asparagus stem blight is similar to that of chemical fungicides.

[0016] The present application also provides a use of the above-mentioned Acinetobacter, bacterial agent, plant growth promoter or biofertilizer in promoting the recovery of microbial community in rhizosphere soil of asparagus. Acinetobacter can affect the microbial community structure in the rhizosphere of asparagus and can restore the diseased soil microbial community to a healthy state.

[0017] The present application also provides a use of the above-mentioned Acinetobacter, bacterial agent, plant growth promoter or biological fertilizer in promoting plant growth. Preferably, the plant is asparagus.

[0018] The present application also provides an application of the above-mentioned Acinetobacter, bacterial agent, plant growth promoter or biological fertilizer in biological nitrogen fixation.

[0019] The present application also provides a method for promoting the recovery of microbial communities in rhizosphere soil of asparagus, the method comprising: applying the above-mentioned Acinetobacter, bacterial agent, plant growth promoter or biological fertilizer to the rhizosphere soil of asparagus.

[0020] The present application also provides a method for promoting the growth of asparagus, the method comprising: applying the above-mentioned Acinetobacter, bacterial agent, plant growth promoter or biological fertilizer to the rhizosphere soil of asparagus.

[0021] The Acinetobacter, bacterial agent, plant growth promoter, biofertilizer and application provided in each embodiment of the present application have been proved through experiments that Acinetobacter can degrade ursolic acid produced by asparagus, inhibit the toxic effects of toxic substances produced by harmful microorganisms in the roots of asparagus; it exceeds the activity of pathogens in preventing and controlling asparagus stem blight and has the effect of resisting pathogens; it can effectively reduce the content of Phomopsis in diseased soil, promote the recovery of diseased soil microbial communities, and make its structure and function closer to the state of healthy soil; it can increase the chlorophyll content of asparagus, the vitality and growth of the root system, and promote plant growth; it can also have the effect of nitrogen fixation; therefore, Acinetobacter can have important application value in the prevention and control of asparagus diseases and growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In the drawings, which are not necessarily drawn to scale, the same reference numerals may describe similar parts in different views. The same reference numerals with letter suffixes or different letter suffixes may represent different instances of similar parts. The accompanying drawings generally illustrate various embodiments by way of example and not limitation, and together with the specification and claims, are used to illustrate the embodiments applied for. When appropriate, the same reference numerals are used throughout the drawings to refer to the same or similar parts. Such embodiments are illustrative and are not intended to be exhaustive or exclusive embodiments of the present apparatus or method.

[0023] Figure 1 Comparison diagrams of different asparagus according to the embodiments of the present application are shown, wherein one is a blank control without disease, two is asparagus after adding Acinetobacter to cause disease, three is asparagus after not applying Acinetobacter and carbendazim in advance to cause disease, and four is asparagus after adding carbendazim to cause disease.

[0024] Figure 2 The PCR comparative detection results of sRNA of rhizosphere soil with and without Acinetobacter according to Example 5 of the present application are shown;

[0025] Figure 3The PCR comparative detection results of ITS of rhizosphere soil with and without application of Acinetobacter according to Example 5 of the present application are shown. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the technical solution of the present application, the present application is described in detail below in conjunction with the accompanying drawings and specific implementation examples. The embodiments of the present application are further described in detail below in conjunction with the accompanying drawings and specific implementation examples, but are not intended to limit the present application.

[0027] The words "first", "second" and similar words used in this application do not indicate any order, quantity or importance, but are only used to distinguish. The words "include" or "comprise" and similar words mean that the elements before the word include the elements listed after the word, and do not exclude the possibility of including other elements.

[0028] Example 1 Acquisition of strains

[0029] (1) In the field of "Champion" asparagus with asparagus stem blight, select the site where asparagus plants with less disease are found, scrape samples with a sterile spoon, take about 2 g of the sample and put it in 15 mL of sterile water, incubate at 200 r / min and 37°C for 20 min to mix it evenly, and dilute the mixture by 10% gradient. 3 times, spread the dilution on LB medium for purification, repeat the purification step 3-4 times until all colonies have the same morphology, and purification is completed;

[0030] (2) Pick the purified single colony obtained in step (1) and place it in 4 mL LB liquid culture medium. Culture it overnight at 200 r / min and 37°C. Mix the bacterial liquid with 50% glycerol in a volume ratio of 1:1 and store it in a -20°C refrigerator or a -80°C refrigerator.

[0031] Example 2 Identification of strains

[0032] (1) Morphological identification

[0033] The purified bacteria obtained in step (1) of Example 1 were selected and placed in 4 mL of LB liquid culture medium. After culturing at 200 r / min and 37°C for 3 weeks, the morphological characteristics of the purified strain were observed and recorded. The colonies were green with a slight yellow tint, club-shaped, and arranged in clusters. When observed under an optical microscope, the mycelium of the strain was smooth.

[0034] (2) Molecular biological identification

[0035] ① DNA extraction and sequencing of strains

[0036] The purified bacteria were picked up and placed in 4 mL LB liquid medium, cultured at 200 r / min and 37°C overnight, bacterial DNA was extracted using a kit, and 16S rDNA was amplified by PCR (the primer sequences for PCR amplification include: 27f and 1492r, wherein the primer sequence of 27f is: 5'-TCCGTAGGTGAACCTGCGG-3', and the gene sequence of the primer sequence of 27f is shown in SEQ ID NO: 1; the primer sequence of 1492r is: 5'-TCCTCCGCTTATTGATATGC-3'), and the gene sequence of 1492r is shown in SEQ ID NO: 2; the PCR amplification system is 50 µL: 25 µL of 2×Tap PCR PreMix, 2 µL of 27F, 2 µL of 1492R, 1 µL of bacterial DNA, 20 µL of ddH2O was added for mixed reaction. After the reaction, the product was tested by 1% agarose gel electrophoresis. The band was sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing. The gene sequence of Acinetobacter sp.11.12.7.6 was obtained as shown in SEQ ID NO: 3. The specific sequence is shown below.

[0037]

[0038] ②BLAST alignment

[0039] The sequence of Acinetobacter sp.11.12.7.6 obtained in step ① was compared by BLAST in the GenBank database. The results showed that its similarity with Acinetobacter rhizobium and Acinetobacter pitei was 100%, and they were tentatively identified as the same genus. Combined with the morphological identification results, it was finally determined that Acinetobacter sp.11.12.7.6 belonged to the genus Acinetobacter.

[0040] ③Strain preservation

[0041] The Acinetobacter sp.11.12.7.6 strain was deposited on December 8, 2023 at the General Microbiology Center of the China Culture Collection Committee, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No.29270.

[0042] Example 3 Application of Degrading Ursolic Acid

[0043] (1) The purified bacteria obtained in step (1) of Example 1 were placed in 4 mL of LB liquid culture medium and cultured overnight at 200 r / min and 37° C. to obtain a bacterial solution;

[0044] Prepare medium 1, medium 2, medium 3, medium 4 and medium 5;

[0045] Culture medium 1: 0.1 mg ursolic acid, 5 g peptone, 0.5 g ammonium sulfate, 20 g complex inorganic salt (NaCl: KCl: MgCl2 = 10:1:1), 1000 ml water, pH 6.8~7.2.

[0046] Culture medium 2: ursolic acid 1 mg, peptone 5 g, ammonium sulfate 0.5 g, complex inorganic salt (NaCl: KCl: MgCl2 = 10:1:1) 20 g, water 1000 ml, pH 6.8~7.2.

[0047] Culture medium 3: ursolic acid 10 mg, peptone 5 g, ammonium sulfate 0.5 g, complex inorganic salt (NaCl: KCl: MgCl2 = 10:1:1) 20 g, water 1000 ml, pH 6.8~7.2.

[0048] Culture medium 4: ursolic acid 100 mg, peptone 5 g, ammonium sulfate 0.5 g, complex inorganic salt (NaCl: KCl: MgCl2 = 10: 1: 1) 20 g, water 1000 ml, pH 6.8~7.2.

[0049] Culture medium 5: ursolic acid 500 mg, peptone 5 g, ammonium sulfate 0.5 g, complex inorganic salt (NaCl: KCl: MgCl2 = 10: 1: 1) 20 g, water 1000 ml, pH 6.8~7.2.

[0050] The Acinetobacter was transferred into the above five culture media and cultured at 35°C and 180 rpm for 7 days. 10 mL of the culture solution was taken and centrifuged at 3500 r / min for 15 min. The precipitate was discarded and the supernatant was extracted with 10 mL of dichloromethane. The extract phase was evaporated to dryness using a vacuum rotary evaporator, and the residue was dissolved with 1 mL of methanol for later use.

[0051] The residual amount of ursolic acid in the sample was detected by high performance liquid chromatography, and the degradation rate was calculated. The parameter conditions were: acetonitrile and water (pH 2.8 adjusted with acetic acid) as the mobile phase, flow rate 1.0 mL / min, column temperature 30°C, detection wavelength 280 nm. Gradient elution was used, 0-30 min, acetonitrile increased from 5% to 40%, 30-40 min, acetonitrile maintained at 40%, 40-45 min, acetonitrile decreased from 40% to 5%.

[0052] The growth of Acinetobacter sp.11.12.7.6 in the presence of different concentrations of ursolic acid autotoxic substances was analyzed by high-performance liquid chromatography. During the 7-day culture period, Acinetobacter showed a significant concentration dependence on the degradation efficiency of ursolic acid. Specifically, when the concentration of ursolic acid was 0.1 mg / L, Acinetobacter showed extremely high degradation efficiency, with a degradation rate of up to 95.4%. As the concentration of ursolic acid increased to 1 mg / L, the degradation efficiency decreased slightly, but still remained at a high level of 90.8%. Further increasing the concentration of ursolic acid to 10 mg / L, the degradation efficiency dropped to 81.7%. When the concentration of ursolic acid reached 100 mg / L, the degradation efficiency of Acinetobacter dropped significantly to 70.4%. Under the condition of high concentration of ursolic acid of 500 mg / L, the degradation efficiency of Acinetobacter was further reduced to 32.6%.

[0053] Example 4 Effect of preventing and controlling asparagus stem blight and nitrogen fixation to promote growth

[0054] The test site is located in Bincheng District, Binzhou City, Shandong Province. The target of prevention and control is 4-year-old champion asparagus, which used to have an incidence rate of about 23%. The treatment time is half a month before picking bamboo shoots (April 20, 2023) and half a month after picking bamboo shoots (June 20). Treatment method and concentration: Pick the purified bacteria obtained in step (1) of Example 1 in 4 mL LB liquid culture medium, culture it at 200 r / min and 37°C overnight to obtain a bacterial solution; use 300-fold diluted Acinetobacter bacterial solution fermentation liquid to irrigate the roots, 500 mL per hole, and the control agent is 800 times 50% compound carbendazim and blank treatment. The area of ​​the plot is 70m 2 Each treatment was repeated 3 times. The survey method was random sampling, 10 plants were surveyed in each plot, the total number of stems, the number of diseased stems, the height and fresh weight of asparagus, and the nitrogen content were counted, and the control effect was calculated by comparing with the control.

[0055] Table 1 Asparagus stem blight and nitrogen fixation growth promotion effect

[0056]

[0057] According to the survey conducted on July 20, 2023, as shown in Table 1, the stem blight was significantly reduced after treatment with Acinetobacter, with an average diseased stem rate of 6.5%, and 6.3% for carbendazim treatment, which is similar to carbendazim, and its control effect also reached 70.35%. This result reveals that Acinetobacter bacterial solution has the potential to be comparable to chemical fungicides in the prevention and control of asparagus stem blight. Although its incidence rate is slightly higher, this slight difference may not be statistically significant in practical applications. In addition, the growth-promoting effect of Acinetobacter bacterial solution on asparagus is also very obvious, as shown in the field visual effect: the asparagus plants in the Acinetobacter treatment area are tall and dense, while the asparagus in the control area is short and sparse. The nitrogen content in the asparagus samples inoculated with Acinetobacter was significantly higher than that in the control group (untreated) and the carbendazim treatment group. Specifically, the nitrogen content of asparagus samples inoculated with Acinetobacter was 37.64 mg / g, which was higher than that of asparagus samples that had not been treated with any treatment, and also higher than that of asparagus samples that had been treated with carbendazim. The five indicators of plant height, number of stems per plant, fresh weight of each stem, nitrogen content of the plant, and incidence of disease were measured. Acinetobacter showed significant improvement over the control.

[0058] pass Figure 1 It can be seen that the asparagus that was not infected with the disease grew well; the asparagus that was not pre-treated with Acinetobacter and carbendazim withered after the disease occurred, and there were basically no fresh branches and leaves; the asparagus that was treated with carbendazim and the asparagus that was treated with Acinetobacter grew well after the disease occurred.

[0059] Example 5 Application in Restoration of Microbial Community in Rhizosphere Soil of Asparagus

[0060] Select the occurrence point of asparagus plants with less severe diseases, scrape samples with a sterile spoon, take about 2 g of the sample and put it in 15 mL of sterile water, incubate it at 200 r / min and 37°C for 20 min to mix it evenly, and dilute the mixture by 10% gradient. 3 times, spread the dilution on LB medium for purification, repeat the purification step 3-4 times until all colonies have the same morphology, and purification is completed;

[0061] The purified bacteria were picked and placed in 4 mL LB liquid medium, cultured overnight at 200 r / min and 37°C to obtain a bacterial solution, which was applied to the above-mentioned asparagus rhizosphere soil. After 72 hours of application, the soil pretreated with Acinetobacter was obtained. The soil pretreated with Acinetobacter and the healthy soil were purified by the above-mentioned purification method to obtain the colony samples of Acinetobacter pretreated diseased soil and the colony samples of healthy soil, which were sent to Qingke Biotechnology together with the colony samples of the diseased soil. The 16S rDNA (bacteria) in the soil was detected by high-throughput sequencing, and the results were obtained. Figure 2 The results are shown. And the ITS (fungi) in the soil was detected by high-throughput sequencing method, and it was concluded Figure 3 The results are shown. Figure 2 and Figure 3 It can be seen that the treatment with Acinetobacter significantly affected the composition of soil microbial communities, especially the relative abundance of pathogenic fungi Phomopsis decreased to a level similar to that of healthy soil.

[0062] in, Figure 2 These include Cladosporiaceae, Sphaeriaceae, Eremomycetaceae, Lasiosphaeriaceae, Nectriaceae, Rhizophlyctidaceae, Asperigillaceae, and Pleosporaceae. Figure 3 These include the vicinamibacterales, gemmatimonadales, Bacillales, Chitinophagales, Rhizobiales, saccharimonadales, burkholderiales, xanthomonadales, and sphingomonadales.

[0063] The content ratio of the diseased soil microbial community after the addition of Acinetobacter was significantly similar to that of the healthy soil. Specifically, the introduction of Acinetobacter significantly promoted the recovery of the diseased soil microbial community, making its structure and function closer to the state of healthy soil. By analyzing the soil microbial community through high-throughput sequencing technology, we observed that the relative abundance of dominant microbial phyla such as Gemmatimonadales and Bacillales in the soil samples treated with Acinetobacter was not significantly different from that in healthy soil. In addition, the functional structure of the microbial community in the soil treated with Acinetobacter was also similar to that of healthy soil, indicating that the introduction of Acinetobacter had limited effects on the function and stability of the soil microbial community and helped to restore soil health.

[0064] The strains of Acinetobacter sp.11.12.7.6 compete with pathogenic bacteria. The strains of Acinetobacter sp.11.12.7.6 multiply and colonize rapidly and in large quantities in the soil environment where asparagus grows, which can prevent the pathogenic microorganisms from multiplying and reproducing, destroy the colonization of pathogenic microorganisms on asparagus, and interfere with the infection of asparagus by pathogenic microorganisms, thereby playing a role in disease prevention and control.

[0065] In addition, although exemplary embodiments have been described herein, the scope includes any and all embodiments based on the present application with equivalent elements, modifications, omissions, combinations (e.g., various embodiments intersecting schemes), adaptations or changes. The elements in the claims are to be interpreted broadly based on the language adopted in the claims, and are not limited to the examples described in this specification or during the practice of this application, and the examples are to be interpreted as non-exclusive. Therefore, this specification and examples are intended to be considered as examples only, and the true scope and spirit are indicated by the following claims and the full scope of their equivalents.

[0066] The above description is intended to be illustrative rather than restrictive. For example, the above examples (or one or more schemes thereof) may be used in combination with each other. For example, a person of ordinary skill in the art may use other embodiments when reading the above description. In addition, in the above-mentioned specific embodiments, various features may be grouped together to simplify the present application. This should not be interpreted as an intention that a feature of an application that does not require protection is necessary for any claim. On the contrary, the subject matter of the present application may be less than all the features of an embodiment of a particular application. Thus, the following claims are incorporated herein in the specific embodiments as examples or embodiments, wherein each claim is independently a separate embodiment, and it is considered that these embodiments may be combined with each other in various combinations or arrangements. The scope of the present invention should be determined with reference to the appended claims and the full scope of equivalent forms granted by these claims.

[0067] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. An Acinetobacter, characterized in that The Acinetobacter was named Acinetobacter sp.11.12.7.

6. The strain was deposited in the General Microbiology Center of China Culture Collection Committee on December 8, 2023. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, Chaoyang District, Beijing, and the deposit number is CGMCC No.29270.

2. A bacterial agent, characterized in that The active ingredient of the bacterial agent comprises the Acinetobacter described in claim 1.

3. A plant growth promoter, characterized in that The plant growth promoter contains the Acinetobacter described in claim 1.

4. A biological fertilizer, characterized in that: The biological fertilizer contains the Acinetobacter described in claim 1.

5. Use of the Acinetobacter described in claim 1 in degrading ursolic acid produced by asparagus.

6. The use of Acinetobacter according to claim 1 in the prevention and treatment of asparagus diseases, characterized in that: Application in the prevention and treatment of asparagus stem blight.

7. Use of the Acinetobacter described in claim 1 in promoting the recovery of microbial communities in rhizosphere soil of asparagus.

8. The use of Acinetobacter according to claim 1 in promoting plant growth, characterized in that: The plant is asparagus.

9. The use of Acinetobacter according to claim 1 in promoting nitrogen fixation in plants, characterized in that: The plant is asparagus.

10. A method for promoting the recovery of microbial communities in rhizosphere soil of asparagus, characterized in that: The method comprises: applying the Acinetobacter of claim 1 to the rhizosphere soil of asparagus.

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