A fungal endophytic Klebsiella michiganensis and its application

Abstract

The present invention discloses an endophytic fungal Klebsiella Michigan and an application thereof. The deposit number of the strain is CCTCC M 2022708, and it was deposited in the China Center for Type Culture Collection on May 24, 2022. The endophytic fungal Klebsiella Michigan of the present application can grow in a nitrogen-free environment and has high nitrogenase activity, thereby providing a nitrogen source for plant growth and development. The endophytic fungal Klebsiella Michigan of the present application expands the resources of nitrogen-fixing strains. At the same time, the endophytic fungal Klebsiella Michigan of the present application has the ability to colonize wheat, synthesize IAA and serve as an iron carrier, thereby increasing the protein content of beans.

Description

Technical Field

[0001] The invention relates to the technical field of fungal endophytic bacteria, in particular to fungal endophytic Klebsiella michiganensis and application thereof. Background Art

[0002] Endophytes are fungi or bacteria that live inside the tissues and organs of plants at certain stages or all stages. They are commonly found in higher plants. Endophytic bacteria can be found in woody plants, herbaceous plants, monocotyledons and dicotyledons. They have become potential microbial pesticides, yield-increasing bacteria or as potential biocontrol vectors in biological control.

[0003] One of the main problems encountered in the development of biofertilizers is the lack of high-efficiency bacterial strain resources. Biofertilizer companies are in urgent need of updating high-efficiency bacterial strains. The sources of strains are generally rhizosphere soil, endophytes of plants and animals, etc. Although corn smut is a pathogenic bacterium that causes corn smut to infect various parts of corn to form tumors, it can also be used as a delicacy and is harmless to humans. It is of great significance to the exploration and development of nitrogen-fixing bacterial strain resources.

[0004] Therefore, it is necessary to provide an expanded plant cultivation strain resource of fungal endophytic Klebsiella michiganensis. Summary of the invention

[0005] In view of this, the present application provides a fungal endophytic Klebsiella michiganensis and its application, which are used to solve the problem of insufficient nitrogen-fixing bacteria strain resources.

[0006] In order to achieve the above technical objectives, this application adopts the following technical solutions:

[0007] In the first aspect, the present application provides a fungal endophytic Klebsiella michiganensis, whose preservation number is CCTCC M2022708, and it was deposited in the China Center for Type Culture Collection on May 24, 2022.

[0008] In a second aspect, the present application provides an endophytic Klebsiella michiganensis agent, which contains the endophytic Klebsiella michiganensis with a deposit number of CCTCC M 2022708.

[0009] In a third aspect, the present application provides a method for isolating endophytic Klebsiella michiganensis from fungi, comprising the following steps:

[0010] S1. The corn smut bacteria were inoculated into YEPSL liquid medium to obtain a fermentation broth, the fermentation broth was transferred to a fresh YEPSL liquid medium for culture, centrifuged, resuspended, the bacteria were collected, the bacteria were added to the lysis solution for incubation, and after centrifugation, protoplasts were obtained;

[0011] S2. Grind the protoplasts and culture them in a nitrogen-free medium, then pick out a single colony for purification and culture to obtain Klebsiella michiganensis.

[0012] Preferably, the corn smut contains endogenous bacteria in the body.

[0013] In a fourth aspect, the present application provides a use of an endophytic Klebsiella michiganensis or an endophytic Klebsiella michiganensis agent in the field of plant cultivation.

[0014] Preferably, the fungal endophytic Klebsiella michiganensis or its culture solution or fermentation solution or a bacterial agent containing the same is used to increase the content of plant protein.

[0015] Preferably, the fungal endophytic Klebsiella michiganensis or its culture solution or fermentation solution or a bacterial agent containing the same is used for colonizing wheat.

[0016] Preferably, the fungal endophytic Klebsiella michiganensis or its culture broth or fermentation broth or a bacterial agent containing the same is used in the synthesis of IAA.

[0017] Preferably, the fungal endophytic Klebsiella michiganensis or its culture solution or fermentation solution or a bacterial agent containing the same is used in plant nitrogen fixation.

[0018] In a fifth aspect, the present application provides a fertilizer comprising endophytic Klebsiella michiganensis or an endophytic Klebsiella michiganensis agent.

[0019] The beneficial effects of this application are as follows:

[0020] The fungal endophytic Klebsiella michiganensis of the present application is isolated from edible fungi and is friendly to humans and the environment;

[0021] The fungal endophytic Klebsiella michiganensis of the present application has a strong nitrogen fixation ability and can grow normally under nitrogen-free conditions. Its nitrogenase activity is 646.25±38.61nmol·mL -1 ·h -1 C2H4, at the same time, also has the ability to colonize wheat, synthesize IAA and act as an iron carrier, which can increase the protein content of beans by 6-8;

[0022] The fungal endophytic Klebsiella michiganensis of the present application has a short production cycle and is easy to use, and has very broad application prospects in the fields of plant growth promotion, biofertilizer, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the fluorescence detection result of Klebsiella michiganensis YZUMF20201 in Ustilago maydis;

[0024] Figure 2The colonies (left) and bacterial morphology of Klebsiella michiganensis YZUMF202001 on LB plates;

[0025] Figure 3 This is the test result of nitrogen fixation ability of Klebsiella michiganensis YZUMF202001;

[0026] Figure 4 This is the colonization ability test result of Klebsiella michiganensis YZUMF202001;

[0027] Figure 5 These are the results of the growth-promoting potential test of Klebsiella michiganensis YZUMF202001. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] The present application provides a fungal endophytic Klebsiella michiganensis, hereinafter referred to as YZUMF202001, whose deposit number is CCTCC M 2022708, and is deposited in the China Center for Type Culture Collection on May 24, 2022; YZUMF202001 is a Gram-negative bacterium, rod-shaped, and the colonies formed on the LB plate are round, opaque, convex, moist, and light yellow. The 16S rDNA and nifH sequencing comparison analysis was performed, and the developmental evolution tree was constructed, and the strain YZUMF202001 was finally determined to be Klebsiella michiganensis.

[0030] As is well known to those skilled in the art, nitrogen is the most abundant element in the air and one of the key elements in the growth process of plants. It is an important component of amino acids, proteins, and nucleic acids. Nitrogen cannot be directly used by most organisms. It needs to go through a complex nitrogen fixation process to convert nitrogen into ammonia. This biological nitrogen fixation process is inseparable from the participation of nitrogenase in nitrogen-fixing bacteria. The fungal endophytic Klebsiella michiganensis of the present application can grow in a nitrogen-free environment and has high nitrogenase activity. Therefore, it can provide a nitrogen source for plant growth and development. The fungal endophytic Klebsiella michiganensis of the present application expands the resources of nitrogen-fixing strains.

[0031] The present application provides an endophytic Klebsiella michiganensis agent, which contains the endophytic Klebsiella michiganensis with a deposit number of CCTCC M2022708.

[0032] The present application provides a method for isolating endophytic Klebsiella michiganensis from fungi, comprising the following steps:

[0033] S1. The corn smut bacteria were inoculated into YEPSL liquid medium to obtain a fermentation broth, the fermentation broth was transferred to a fresh YEPSL liquid medium for culture, centrifuged, resuspended, the bacteria were collected, the bacteria were added to the lysis solution for incubation, and after centrifugation, protoplasts were obtained;

[0034] S2. Grind the protoplasts and culture them in a nitrogen-free medium, then pick out a single colony for purification and culture to obtain Klebsiella michiganensis.

[0035] The YZUMF202001 in the present application is isolated from the corn smut. First, the corn smut is cultured on a nitrogen-free medium, and the hyphae and spores are collected to prepare them into protoplasts. The protoplasts are ground into a homogenous slurry by adding an appropriate amount of sterilized quartz sand. After enrichment culture in a nitrogen-free medium, the protoplasts are spread on nitrogen-free plates for screening and single colonies are picked for purification.

[0036] Corn smut contains endophytic bacteria. In the present application, fluorescence in situ hybridization and SYTO-9 staining are used to jointly detect the endophytic bacteria in corn smut, and the fluorescence phenomenon is used to screen out the fungal strains containing endophytic bacteria for subsequent separation.

[0037] The present application provides an application of endophytic Klebsiella michiganensis or an endophytic Klebsiella michiganensis agent in the field of plant cultivation, including: application of endophytic Klebsiella michiganensis or its culture solution or fermentation solution or an agent containing it in increasing the protein content of plants; application of endophytic Klebsiella michiganensis or its culture solution or fermentation solution or an agent containing it in colonizing wheat; application of endophytic Klebsiella michiganensis or its culture solution or fermentation solution or an agent containing it in synthesizing IAA; application of endophytic Klebsiella michiganensis or its culture solution or fermentation solution or an agent containing it in nitrogen fixation of plants.

[0038] The present application provides a fertilizer comprising endophytic Klebsiella michiganensis or an endophytic Klebsiella michiganensis agent.

[0039] The present solution is further described below through specific embodiments.

[0040] Source of raw materials

[0041] The corn smut fungus of the present application is obtained by the following steps:

[0042] Fluorescence in situ hybridization: Corn smut (from the Plant Pathology Laboratory of Yangtze University) was inoculated in a nitrogen-free culture medium and cultured at 28°C for 7 days. An appropriate amount of mycelium was collected in a sterile 1.5 mL centrifuge tube, and the bacteria were resuspended in 1 mL of phosphate buffer. The bacteria were immediately collected by centrifugation at 8000 rpm for 5 min, and the waste liquid was discarded. Then 1 mL of 4% formaldehyde fixative was added and fixed at 4°C for 3 h. The bacteria were resuspended in 1 mL of phosphate buffer and immediately centrifuged at 8000 rpm for 5 min. The waste liquid was discarded. This step was repeated once, and a gradient of 50%, 70%, and 95% ethanol solutions was used for dehydration for 3 min. 100 μL of probe solution (40% formamide, 35% DEPC-treated water, 25% EDTA and 0.01 μM oligonucleotide probe) was added and incubated at 46°C in the dark for 1.5 h, and then washed twice with a washing solution (NaCl 50 mM, SDS 0.01%, Tris-HCl 20 mM, pH=7.2) and prepared for observation.

[0043] SYTO-9 staining: refer to the instruction manual of STYO-9 Green-Fluorescent Nucleic Acid Stains, inoculate corn smut into nitrogen-free culture medium and culture at 28°C for 7 days, collect appropriate amount of mycelium in a sterile 1.5mL centrifuge tube, add 1mL of 0.85% NaCl to resuspend the bacteria, immediately centrifuge at 8000rpm for 5min, discard the waste liquid, repeat this step twice, add 100μL of 0.85% NaCl to gently resuspend the bacteria, then add 100μL of SYTO-9 (10μmol / L) green fluorescent nucleic acid stain, mix well, incubate in the dark for 15min, prepare slides for observation.

[0044] Endophytic bacteria in corn smut were detected by fluorescence in situ hybridization and SYTO-9 staining, and fungal strains containing endophytic bacteria were selected as corn smut bodies used in this application by using fluorescence phenomenon. Figure 1 The results of the detection of Klebsiella michiganensis YZUMF202001 in corn smut are shown, among which, Figure 1 a. Figure 1 b. Figure 1 c is the result of fluorescence in situ hybridization observation. Figure 1 d is the result of SYTO-9 staining observation, indicating that the corn smut used in the present application contains endophytic bacteria.

[0045] Example 1

[0046] A method for isolating endogenous Klebsiella michiganensis from fungi comprises the following steps:

[0047] S1. The corn smut was inoculated into YEPSL liquid medium (4 g yeast extract, 4 g peptone, 20 g sucrose) and placed in a 28°C constant temperature shaker (150 rpm) for 2 days, 1 mL of the fermentation broth was transferred to a fresh YEPSL liquid medium and cultured under the same conditions for 12 h, 50 mL of the fermentation broth was centrifuged at 7000 rpm for 10 min to collect fresh cells, the waste liquid was discarded, 10 mL of NaCl (0.8 M) was added to resuspend the cells and centrifuged at 7000 rpm for 10 min to collect the cells, this step was repeated once, 20 mL of lysis solution (20 mg / mL crash enzyme, 20 mg / mL wall lysing enzyme, 0.22 μm filter was filtered to remove impurities) was added and placed at 28°C (75 rpm) for incubation for 3 h, then centrifuged at 4000 rpm for 10 min to collect protoplasts, and 10 mL of STC solution (1 M sorbitol, 10 mM The cells were washed twice with Tris-HCl, 50 mM CaCl2, pH = 7.5, and finally resuspended in 2 mL STC solution for later use;

[0048] S2. Add the protoplasts of corn smut into a sterile mortar, add an appropriate amount of quartz sand, and grind into a homogenate. Use a pipette to draw 100 μL of the homogenate and spread it on a nitrogen-free culture medium, place it in a 28°C incubator, and after the colonies grow, pick a single colony for purification and culture, and store it in 15% glycerol for later use, which is the strain YZUMF202001.

[0049] The above strain YZUMF202001 was identified: the morphological observation of the isolated strain YZUMF202001 was as follows Figure 2 As shown, Figure 2 a is the colony morphology of Klebsiella michiganensis YZUMF202001 on the LB plate, Figure 2 b is the bacterial morphology of Michigan Klebsiella YZUMF202001 on the LB plate (right), which was determined to be Klebsiella genus. Then the total DNA of the strain was extracted, and the 16S rDNA and nifH genes were amplified and sequenced using the universal primers for bacterial identification. According to the phylogenetic tree results, the strain YZUMF20201 was identified as Michigan Klebsiella, and preserved in the patent strain library of Wuhan University, and the fungal endophytic Michigan Klebsiella with the deposit number of CCTCC M 2022708 was obtained.

[0050] Test and Evaluation

[0051] The nitrogen fixation ability of the YZUMF202001 strain of the present application was tested by plate detection and acetylene reduction method.

[0052] Plate test: The isolated Michigan Klebsiella strain YZUMF202001 and Escherichia coli DH5α were inoculated in LB and cultured at 28°C (150 rpm) for 12 h, washed three times with sterile water, adjusted to OD600 = 1, and 10 μL was inoculated at each position on the nitrogen-free medium to test its nitrogen fixation ability.

[0053] Acetylene reduction method: The isolated Michigan Klebsiella strain YZUMF202001 and Escherichia coli DH5α were inoculated into Nfb medium and cultured at 28°C (150 rpm) for 24 h, adjusted to OD600 = 1, inoculated into fresh Nfb medium at 1% and cultured at 28°C (150 rpm) for 24 h, replaced with acetylene gas at 10% (v / v) and cultured at 28°C (150 rpm) for 24 h, and the gas was collected and detected by GC-MS (MDGCMSMS 8050).

[0054] The results are as follows Figure 3 As shown, Figure 3 As shown in a, it can be clearly observed on the plate that strain YZUMF202001 grows well, with bulges and moisture, while the control Escherichia coli cannot grow normally, indicating that YZUMF202001 has the ability to fix nitrogen; Figure 3 b, nitrogenase activity of strain YZUMF202001 was 646.25±38.61nmol·mL -1 ·h -1 C2H4, while the enzyme activity of E. coli was weak, indicating that strain YZUMF202001 had strong nitrogen fixation ability; at the same time, Figure 3 As shown in c, YZUMF202001 has a complete nitrogen fixation gene cluster of 23.4 kb.

[0055] The colonization ability of the YZUMF202001 strain of the present application was tested.

[0056] IAA detection method:

[0057] Qualitative analysis: Pick a single colony of the isolated endophytic bacteria and inoculate it into LB medium containing 100 mg / L tryptophan, culture it in a constant temperature shaker at 28°C at 200 r / min for 48 hours, pipette 5 mL of bacterial solution into a 10 mL centrifuge tube, and centrifuge it at 10000 r / min for 10 minutes. Take 3 mL of the supernatant and mix it with an equal amount of Salkowski reagent, and react it in the dark for 30 minutes. If the color of the liquid turns pink, it means that the fungal endophytic bacteria have the ability to secrete IAA, otherwise, they have no ability to secrete IAA.

[0058] Quantification: Pick a single colony of the isolated endophytic bacteria and inoculate it into 100mL LB medium containing 100mg / L tryptophan, culture it in a constant temperature shaker at 28℃ at 200r / min, take 5mL of bacterial solution every day, centrifuge it at 10000r / min for 10min, take 3mL of supernatant and add it to a 10mL test tube, add an equal amount of Salkowaski reagent and mix it, and repeat the above operation with uninoculated LB medium. After standing in the dark for 30min, measure the absorbance at 530nm with a UV-visible spectrophotometer, and adjust it to zero with uninoculated LB medium as a control. Repeat the operation three times, record the data, and use the IAA standard curve to calculate the content of IAA produced by fungal endophytic bacteria every day.

[0059] After testing, the results are as follows Figure 4 As shown in a, it is the result of biofilm formation detection, indicating that YZUMF202001 has the ability to produce biofilm, indicating that it has colonization potential. Michigan Klebsiella YZUMF202001 was GFP-labeled by conjugation transfer, as shown in Figure 4 As shown in b, in the wheat germination promotion experiment, after sectioning and observation using a laser confocal microscope, it was shown that YZUMF202001 has the ability to colonize wheat.

[0060] Biofilm detection method: The growth-promoting bacteria were cultured in LB medium with shaking until OD600 = 0.2, 0.4, 0.6, 0.8, 1.0, 5 ml of bacterial solution of different concentrations were taken and continued to be cultured in glass test tubes for 48 hours, and then 5 ml of crystal violet solution was added to react for 15 minutes, and the excess liquid was poured off to observe whether purple ring marks appeared on the wall of the glass test tube. 5 ml of 95% anhydrous ethanol was added to dissolve the biofilm, and the absorbance value at OD570 was measured using a UV spectrophotometer to record the amount of biofilm produced by the growth-promoting bacteria.

[0061] The growth-promoting potential of the YZUMF202001 strain of the present application was tested.

[0062] The IAA method was used to detect the Scharkovsky reaction on YZUMF202001. The results are as follows Figure 5 As shown in a, the results show that it is yellow-brown and the substance produced is tryptamine, which is an intermediate product in the IAA synthesis pathway and has the potential for IAA synthesis. Iron carrier detection is performed, and the results are as follows Figure 5 As shown in b, it was found to have the ability to produce siderophore.

[0063] The YZUMF202001 strain of Example 1 was added to soybean meal and wheat bran for fermentation, and compared with other strains. The fermentation results were tested after 5 days, as follows:

[0064] The changes in the content of each component of soybean meal before and after fermentation are shown in Table 1.

[0065] The results of the changes in the contents of various components in wheat bran before and after fermentation are shown in Table 2.

[0066] Table 1 Results of soybean meal fermentation before and after adding strains

[0067]

[0068] The results in Table 1 show that the protein increase of the fermented soybean meal by the YZUMF202001 strain is: [36.23% (adding the yml strain) - 28.91% (normal production of fermented soybean meal)] ÷ 28.91% = 35.23%, indicating that Klebsiella michiganensis can significantly increase the protein content in soybean meal.

[0069] Table 2 Wheat bran fermentation results

[0070]

[0071] In Table 2, sample 3 was added with Klebsiella Michiganensis YZUMF202001 to participate in the fermentation of wheat bran. The experiment shows the contribution of Klebsiella Michiganensis YZUMF202001 to the fermentation protein of wheat bran. Samples 1, 2, and 4 were round brown nitrogen-fixing bacteria (representative strain of self-generated nitrogen-fixing bacteria, purchased from "China General Bacteria Library" CGMCC1.502), Escherichia coli, and no additional strains were added. The results show that: (14.03%-10.01%) ÷ 10.01% = 40.16%, and Klebsiella Michiganensis can contribute 40.16% of protein to fermented wheat bran.

[0072] The above are only preferred specific implementations of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A fungal endophytic Klebsiella michiganensis, characterized in that Its deposit number is CCTCC M 2022708, and it was deposited in the China Center for Type Culture Collection on May 24, 2022.

2. A fungal endophytic Klebsiella michiganensis inoculant, characterized in that: It contains the fungal endophytic Klebsiella michiganensis with the deposit number of CCTCC M2022708.

3. Use of the fungal endophytic Klebsiella michiganensis as claimed in claim 1 or the fungal endophytic Klebsiella michiganensis agent as claimed in claim 2 in the fields of increasing plant protein content, increasing plant protein content, synthesizing IAA or plant nitrogen fixation.

4. The use according to claim 3, characterized in that: The fungal endophytic Klebsiella michiganensis or its culture solution or fermentation solution or a bacterial agent containing the same is used for increasing the content of plant protein.

5. The use according to claim 3, characterized in that: The fungal endophytic Klebsiella michiganensis or its culture solution or fermentation solution or a bacterial agent containing the same is used in colonizing wheat.

6. The use according to claim 3, characterized in that: The fungal endophytic Klebsiella michiganensis or its culture solution or fermentation solution or a bacterial agent containing the same is used in synthesizing IAA.

7. The use according to claim 3, characterized in that: The fungal endophytic Klebsiella michiganensis or its culture solution or fermentation solution or a bacterial agent containing the same is used in plant nitrogen fixation.

8. A fertilizer, characterized in that The method comprises the endophytic Klebsiella michiganensis of claim 1 or the endophytic Klebsiella michiganensis agent of claim 2.

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