Klebsiella oxytoca as well as fungicide and application thereof

Abstract

The invention provides klebsiella oxytoca as well as a fungicide and application thereof, and relates to the field of microorganisms. The Klebsiella oxytoca is specifically Klebsiella oxytoca NA-K1, and the preservation number of the Klebsiella oxytoca NA-K1 is CGMCC (China General Microbiological Culture Collection Center) No.32130. The Klebsiella oxytoca NA-K1 can be used for preparing the acid-producing The Klebsiella oxytoca NA-K1 provided by the invention has a relatively comprehensive growth promoting function: the Klebsiella oxytoca NA-K1 can produce heteroauxin at high yield, also has the capability of producing gibberellin, can effectively solubilize phosphorus and potassium, and also has the capability of fixing nitrogen. In addition, the klebsiella oxytoca NA-K1 can resist extreme environments such as acid and alkali, high salt and the like, and is high in stress resistance. Based on a relatively comprehensive growth promoting function and relatively strong stress resistance of the strain, soil nutrients can be increased, the fertilizer utilization rate can be increased, the absorption of plants to the soil nutrients can be promoted, experiments prove that the strain can be successfully planted in rhizosphere soil of cucumbers and spinaches, the growth of the cucumbers and the spinaches can be promoted, the yield can be increased, and the strain has great application potential.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to an acid-producing Klebsiella and a bacterial agent and application thereof. Background Art

[0002] Growth-promoting bacteria mainly refer to a type of beneficial bacteria that live freely in the soil or attach to plant roots, which can promote plant growth and its absorption and utilization of mineral nutrients, produce metabolites to promote plant growth and development, increase crop yields, and inhibit harmful microorganisms to prevent and control diseases. The growth-promoting mechanisms of microorganisms mainly include: biological nitrogen fixation, phosphorus and potassium solubilization, production of plant growth regulators, induction of plant systemic stress resistance, etc. Currently, more and more research focuses on obtaining strains and microbial agents with multi-faceted growth-promoting mechanisms, in order to play an important role in increasing crop yields.

[0003] Klebsiella oxytoca ( Klebsiella oxytoca ) is a very promising plant growth-promoting bacterium. However, its current growth-promoting mechanism is rather one-sided, and its outstanding growth-promoting index yield is not high, and its stress resistance also needs to be improved. For example, the non-patent document "Mechanism of Acidogenic Klebsiella to Improve the Salt-alkali Stress Tolerance of Corn Seedlings" discloses a strain of acidogenic Klebsiella, which produces indoleacetic acid (IAA) at a concentration of 256.57 mg / L. Although it has achieved a relatively high IAA yield, it still needs to be improved, and the acidogenic Klebsiella disclosed in the prior art do not have the function of producing gibberellins.

[0004] Therefore, it is still an urgent need for technicians in this field to obtain a strain of Klebsiella oxytoca with more comprehensive growth-promoting functions and stronger stress resistance. Summary of the invention

[0005] Therefore, the purpose of the present invention is to provide a strain and a bacterial agent with more comprehensive growth-promoting functions and stronger stress resistance, which can produce high indoleacetic acid, have the ability to produce gibberellins, can effectively dissolve phosphorus and potassium, and have the ability to fix nitrogen. In addition, it can also withstand extreme environments such as acid and alkali, high salt, and has strong stress resistance.

[0006] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a kind of oxytoca Klebsiella, and the oxytoca Klebsiella is specifically oxytoca Klebsiella ( Klebsiella oxytoca )NA-K1, on September 30, 2024, was deposited in the General Microbiology Center of China Culture Collection Committee. The address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 32130.

[0007] In a second aspect, the present invention provides an acid-producing Klebsiella agent, comprising an acid-producing Klebsiella ( Klebsiella oxytoca )NA-K1.

[0008] In a third aspect, the present invention provides a method for preparing the Klebsiella oxytoca agent, comprising the following steps: (1) strain activation: activating the Klebsiella oxytoca NA-K1 in an activation medium to obtain NA-K1 bacteria; (2) Seed solution preparation: picking up the NA-K1 bacteria and culturing them in a seed culture medium to obtain a seed solution; (3) Preparation of bacterial agent: inoculating the seed liquid into a liquid fermentation medium for cultivation to obtain the Klebsiella oxytoca inoculum.

[0009] In step (1), the activation medium is NA medium, and the activation conditions are: culturing at 30-35°C for 20-24h; In step (2), the seed culture medium is LB liquid culture medium, and the culture conditions are: 30-35°C, 180-200 rpm shaking to the logarithmic phase; In step (3), the liquid fermentation medium is composed of: 4% glucose, 1.2% corn steep liquor powder, 0.3% (NH4)2SO4, 0.4% NaH2PO4, 0.05% MnSO4, 0.08% CaCO3, 0.05% KCl, 0.01% MgSO4, 0.01% FeSO4, pH value 7.2-7.4; the culture conditions are: 30-35°C, 180-200rpm, 20-24h.

[0010] Furthermore, in step (1), the activation medium is NA medium, and the activation conditions are: culturing at 30-35°C for 20-24h, preferably culturing at 30°C for 24h.

[0011] Furthermore, in step (2), the seed culture medium is LB liquid culture medium, and the culture conditions are: 30-35°C, 180-200 rpm shaking to the logarithmic phase, preferably 30°C, 200 rpm shaking to the logarithmic phase.

[0012] Furthermore, in step (3), the liquid fermentation medium is composed of: 4% glucose, 1.2% corn steep liquor powder, 0.3% (NH4)2SO4, 0.4% NaH2PO4, 0.05% MnSO4, 0.08% CaCO3, 0.05% KCl, 0.01% MgSO4, 0.01% FeSO4, and a pH value of 7.2-7.4; the culture conditions are: 30-35°C, 180-200rpm, 20-24h, preferably 30°C, 200rpm, 24h.

[0013] In a fourth aspect, the present invention provides the use of the oxytoca Klebsiella or the oxytoca Klebsiella agent or the oxytoca Klebsiella agent obtained by the preparation method in high production of indoleacetic acid and / or gibberellin.

[0014] In a fifth aspect, the present invention provides the use of the Klebsiella oxytoca or the Klebsiella oxytoca agent or the Klebsiella oxytoca agent obtained by the preparation method in phosphate solubilization, wherein the phosphate solubilization includes inorganic phosphorus solubilization and / or inorganic phosphorus solubilization.

[0015] In a sixth aspect, the present invention provides use of the Klebsiella oxytoca or the Klebsiella oxytoca agent or the Klebsiella oxytoca agent obtained by the preparation method in potassium solution.

[0016] In a seventh aspect, the present invention provides use of the Klebsiella oxytoca or the Klebsiella oxytoca agent or the Klebsiella oxytoca agent obtained by the preparation method in nitrogen fixation.

[0017] In an eighth aspect, the present invention provides use of the Klebsiella oxytoca or the Klebsiella oxytoca agent or the Klebsiella oxytoca agent obtained by the preparation method in promoting plant growth.

[0018] Furthermore, the Klebsiella oxytoca or the Klebsiella oxytoca agent is used to promote the growth of vegetables; the vegetables include cucumbers and lettuce.

[0019] The technical solution of the present invention has the following advantages: The present invention provides the oxytoca Klebsiella ( Bacillus halodurans )NA-K1 has relatively comprehensive growth-promoting functions: it can produce high levels of indoleacetic acid (IAA), and it also has the ability to produce gibberellins. It can effectively convert insoluble potassium silicates into soluble potassium salts (i.e., potassium dissolution), convert organic phosphorus compounds into phosphates (dissolution of organic phosphorus), and convert insoluble inorganic phosphorus into soluble phosphorus (i.e., dissolution of inorganic phosphorus). It also has the ability to fix nitrogen. In addition, Klebsiella acidophilus NA-K1 is resistant to extreme environments such as acid and alkali, and high salt, and has strong stress resistance. Based on the relatively comprehensive growth-promoting functions and strong stress resistance of this strain, it can increase soil nutrients, improve fertilizer utilization, and promote plant absorption of soil nutrients. Experiments have shown that it can be successfully colonized in the rhizosphere soil of cucumbers and spinach, promote the growth of cucumbers and spinach, and increase yields. It has great application potential.

[0020] Specifically: 1. Klebsiella oxytoca NA-K1 can produce high IAA yields, with an outstanding yield of up to 350.40 μg / mL, which is significantly higher than the existing IAA yields of Klebsiella oxytoca; 2. Klebsiella oxytoca NA-K1 has the ability to produce gibberellins, with a yield of 30.57 mg / L; 3. Klebsiella oxytoca NA-K1 has a strong ability to degrade inorganic phosphorus. The qualitative determination of D / d value is 1.63, and the quantitative determination of the soluble phosphorus content after degrading phosphorus is 1486.96 mg / L. 4. Klebsiella oxytoca NA-K1 has a strong ability to degrade organic phosphorus, and the D / d value is 4.13 according to qualitative determination; 5. Klebsiella oxytoca NA-K1 has a strong potassium-solubilizing ability, and the qualitative determination D / d value is 2.63; 6. Klebsiella oxytoca NA-K1 has the ability to fix nitrogen and can grow on nitrogen-fixing medium; 7. Klebsiella acidophilus NA-K1 has strong stress resistance, strong acid-base tolerance, and a wide range of salt tolerance. The strain can still grow at pH=2, pH=12, and salt content of 25%; 8. Klebsiella oxytoca NA-K1 can successfully colonize the roots of cucumber and lettuce. The number of colonization in cucumber roots reached the highest peak on the 5th day, and the rhizosphere bacterial activity was 1.98×10 9 cfu / g; the number of colonization in the roots of lettuce reached its peak on the 10th day, and the activity of rhizosphere bacteria was 4.81×10 9 cfu / g; 9. The bacterial agent of Klebsiella acidophilus NA-K1 has a significant growth-promoting effect on cucumber and lettuce. Among them, it has a significant growth-promoting effect on plant height, stem diameter and single leaf area of ​​cucumber, with increases of 25.90%, 10.71% and 26.97% respectively; it has a significant growth-promoting effect on plant height and single leaf area of ​​lettuce, with increases of 16.22% and 16.01% respectively, and has little effect on stem diameter and leaf number. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The present invention provides a colony morphology of Klebsiella oxytoca NA-K1 on NA culture medium and a bacterial morphology diagram under an optical microscope; Figure 2 This is a qualitative test result diagram of the decomposition of inorganic phosphorus by Klebsiella oxytoca NA-K1 provided by the present invention; Figure 3This is a qualitative detection result diagram of the decomposition of organic phosphorus by Klebsiella oxytoca NA-K1 provided by the present invention; Figure 4 This is a qualitative test result diagram of potassium solubilization of Klebsiella oxytoca NA-K1 provided by the present invention; Figure 5 It is a qualitative detection result diagram of IAA production of Klebsiella oxytoca NA-K1 and a control group provided by the present invention; Figure 6 It is a qualitative test result diagram of gibberellin production of Klebsiella oxytoca NA-K1 and a control group provided by the present invention; Figure 7 This is a qualitative detection result diagram of nitrogen fixation by Klebsiella oxytoca NA-K1 provided by the present invention. DETAILED DESCRIPTION

[0023] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0024] The inventors isolated and screened three strains of Klebsiella oxytoca (KLEB) from the rhizosphere soil of cherry trees in different cherry orchards in Shanhaiguan District, Hebei Province. Klebsiella oxytoca ), named NA-K1, NA-K2, and NA-K3 respectively. The separation process is as follows: weigh 10g of soil sample and add it to 100mL of water, shake at 200r / min for 30min, draw 5mL of sample solution and add it to 45mL of sterile water to obtain 10 -2 dilution, dilute step by step to get 10 -4 , 10 -5 , 10 -6 The dilution was then evenly spread on the NA solid medium plate and cultured at 35°C. After 1 day, a single colony was picked and continued to be cultured on the NA solid medium. After 1 day of culture in a 35°C constant temperature incubator, the colony morphology was observed and the bacterial morphology was observed under an optical microscope.

[0025] like Figure 1 As shown, the morphological characteristics of Klebsiella oxytoca NA-K1 are micro-rod-shaped, approximately spherical; the surface of the colony is smooth and opaque, milky white or grayish white, and the surface is moist.

[0026] The identification results of NA-K1, NA-K2, and NA-K3 strains were all Klebsiella oxytoca ( Klebsiella Oxytoca), and the strain NA-K1 with the best performance was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit address of the depository is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 30, 2024, and the deposit number is CGMCC No.32130.

[0027] The culture medium of the present invention comprises: NA medium: peptone 10g, beef powder 3g, NaCl 5g, distilled water 1L, pH about 7.2; LB liquid medium: 5 g yeast powder, 10 g tryptone, 10 g sodium chloride, 20 g agar, pH 7; Meng Jinna inorganic phosphorus liquid medium: glucose 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, calcium phosphate 5g, pH 7.0~7.2; Meng Jinna inorganic phosphorus solid medium: glucose 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, calcium phosphate 5g, agar 20g, pH 7.0~7.2; Meng Jinna organophosphorus liquid medium: glucose 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, egg lecithin 0.2g, calcium carbonate 1g, yeast powder 0.5g, pH 7.0~7.2; Meng Jinna organophosphorus solid medium: glucose 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, egg lecithin 0.2g, calcium carbonate 1g, yeast powder 0.5g, agar 20g, pH 7.0~7.2; Potassium-dissolving solid medium: glucose 10g, disodium hydrogen phosphate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.2g, calcium sulfate 0.2g, calcium carbonate 5g, agar 20g, potassium feldspar powder (150 mesh) 2.5g, water 1L, pH 7.2; Nitrogen-fixing medium: mannitol 10, potassium hydrogen phosphate 0.2, sodium chloride 0.2, magnesium sulfate 0.2, calcium sulfate 0.1, calcium carbonate 5, agar 20, pH 7.2; The composition of the liquid fermentation medium used in the preparation of the bacterial agent is: glucose 4%, corn steep liquor powder 1.2%, (NH4)2SO4 0.3%, NaH2PO4 0.4%, MnSO4 0.05%, CaCO3 0.08%, KCl 0.05%, MgSO4 0.01%, FeSO4 0.01%, pH value 7.2~7.4.

[0028] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the art can be used. The raw materials or instruments used are all conventional products that can be obtained commercially, including but not limited to the raw materials or instruments used in the examples of this application.

[0029] Example 1 Strain cloning identification The DNA of strain NA-K1 was extracted using the TRAN genomic DNA extraction kit (Beijing Quanshijin Biotechnology). Primers were designed based on the most conserved sequence in bacterial 16S rDNA: Primer 27F: 5′-AGA GTT TGA TCC TGG CTCA-3′; Primer 1492R: 5′-GGT TAC CTT GTT ACG ACTT-3′.

[0030] The primers were synthesized by Beijing Liuhe BGI Genomics Co., Ltd.

[0031] PCR reaction conditions: 94°C, 4 min, 94°C, 30 s, 60°C, 30 s, 72°C, 30 s, 30 cycles, and final extension at 72°C for 10 min. The PCR amplification product was connected to the plasmid at 25°C for 15 min, then heat-shocked in a water bath for 45 s and transferred into competent E. coli for cloning and culture. The strain containing the target gene was obtained by blue-white screening and sent to Beijing Liuhe BGI Gene Technology Co., Ltd. for sequencing.

[0032] After bidirectional splicing of the NT sequencing results, the obtained 16S rDNA sequence was compared on the NCBI website, and the result was Klebsiella oxytoca Klebsiella oxytoca , the homology is 100%.

[0033] The 16S rDNA sequence is shown in SEQ ID No. 1: Example 2 Physiological and biochemical identification of strains Physiological and biochemical identification was performed with reference to the relevant contents of the Manual of Identification of Common Bacterial Systems and the Bergey's Manual of Bacterial Identification (8th Edition).

[0034] Klebsiella oxytoca NA-K1 is a Gram-negative bacterium, and its physiological and biochemical characteristics are shown in Table 1.

[0035] Table 1 Physiological and biochemical characteristics of Klebsiella oxytoca NA-K1

[0036] Example 3 Qualitative detection of growth-promoting function of strains 1. Detection method ①Inorganic phosphorus detection 60 µL of NA-K1 LB liquid culture medium fermentation broth which had been cultured with shaking (35°C, 200 rpm) for 3 days was inoculated into holes on the Montana inorganic phosphate solid culture medium. 60 µL of LB liquid culture medium was inoculated as a control. The culture was carried out at 35°C for 6 days, and the transparent zone was observed.

[0037] ②Solution of organic phosphorus detection 60 µL of NA-K1 LB liquid culture medium fermentation broth which had been cultured with shaking (35°C, 200 rpm) for 3 days was inoculated into a hole on the Montana organophosphate solid culture medium. 60 µL of LB liquid culture medium was inoculated as a control. The culture was carried out at 35°C for 6 days, and the transparent zone was observed.

[0038] ③ Potassium dissolution test Punch a hole on the potassium-dissolving solid medium and inoculate 60 µL of NA-K1 LB liquid culture medium fermentation broth that has been shaken (35°C, 200 rpm) for 2 days. Inoculate 60 µL of LB liquid culture medium as a control. Culture at 35°C for 6 days and observe the transparent zone.

[0039] ④IAA detection No liquid was added to well 1 as a blank control; 50 µL of LB liquid culture medium and 50 µL of color developer were added to well 2 as a negative control; 50 µL of IAA standard solution (100 µg / mL) and 50 µL of color developer were added to well 3 as a positive control; 50 µL of LB liquid culture medium fermentation broth (with 500 µg / mL tryptophan) of Klebsiella oxytoca NA-K1 cultured with shaking culture (35°C, 200 rpm) for 3 days and 50 µL of color developer were added to well 4; 50 µL of LB liquid culture medium fermentation broth (with 500 µg / mL tryptophan) of Klebsiella oxytoca NA-K2 cultured with shaking culture (35°C, 200 rpm) for 3 days and 50 µL of color developer were added to well 5; 50 µL of LB liquid culture medium fermentation broth (with 500 µg / mL tryptophan) of Klebsiella oxytoca NA-K3 cultured with shaking culture (35°C, 200 rpm) for 3 days and 50 µL of color developer were added to well 6. LB liquid culture medium fermentation liquid (add tryptophan 500µg / mL) 50µL and color developer 50µL. Place the plate away from light for 30 minutes and observe the color reaction.

[0040] ⑤ Gibberellin detection Add 500 µL of NA-K1 LB liquid culture medium fermentation broth that has been shaken (35°C, 200 rpm) for 3 days to the test tube, and add 500 µL of LB liquid culture medium as a control. Also add 1.5 mL each of Folin's reagent and concentrated hydrochloric acid to the test tube, and observe the color reaction.

[0041] ⑥ Nitrogen fixation detection NA-K1 was streaked onto nitrogen-fixing medium and cultured at 35°C for 3 days to observe whether the strain grew.

[0042] 2. Results ①Interpretation of inorganic phosphorus test results After 4 days of incubation at 35℃, a transparent zone appeared. Figure 1 As shown, the diameter D of the transparent ring is 13 mm, the diameter d of the hole is 8 mm, and the D / d value is 1.63.

[0043] ②Interpretation of organophosphorus test results After 4 days of incubation at 35℃, a transparent zone appeared. Figure 2 As shown, the diameter D of the transparent ring is 33 mm, the diameter d of the hole is 8 mm, and the D / d value is 4.13.

[0044] ③ Potassium dissolution test results After 4 days of incubation at 35℃, a transparent zone appeared. Figure 3 As shown, the diameter D of the transparent ring is 21 mm, the diameter d of the hole is 8 mm, and the D / d value is 2.63.

[0045] ④IAA test results The qualitative test results of IAA production of three strains of Klebsiella oxytoca, NA-K1, NA-K2, and NA-K3 are as follows: Figure 4As shown, it can be seen that the well containing NA-K1 bacterial solution has the darkest color, proving that its IAA production is significantly higher.

[0046] ⑤ Gibberellin test results The qualitative test results of gibberellin production by Klebsiella oxytoca NA-K1 are as follows Figure 5 As shown, it can be seen that the test tube on the right containing NA-K1 bacterial solution appears green, and the color is darker than that of the control test tube on the left, proving that Klebsiella oxytoca NA-K1 has the ability to produce gibberellins.

[0047] ⑥Nitrogen fixation test results like Figure 6 As shown, after NA-K1 was cultured at 35°C on nitrogen-fixing medium for 3 days, the strain grew significantly, proving that the strain has the ability to fix nitrogen.

[0048] Example 4 Quantitative determination of growth-promoting function of strains 1. Detection method ①Quantitative determination of inorganic phosphorus a. Phosphorus standard curve determination: Phosphorus standard stock solution: Accurately weigh 0.2195 g of potassium dihydrogen phosphate that has been dried at 105°C for 2 hours, dissolve it in distilled water, add 5 mL of sulfuric acid solution (ρ=1.84), cool it, and calibrate it to 1000 mL to obtain a phosphorus standard stock solution with a phosphorus (P) concentration of 50 μg / mL (500 ppm).

[0049] Potassium antimony tartrate solution: Take 0.5 g of potassium antimony tartrate and dissolve it in 100 mL of water to obtain solution A.

[0050] Ammonium molybdate-sulfuric acid solution: Weigh 10 g of ammonium molybdate, dissolve it in 450 mL of water, slowly add 153 mL of concentrated sulfuric acid while stirring to obtain solution B.

[0051] Molybdenum antimony sulfate stock solution: Add the above solution A to solution B, and finally add water to 1L, shake well, and store in a brown bottle to obtain molybdenum antimony sulfate stock solution.

[0052] 7.5N molybdenum antimony sulfate anti-color developing agent: Add 1.5g L-ascorbic acid to 100mL molybdenum antimony sulfate stock solution. This reagent is valid for 24 hours and should be prepared before use.

[0053] Pipette 0, 100, 200, 300, 400, and 500 μL of 500 ppm phosphorus standard stock solution into a 50 mL volumetric flask (corresponding to concentrations of 0, 1, 2, 3, 4, and 5 ppm), add 5 mL of 7.5 N molybdenum antimony sulfate anti-color developer, add ddH2O to make up to the scale, shake well, let stand for 30 min, and determine the OD660 value; draw a phosphorus standard curve with concentration as the horizontal axis and the corresponding OD660 value as the vertical axis.

[0054] b. Quantitative determination of inorganic phosphorus: A single NA-K1 colony was picked and inoculated into LB liquid culture medium, and cultured at 35°C and 200rpm for 3 days. The bacterial solution was centrifuged at 10000rpm for 10 minutes, the bacteria were collected, washed 3 times with physiological saline, resuspended in Meng Jinna inorganic phosphorus liquid culture medium, and cultured at 35°C and 200rpm for 7 days; the bacterial solution was centrifuged at 10000rpm for 10 minutes, 0.1mL of the supernatant was taken into a 100mL volumetric flask, 5mL of 7.5N molybdenum antimony sulfate anti-color developing agent was added, ddH2O was added to the scale, the volume was fixed to the mark, shaken, and allowed to stand for 30 minutes, and the OD660 value was determined; the content of soluble phosphorus in the bacterial solution was calculated based on the measured OD660 value and the phosphorus standard curve.

[0055] ② Determination of the ability to produce indoleacetic acid a. IAA standard curve determination: IAA standard solutions with concentrations of 0, 10, 30, 40, 50, and 60 μg / mL were prepared and mixed with an equal volume of Salkowski colorimetric solution. The solutions were placed at room temperature and away from light for 30 min, and then the OD530 values ​​of the solutions at each concentration were measured (the mixed solution of distilled water and an equal volume of Salkowski colorimetric solution was used as the blank control). The IAA standard curve was drawn with the IAA concentration as the horizontal axis and the corresponding OD530 value as the vertical axis.

[0056] b. Determination of IAA concentration in bacterial solution: Three strains of Klebsiella oxytoca, NA-K1, NA-K2 and NA-K3, were activated and inoculated into LB liquid culture medium (containing L-tryptophan at a final concentration of 500 mg / L), respectively. After culturing at 35°C for 3 days, the culture medium was taken out and centrifuged at 6000 rpm for 15 min. Then, 4 mL of the supernatant was mixed with an equal volume of Salkowski colorimetric solution and placed in the dark at room temperature for 30 min. The OD530 value was determined (the mixed solution of uninoculated LB liquid culture medium and an equal volume of Salkowski colorimetric solution was used as a control). The IAA concentration in the bacterial solution was calculated based on the measured OD530 value and the IAA standard curve.

[0057] ③ Determination of the ability to produce gibberellins a. Determination of gibberellin standard curve: Gibberellic acid standard solution (200ppm): Add 20mg of gibberellin into a 100mL volumetric flask and add anhydrous ethanol to make up to volume.

[0058] Gibberellic acid standard curve: pipette 0, 0.1, 0.2, 0.3, 0.4mL (equivalent to 0, 20, 40, 60, 80μg of gibberellin) of gibberellin standard solution into test tubes in turn, heat the test tubes in a boiling water bath for 5-6min, add 1.5mL each of Folin reagent and concentrated hydrochloric acid to the test tubes, mix and let stand for 10min, continue to add water to 5mL to the test tubes, mix well, and measure the OD680 value; draw the gibberellin standard curve with the gibberellin concentration as the horizontal axis and the corresponding OD680 value as the vertical axis.

[0059] b. Determination of gibberellin concentration in bacterial solution: Activate Klebsiella oxytoca NA-K1, inoculate it in LB liquid culture medium, culture it at 35℃ for 3 days, take it out, aspirate 1mL of fermentation broth, add 9mL of water, plug the rubber stopper and shake for 1min, filter, transfer 2mL into a separatory funnel, add 4mL of ethyl acetate and shake for 2min to extract, let it stand for 5min to separate the layers, and discard the lower layer; aspirate 0.5mL of the upper ethyl acetate extract and determine its OD680 value; calculate the gibberellin concentration in the bacterial solution based on the measured OD680 value and the gibberellin standard curve.

[0060] 2. Results ①IAA quantitative test results The results are shown in Table 2. NA-K1 has a strong ability to produce IAA, and its IAA production is 6 to 11 times that of the other two strains of acid-producing Klebsiella.

[0061] Table 2 IAA quantitative detection results

[0062] ② Quantitative test results of gibberellins and inorganic phosphorus The results are shown in Table 3. NA-1 has a strong ability to produce gibberellins and decompose inorganic phosphorus.

[0063] Table 3 Quantitative detection results of gibberellins and inorganic phosphorus

[0064] Example 5: Detection of strain stress resistance 1. Detection method ① Acid and alkali resistance test of strains Pick an appropriate amount of NA-K1 bacteria and inoculate it into 100 mL LB liquid culture medium. Incubate it at 35 ° C and 200 r / min with a shaker for 24 h to obtain the seed solution. Inoculate it at a 1% inoculum amount into LB liquid culture medium adjusted to pH values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively. Incubate it at 35 ° C and 200 r / min with a shaker for 1 day, and determine the OD600 value.

[0065] ②Strain salt tolerance test Pick an appropriate amount of NA-K1 bacteria and inoculate it into 100 mL LB liquid culture medium. Culture it at 35 ° C and 200 r / min in a shaking incubator for 24 h to obtain the seed solution. Inoculate it at a 1% inoculum amount into LB liquid culture medium with NaCl contents of 1%, 5%, 10%, 15%, 20%, and 25%, respectively. Culture it at 35 ° C and 200 r / min in a shaking incubator for 1 day, and determine its OD600 value.

[0066] 2. Results ①Acid and alkali resistance test results As shown in Table 4, the pH tolerance range of NA-K1 is 2-12. The strain can grow in this range and has high acid-base tolerance.

[0067] Table 4 Acid and alkali resistance test results

[0068] ②Salt tolerance test results As shown in Table 5, NA-K1 can still grow under conditions with a salt content as high as 25%, showing strong salt tolerance.

[0069] Table 5 Salt tolerance test

[0070] Example 6 Preparation of Klebsiella oxytoca inoculum This embodiment provides a method for preparing an acid-producing Klebsiella agent, and the specific steps are as follows: (1) Strain activation: Klebsiella oxytoca NA-K1 was activated in NA medium and cultured at 30°C for 24 h to obtain NA-K1 bacteria; (2) Seed solution preparation: Pick NA-K1 bacteria and culture them in LB liquid medium with a liquid volume of 100 mL / 500 mL. Oscillate at 30°C and 200 rpm until the logarithmic phase to obtain seed solution. (3) Preparation of inoculant: The seed solution was inoculated into a liquid fermentation medium and cultured at 30°C and 200 rpm for 24 h to obtain an acidogenic Klebsiella inoculant with a bacterial activity of 9.41×10 8 cfu / mL.

[0071] Example 7 Colonization dynamics of strains in cucumber and lettuce roots 1. Detection method ①Seed treatment The cucumber and lettuce seeds were soaked in 50°C warm water for 10 minutes, disinfected with 1% sodium hypochlorite disinfectant for 3 minutes, rinsed with sterile water for 3 to 5 times, and air-dried. The seeds were soaked with the bacterial agent prepared in Example 6 at a ratio of 1:50, and the seeds were soaked with blank liquid fermentation culture as a control. After air-drying at room temperature, the seeds were divided into two parts, one of which was used to determine the initial bacterial load of the seeds by plate count method, and the other was used for sowing.

[0072] ② Determination of initial seed bacterial load Take 20 cucumber and lettuce seeds treated with the fungicide, put them into Erlenmeyer flasks containing 50 mL of sterile water, and shake them on a shaker at 200 r / min for 30 min. After serial dilution with sterile water, take 0.1 mL and spread it on an LB medium plate. Repeat each treatment 3 times. After culturing at 30°C for 2 days, use the plate count method to count the initial number of bacteria on the seeds.

[0073] ③Sowing and management Seeds treated with the fungicide were planted in sterilized nutrient soil (consisting of perlite, vermiculite and peat, containing 14.85% organic matter, 0.4% total nitrogen, 2.62% total potassium, 0.16% total phosphorus, pH 6.24), and seeds soaked in blank liquid fermentation culture solution were used as controls. Six pots were treated per time, with 20 seeds in each pot. They were cultured at room temperature (20-25°C) and watered regularly (100 mL / pot, once a week). Plant growth was observed after germination, and sampling began on the 6th day after germination.

[0074] ④Detection Shake off the large pieces of soil on the roots, retain the attached soil, weigh it and put it into a conical flask containing 50mL of sterile saline, place it on a shaker at 200r / min for 30min, dilute it in series with sterile water, take 0.1mL of the dilutions of different concentrations and spread it on the LB culture medium plate, repeat each treatment 3 times, and culture it at 30℃ for 2d. The control plants were separated in the same way as above, and the colonization of the labeled strain in the rhizosphere of cucumber and lettuce was detected by the plate count method.

[0075] 2. Test results ① Colonization dynamics of strains in cucumber roots The colonization amount of Klebsiella oxytoca NA-K1 in cucumber roots is shown in Table 6. The colonization amount reached the peak on the 5th day, and showed a significant downward trend on the 20th day, decreasing by one order of magnitude on the 35th day.

[0076] Table 6 Colonization dynamics of strains on cucumber roots

[0077] ② Colonization dynamics of strains in lettuce roots The colonization amount of Klebsiella oxytoca NA-K1 in the roots of lettuce is shown in Table 7. The colonization amount reached the peak on the 10th day, and showed a significant downward trend on the 30th day, decreasing by one order of magnitude on the 35th day.

[0078] Table 7 Colonization dynamics of strains in lettuce roots

[0079] This shows that Klebsiella oxytoca NA-K1 can successfully colonize the roots of cucumber and lettuce.

[0080] Example 8 Detection of the growth-promoting effect of strains on vegetables 1. Detection method ① The growth-promoting effect of the strain on cucumber growth Each greenhouse planting area is 4.5m long, 3.5m wide and 15.75m in area. 2 , randomly arranged. Each plot has 6 rows, with 10 trees planted in each row (equivalent to 38,000 plants / hm2 2 ), 15 days after planting, root inoculation: the control group was 30 mL / tree of liquid fermentation medium (diluted 500 times); the experimental group was irrigated with an equal volume of the bacterial agent prepared in Example 6 (the number of viable bacteria was 10 8 cfu / mL, diluted 500 times), watered once every 10 days, for a total of 3 times, to strengthen the colonization of bacteria in the rhizosphere. Other management measures were the same as those of the local cold greenhouse.

[0081] The middle 4 rows excluding the side rows were selected as test samples. After 60 days of planting, plant height, stem diameter and single leaf area were measured.

[0082] ②The growth-promoting effect of the strain on lettuce Each greenhouse planting area is 4.5m long, 3.5m wide and 15.75m in area. 2 , randomly arranged. Each plot has 10 rows, and each row is sown and sown. Later, thinning is performed with a spacing of 15 cm between each tree. Root inoculation after 20 days of planting: the control group is 30 mL / tree of liquid fermentation medium (diluted 500 times); the experimental group is irrigated with an equal volume of the bacterial agent prepared in Example 5 (the number of viable bacteria is 10 8 cfu / mL, diluted 500 times), watered once every 10 days, for a total of 3 times, to strengthen the colonization of bacteria in the rhizosphere. Other management measures were the same as those of the local cold greenhouse.

[0083] The middle 8 rows excluding the side rows were selected as test samples. After 60 days of planting, plant height, stem diameter, single leaf area and leaf number were measured.

[0084] 2. Test results ① The growth-promoting effect of the strain on cucumber The growth-promoting effects of Klebsiella oxytoca NA-K1 on cucumber plant height, stem diameter, and single leaf area are shown in Table 8: Table 8 Growth-promoting effect of Klebsiella oxytoca NA-K1 on cucumber

[0085] ②The growth-promoting effect of the strain on lettuce The growth-promoting effects of Klebsiella oxytoca NA-K1 on lettuce plant height, stem thickness, single leaf area, and leaf number are shown in Table 9: Table 9 Growth-promoting effect of Klebsiella oxytoca NA-K1 on lettuce

[0086] It can be seen that the bacterial agent of Klebsiella acidophilus NA-K1 has a significant growth-promoting effect on cucumber and lettuce. Among them, it has a significant growth-promoting effect on plant height, stem diameter and single leaf area of ​​cucumber, with increases of 25.90%, 10.71% and 26.97% respectively; it has a significant growth-promoting effect on plant height and single leaf area of ​​lettuce, with increases of 16.22% and 16.01% respectively, and has little effect on stem diameter and leaf number.

[0087] In summary, the present invention provides Klebsiella oxytoca ( Bacillus halodurans )NA-K1 has relatively comprehensive growth-promoting functions: it can produce high levels of indoleacetic acid (IAA), and it also has the ability to produce gibberellins. It can effectively convert insoluble potassium silicates into soluble potassium salts (i.e., potassium dissolution), convert organic phosphorus compounds into phosphates (dissolution of organic phosphorus), and convert insoluble inorganic phosphorus into soluble phosphorus (i.e., dissolution of inorganic phosphorus). It also has the ability to fix nitrogen. In addition, Klebsiella acidophilus NA-K1 is resistant to extreme environments such as acid and alkali, and high salt, and has strong stress resistance. Based on the relatively comprehensive growth-promoting functions and strong stress resistance of this strain, it can increase soil nutrients, improve fertilizer utilization, and promote plant absorption of soil nutrients. Experiments have shown that it can be successfully colonized in the rhizosphere soil of cucumbers and spinach, promote the growth of cucumbers and spinach, and increase yields. It has great application potential.

[0088] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A Klebsiella oxytoca, characterized in that The oxytoca Klebsiella is specifically oxytoca Klebsiella ( Klebsiella oxytoca )NA-K1, the deposit number is CGMCC No.32130.

2. A Klebsiella oxytoca agent, characterized in that Contains Klebsiella oxytoca (CGMCC No. 32130) Klebsiella oxytoca )NA-K1.

3. The method for preparing the Klebsiella oxytoca inoculum according to claim 2, characterized in that: The following steps are involved: (1) strain activation: activating the Klebsiella oxytoca NA-K1 in an activation medium to obtain NA-K1 bacteria; (2) Seed solution preparation: picking up the NA-K1 bacteria and culturing them in a seed culture medium to obtain a seed solution; (3) Preparation of bacterial agent: inoculating the seed liquid into a liquid fermentation medium for cultivation to obtain the Klebsiella oxytoca inoculum.

4. The method for preparing the Klebsiella oxytoca inoculum according to claim 3, characterized in that: In step (1), the activation medium is NA medium, and the activation conditions are: culturing at 30-35°C for 20-24h; In step (2), the seed culture medium is LB liquid culture medium, and the culture conditions are: 30-35°C, 180-200 rpm shaking to the logarithmic phase; In step (3), the liquid fermentation medium is composed of: 4% glucose, 1.2% corn syrup powder, 0.3% (NH4)2SO4, 0.4% NaH2PO4, 0.05% MnSO4, 0.08% CaCO3, 0.05% KCl, 0.01% MgSO4, 0.01% FeSO4, pH value 7.2-7.4; the culture conditions are: 30-35°C, 180-200rpm for 20-24h.

5. Use of the oxytoca Klebsiella according to claim 1, the oxytoca Klebsiella agent according to claim 2, or the oxytoca Klebsiella agent obtained by the preparation method according to claim 3 or 4 in high production of indoleacetic acid and / or gibberellin.

6. Use of the oxytoca Klebsiella according to claim 1, the oxytoca Klebsiella agent according to claim 2, or the oxytoca Klebsiella agent obtained by the preparation method according to claim 3 or 4 in phosphate solubilization, wherein the phosphate solubilization includes inorganic phosphorus solubilization and / or inorganic phosphorus solubilization.

7. Use of the oxytoca Klebsiella according to claim 1, the oxytoca Klebsiella agent according to claim 2, or the oxytoca Klebsiella agent obtained by the preparation method according to claim 3 or 4 in potassium solution.

8. Use of the oxytoca Klebsiella according to claim 1, the oxytoca Klebsiella agent according to claim 2, or the oxytoca Klebsiella agent obtained by the preparation method according to claim 3 or 4 in nitrogen fixation.

9. Use of the Klebsiella oxytoca according to claim 1, the Klebsiella oxytoca agent according to claim 2, or the Klebsiella oxytoca agent obtained by the preparation method according to claim 3 or 4 in promoting plant growth.

10. The use according to claim 9, characterized in that: The Klebsiella oxytoca or the Klebsiella oxytoca agent is used for promoting the growth of vegetables; the vegetables include cucumbers and lettuce.

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