Cancer-generating enterobacter with potassium-dissolving and growth-promoting effects and application of cancer-generating enterobacter

Abstract

The invention discloses a cancerogenic enterobacter with potassium dissolving and growth promoting effects and application thereof, the cancerogenic enterobacter is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.30972. The strain can activate insoluble or insoluble potassium in soil to be absorbed and utilized by plants and promote crop growth. The enterobacter cancerogenic strain has stable flora number, can be effectively colonized in rhizosphere soil, has a rapidly available potassium content of 2.7 [mu] g / mL in a culture solution after 4 d of culture, has good potassium dissolving ability, can activate insoluble or insoluble potassium in soil, and improves potassium element conversion and absorption of crops. The enterobacter cancerogenic has a potassium dissolving effect, activates insoluble or indissolvable potassium, improves potassium element conversion and absorption of crops, and promotes plant growth and nutrient element absorption, so that the yield and quality of the crops are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a cancer-causing enterobacterium with potassium-dissolving and growth-promoting effects and application thereof. Background Art

[0002] Potassium is an indispensable element in intensive agricultural production systems. It is necessary for plant metabolism, such as cell synthesis, enzyme activity, protein and vitamin production. At the same time, it can not only promote plant photosynthesis and induce the synthesis of proteins required for plant growth, but also improve plant quality and increase plant drought and cold resistance. Potassium deficiency can lead to unhealthy growth and development of crops, affect the stress resistance of crops, and cause crop yield reduction. Although the soil is rich in potassium, most of it exists in the form of mineral potassium and fixed potassium. Potassium is difficult to release and cannot be directly absorbed by plant roots. Therefore, how to release the insoluble potassium in the soil in the form of K+ and absorb and utilize it by plants to improve the yield and quality of crops is an important area of ​​research in environmentally friendly agricultural technology.

[0003] Microbial fertilizers made from rhizosphere growth-promoting bacteria have important application value in environmentally friendly agriculture. Potassium-solubilizing bacteria are a type of plant rhizosphere growth-promoting bacteria. Potassium-solubilizing bacteria directly promote the weathering of potassium-containing minerals by producing organic and inorganic acids that can lower the pH of the surrounding environment, leading to the slow release of exchangeable potassium and increasing the content of effective potassium in the environment, thereby improving the absorption and utilization of potassium by crops and promoting crop growth. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a cancer-causing Enterobacterium with potassium-solubilizing and growth-promoting effects. The strain has a good potassium-solubilizing function, can activate insoluble or poorly soluble potassium in the soil for plant absorption and utilization, and promote crop growth.

[0005] The first object of the present invention is to provide a cancer-causing Enterobacter cancerogenus with potassium-dissolving and growth-promoting effects. The cancer-causing Enterobacter cancerogenus is deposited in the General Microbiological Center of China National Committee for the Preservation and Administration of Microbiological Cultures with a deposit number of CGMCC No.30972.

[0006] The second object of the present invention is to provide a bacterial solution of Enterobacter cancerogenus obtained by expanding and culturing the above-mentioned Enterobacter cancerogenus CGMCC No.30972 strain.

[0007] Preferably, the expanded culture is specifically: inoculating the Enterobacter cancerogenus CGMCC No.30972 strain into a culture medium, and culturing at 28±1° C. on a shaking table; preferably, the culture medium is LB liquid culture medium.

[0008] The third object of the present invention is to provide the use of the above-mentioned Enterobacter cancerogenus or Enterobacter cancerogenus bacterial solution in the preparation of potassium-solubilizing microbial agent products.

[0009] The fourth object of the present invention is to provide the use of the above-mentioned Enterobacter cancerogenus or Enterobacter cancerogenus bacterial solution in the preparation of products for improving soil physical and chemical properties and promoting crop growth.

[0010] The fifth object of the present invention is to provide Enterobacter cancerogenus fermentation liquid, which is prepared by fermenting the above-mentioned Enterobacter cancerogenus CGMCC No.30972 strain.

[0011] Preferably, the fermentation preparation is specifically as follows: inoculating the Enterobacter cancerogenus CGMCC No.30972 strain into a preferred fermentation formula liquid, sealing with a sealing film and culturing on a shaker, the culture conditions are 27°C, 2 days, 180r / min, pH=7, and shading.

[0012] Preferably, the Solanaceae vegetables include tomatoes; the leafy vegetables include baby cabbage, coriander and lettuce; and the perennial vegetables include Lanzhou lily.

[0013] The colony number of the cancer-causing Enterobacter K4 of the present invention is relatively stable, and it can effectively colonize in the rhizosphere soil. After culturing for 4 days, the content of available potassium in the culture solution reaches 2.7 μg / mL, and the potassium-solubilizing ability is good, and the insoluble or poorly soluble potassium in the soil can be activated, thereby improving the conversion and absorption of potassium by crops. After watering baby cabbage and tomato with the cancer-causing Enterobacter K4 strain suspension, and watering coriander, lettuce, and Lanzhou lily with the cancer-causing Enterobacter K4 fermentation liquid, the plant growth is promoted.

[0014] The cancer-causing Enterobacter K4 of the present invention has the function of dissolving potassium, activating insoluble or poorly soluble potassium, improving the conversion and absorption of potassium by crops, promoting plant growth and absorption of nutrient elements, thereby improving the yield and quality of crops.

[0015] Cancerogenic Enterobacter is an important member of plant rhizosphere growth-promoting bacteria. The present invention isolates cancerogenic Enterobacter from the rhizosphere soil of continuously planted Lanzhou lily (Lilium davidii var. unicolor). After identification, strain K4 belongs to cancerogenic Enterobacter cancerogenus, which has the effect of potassium dissolving and promoting growth. The cancerogenic Enterobacter cancerogenus strain involved in the present invention is isolated from the rhizosphere soil of Lanzhou lily, can be stably colonized in the soil, has the function of potassium dissolving, and has a good growth-promoting effect on plants such as baby cabbage, tomato, coriander, lettuce, Lanzhou lily, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0017] Figure 1 This is the colony morphology of the cancer-causing Enterobacter K4 strain. A is the primary screening culture medium; B is the potassium-lysis circle on the Alexander silicate medium; C is cultured on the LB medium; D is Gram staining.

[0018] Figure 2 PCR amplification of the 16S rDNA gene fragment of Enterobacter carcinogen K4 strain.

[0019] Figure 3 This is the molecular phylogenetic tree of the cancer-causing Enterobacter K4 strain.

[0020] Figure 4 The influence of soil colonization on the cancer-causing Enterobacter K4 strain.

[0021] Figure 5 The invention discloses the effect of the cancer-causing Enterobacter K4 strain on the growth of baby cabbage plants.

[0022] Figure 6 The invention discloses the influence of the cancer-causing Enterobacter K4 strain on the root system of baby cabbage.

[0023] Figure 7 The invention discloses the effect of the cancer-causing Enterobacter K4 strain on the growth of TOM tomato plants.

[0024] Figure 8 The invention discloses the influence of the fermentation liquid of the cancer-causing Enterobacter K4 strain on the growth of coriander plants.

[0025] Fig. 9 The invention discloses an effect of the fermentation liquid of the cancer-causing Enterobacter K4 strain on the growth of lettuce plants.

[0026] Fig.10The invention discloses the influence of the fermentation liquid of the cancer-causing Enterobacter K4 strain on the growth of the bulb of Lanzhou lily.

[0027] Fig.11 The invention discloses the influence of the fermentation liquid of the cancer-causing Enterobacter K4 strain on the growth of Lanzhou lily in the field. DETAILED DESCRIPTION

[0028] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0029] Example 1

[0030] 1. Culture medium used

[0031] MS liquid medium, Alexander silicate bacterial medium, potassium-deficient liquid medium, LB medium.

[0032] 2. Preparation of reagents and indicators

[0033] 2.1 Preparation of solutions required for determination of potassium dissolving capacity

[0034] (1) 100 μg / mL potassium standard stock solution: 0.1907 g KCl (dried at 110°C for 1 h) dissolved in 1 L potassium-deficient liquid culture medium.

[0035] (2) 4% H2O2

[0036] 2.2 Indicators required for physiological and biochemical tests

[0037] (1) Methyl red solution.

[0038] (2) 6% a-naphthol ethanol solution.

[0039] (3) 40% KOH solution.

[0040] (4) Gram staining solution (produced by Solebold Co., Ltd.).

[0041] (5) 3% to 10% H2O2 solution.

[0042] (6) Indole test solution.

[0043] 2.3 Kit

[0044] The bacterial genomic DNA extraction kit was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0045] 2.4 Main test instruments and equipment

[0046] UV-1780 UV-visible spectrophotometer, high-pressure steam sterilizer, water-proof constant temperature incubator, constant temperature shaker, clean bench, high-speed refrigerated centrifuge, etc.

[0047] 3. Obtaining strains and preparing bacterial suspension

[0048] 3.1 Obtaining the strain: Take soil samples from 5 cm of the cultivated layer of Lanzhou lily, collect the rhizosphere soil of the plant by shaking the roots, and put it into a sterilized ziplock bag; mix all the rhizosphere soil together, sieve it with a 1 mm sieve, remove larger particles of soil and residual roots, spread it thinly on sterile newspapers, dry it in the shade, put it into a sterile ziplock bag, and store it in a 4°C refrigerator. Take 3 250 mL conical flasks, fill each with 100 mL of MS liquid culture medium, and then add 1 g of soil sample to each conical flask, and shake and culture it on a constant temperature shaker (28°C, 180 rpm) for 3 days. Then aspirate the soil suspension for transfer to enrich potassium-solubilizing bacteria. Take 1 mL of the above soil suspension and dilute it to 10 -3 g / mL, 10 -4 g / mL, 10 -5 g / mL, 10 -6 g / mL, 10 -7 g / mL. Take 100 μL of each concentration gradient with a pipette and apply it on the plate of Alexander silicate bacterial culture medium with a sterilized applicator. Repeat 3 times each. Set the temperature of the incubator to 28°C and invert the plate for 4 days. Pick a single colony with a potassium-solubilizing circle from the Alexander silicate bacterial culture medium with an appropriate dilution multiple, and purify it on LB medium 4 to 5 times using the three-line drawing method to obtain a pure single colony to screen out the potassium-solubilizing strain Enterobacter cancerogenus. After identification, the strain was named Enterobacter cancerogenus and was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC) on June 17, 2024. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC No. 30972.

[0049] 3.2 Preparation of bacterial suspension: OD 600 =1. Prepare LB liquid medium, inoculate strain K4 in the medium, and culture overnight at 28°C and 180rpm; take out the cultured bacterial liquid, centrifuge at 11000rpm for 5min, pour out the supernatant, and then shake and mix with sterile distilled water; measure OD with a UV-visible spectrophotometer 600 Value, prepared as OD 600 = 1 and keep it for later use.

[0050] 4. The cancer-causing Enterobacterium K4 CGMCC No.30972 of the present invention has the following biological characteristics:

[0051] 4.1 Morphological and cultural characteristics:

[0052] Depend on Figure 1 It can be seen that on the LB plate, the colonies can be observed to be grayish white, with round protrusions, smooth surface and complete edges; on the Alexandrium silicate bacterial culture medium, the colonies can be observed to be large, transparent protrusions like glass balls, very sticky, and with complete edges; Gram staining can be seen under the microscope that K4 is rod-shaped and Gram-negative bacteria. Other physiological and biochemical reactions are: methyl red reaction, indole reaction, hydrogen peroxide test are positive; diacetyl (VP) reaction, starch hydrolysis test are negative.

[0053] 4.2 Genetic characteristics (strain 16srRNA sequence): The present invention measured the 16srRNA sequence of the strain, which is as follows, with a total length of 1349 bp.

[0054] GCTACCTACTTCTTTTGCACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCTAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACTCCTCAAGGGAACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGAGTAACGTCAATCACCAAGGTTATTAACCTTAATGCCTTCCTCCTCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCATTACCCCACCTACTAGCTAATCCCATCTGGGCACATCTGATGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCGCTCGTCACC

[0055] Molecular systematic identification of strain 4.3

[0056] 4.3.1 PCR results

[0057] Inoculate strain K4 in LB liquid medium and shake the bacteria overnight at 180 rmp. The total DNA extraction steps refer to the bacterial DNA extraction kit.

[0058] (1) Add 250 μl of Buffer BL to a centrifuge tube, place it on a collection tube, and centrifuge at 12,000 rpm / min for 1 min to activate the silica membrane;

[0059] (2) Take less than 20 mg of sample dry tissue, grind it thoroughly with liquid nitrogen and put it into a 1.5 ml centrifuge tube, then add 400 μl of Buffer gP1, shake it with a vortexer for 1 min, and let it stand in a 65°C water bath for 10 to 30 min;

[0060] (3) Add 150 μl of Buffer gP2, vortex for 1 min, and place on ice for 5 min;

[0061] (4) Centrifuge at 12000 rpm / min for 5 min, and then pipette the supernatant into a new centrifuge tube;

[0062] (5) Add an equal amount of anhydrous ethanol to the above liquid into a new centrifuge tube, mix well, and finally pour all of it into the centrifuge column. Centrifuge at 12000 rpm / min for 30 seconds and discard the waste liquid;

[0063] (6) Add 500 μl of Buffer Pw to the centrifuge column (with anhydrous ethanol added), centrifuge at 12,000 rpm / min for 30 s, and discard the waste liquid;

[0064] (7) Add 500 μl of Wash Buffer to the centrifuge column (with anhydrous ethanol added), centrifuge at 12000 rpm / min for 30 s, and discard the waste liquid;

[0065] (8) Repeat step 7;

[0066] (9) Place the centrifuge column back into the collection tube, centrifuge at 12,000 rpm for 2 min, and open the lid to dry for 1 min;

[0067] (10) Remove the centrifuge column and place it in a clean centrifuge tube. Add 50–100 μl of TE Buffer (preheated at 65°C) to the center of the centrifuge column adsorption membrane. Let stand at room temperature for 2 min and centrifuge at 12,000 rpm / min for 2 min.

[0068] Bacterial universal primers 27F (5'-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The extracted DNA samples were diluted appropriately and used as PCR templates. Amplification was performed using Qingke 1×TSE101 Gold Mix. The components of the amplification system are as follows:

[0069]

[0070] The PCR reaction procedure is as follows:

[0071]

[0072] The DNA of strain K4 with significant potassium-dissolving effect obtained through repeated screening was used as a template, and PCR amplification was performed using bacterial universal primers (27F / 1492R). The final product was stained with bromophenol blue staining solution and detected by electrophoresis and gel imaging analyzer. The amplified band fragment was about 1500bp ( Figure 2 ), with clearly visible bands and no extra miscellaneous bands.

[0073] The sequencing of strain K4 was compared with the NCBI database, and the maximum likelihood method was used to construct an evolutionary tree for the strain K4 sequence and the sequences of strains with higher homology using MEGA7.0 software ( Figure 3 ). The results showed that strain K4 was identified as Enterobacter cancerogenus.

[0074] Example 2 Potassium-releasing function of the cancer-causing Enterobacter strain CGMCC No.30972 of the present invention

[0075] 1. Potassium-solubilizing ability of potassium-solubilizing bacteria K4

[0076] Determination of potassium-solubilizing ability of strains: The fermentation broth was treated with hydrogen peroxide ashing method, and the content of effective potassium in the fermentation supernatant was determined by reference to flame photometry.

[0077] (1) Potassium series standard solutions: Accurately pipette 0, 2.5, 5, 10, 15, and 20 mL of 100 μg / mL potassium standard solution into a 100-mL volumetric flask, and make up to volume with potassium-deficient culture medium.

[0078] (2) Pick a single colony of the strain screened above and inoculate it into 100mL of liquid Alexandria silicate bacterial culture medium, shake and culture it on a constant temperature shaker (28°C, 180rpm) for 3d, and finally detect the content of fast-acting potassium in the supernatant. Add 4% H2O2 to the fermentation broth and digest it at 121°C for 30min until the fermentation broth is clear. Then centrifuge it at 11000r / min for 6min, pour the supernatant into a 100mL volumetric flask, and make up the volume with sterile water. The control is a culture medium without bacteria. Then use a flame spectrophotometer to measure the content of fast-acting potassium in the supernatant, and finally select a strain with vigorous growth and strong degradation ability.

[0079] Relative increase of available potassium (%) = (content of available potassium in sample - content of available potassium in control) / content of available potassium in control * 100%

[0080] The soil suspensions at different dilution gradients were applied to initially obtain 35 single colonies, which were then purified 4-5 times (LB medium), and then the potassium-solubilizing ability was determined. Finally, a bacterium with the best potassium-solubilizing ability was screened out for the following test. As shown in Table 1, the potassium-solubilizing bacteria and strain K4 in the present invention have the largest content of available potassium in the fermentation broth, reaching 2.7 μg / mL, which is 93% higher than the blank control, and has a strong potassium-solubilizing ability.

[0081] Table 1 The available potassium content produced by potassium-solubilizing bacteria K4

[0082]

[0083] 2. Determination of soil colonization ability of potassium-solubilizing bacteria K4

[0084] In order to verify whether strain K4 can exert its potassium-dissolving effect, the most important thing is that it can effectively colonize in the rhizosphere soil. Figure 4 It can be seen that after 40 days of inoculation, the number of potassium-dissolving bacteria in the soil decreased with the extension of time. The number of bacteria decreased sharply within 2 days of cultivation, and then decreased slowly in the later period. It can be seen that the number of K4 bacteria increased on 9 days, 20 days, and 32 days compared with the previous day, and finally stabilized at 1.5×10 5 CFU / g. Overall, the number of microorganisms in strain K4 was relatively stable, and the colonization effect was good.

[0085] Conclusion: The cancer-causing Enterobacter strain K4 of the present invention has a strong potassium-solubilizing ability, can effectively colonize in the soil, and has a persistent effect.

[0086] Example 3 Growth-promoting effect of the cancer-causing Enterobacter K4 strain CGMCC No. 30972 of the present invention

[0087] Case 1: Promoting the growth of baby cabbage by watering with K4 bacterial solution under complete nutrient matrix cultivation conditions

[0088] In this case, the K4 bacterial suspension of carcinogenic Enterobacter was irrigated on baby cabbages to verify its growth-promoting effect on the baby cabbages.

[0089] Two treatment groups were designed in this case: CK group (sterile water) and K4 group (K4 bacterial solution of the present invention), with 15 strains in each treatment and 3 replicates.

[0090] The preparation method of K4 bacterial solution of the present invention is the same as that in step 3.2 of Example 1. The concentration of K4 bacterial solution in each treatment group is OD 600 =1.

[0091] The plug tray was disinfected and sterilized with 75% alcohol in advance, and then the seedling medium was placed. Then the baby cabbage seeds were planted in the plug tray and placed in an artificial climate box (light temperature / dark temperature is 25℃ / 20℃, light time / dark time is 16h / 8h), watered regularly, and maintained. After 3-4 true leaves grow, select seedlings with consistent growth and transplant them into disinfected and sterilized flower pots, one plant per pot. 50mL of the above bacterial suspension was poured on the roots of the plants for a total of 8 times. The control was added with an equal amount of sterile water, and then placed in an artificial climate box (light temperature / dark temperature is 25℃ / 20℃, light time / dark time is 16h / 8h), and watered regularly and quantitatively. Observe and record the situation after 40d.

[0092] Observe and record the plant's root length, plant height, stem diameter, leaf area, above-ground weight, underground weight and other morphological indicators. The experimental results are shown in Tables 2-3 and Figure 5-6 .

[0093] Table 2 Effect of strain K4 on the growth of baby cabbage

[0094]

[0095] Note: Different lowercase letters indicate significant differences at the P<0.05 level, the same below

[0096] Table 3 Effect of strain K4 on the biomass of baby cabbage

[0097]

[0098]

[0099] pass Figure 5 and Figure 6 , it can be seen intuitively that there are obvious differences between the control group and the experimental group. Compared with the control group CK, the baby cabbage treated with strain K4 is taller and has more and larger leaves. After 40 days of cultivation, the morphological indicators such as plant root length, plant height, stem thickness, leaf area, aboveground weight and underground weight were observed and recorded. As shown in Table 2, the indicators of plant height, root length and leaf area are 6.07cm, 23.68cm and 14.70cm respectively. 2 Compared with the control, the weight of the stem was significantly increased by 22.63%, 38.89% and 48.86%, respectively. The stem diameter was 1.94 mm, which was not significantly different from the control and increased by 9.61%. As shown in Table 3, the aboveground fresh weight, aboveground dry weight, underground fresh weight and underground dry weight were 1.21 g, 0.11 g, 0.10 g and 0.02 g, respectively, which were significantly increased by 65.75%, 57.14%, 100% and 100% compared with the control.

[0100] Conclusion: The potassium-solubilizing bacteria identified in this experiment is Enterobacter cancerogenus. Under the condition of complete nutrient matrix cultivation, it can increase the plant height, stem thickness, root length, leaf area, aboveground fresh weight, aboveground dry weight, underground fresh weight and underground dry weight of baby cabbage and promote plant growth and development.

[0101] Case 2: Promoting tomato growth by watering with K4 bacterial solution under the condition of lack of available potassium in the nutrient matrix

[0102] Plants will grow poorly when the soil is deficient in potassium or low in potassium. By watering with K4 bacterial solution, the insoluble potassium in the cultivation medium can be converted into soluble potassium, thereby increasing the level of available potassium supply in the soil and promoting plant growth. Based on this, in this case, the TOM tomatoes cultivated under the condition of lack of available potassium were watered with the bacterial suspension of Enterobacter carcinogenes K4 to study the growth-promoting effect of potassium-solubilizing bacteria on TOM tomatoes under low potassium conditions.

[0103] This study designed a two-factor experiment, specifically: factor K was potassium-solubilizing bacteria, and two concentration levels were set, namely K0 (no potassium-solubilizing bacteria, watering with sterile water) and K1 (watering with potassium-solubilizing bacteria, OD value = 1); factor H was the potassium element in the nutrient solution, and two concentration levels were set: H0 (the substrate was a low potassium level, which was achieved by watering with Hoagland's nutrient solution without potassium), and H1 (the substrate was a normal potassium level, which was achieved by watering with Hoagland's nutrient solution containing 100% potassium). There were 4 treatments in total, 35 plants in each treatment, and 3 repetitions.

[0104] The preparation method of potassium-dissolving bacteria solution is as follows: The preparation method of K4 bacteria solution of the present invention is the same as that in step 3.2 of Example 1, and the concentration of K4 bacteria solution is OD value = 1 (bacterial number 2×10 8 cfu / ml).

[0105] The preparation method of Hogland nutrient solution with different concentrations of potassium is:

[0106] H0 nutrient solution formula: that is, Hoagland's solution without potassium nitrate. The specific formula is: calcium nitrate 945mg / L, ammonium phosphate 115mg / L, magnesium sulfate 493mg / L, iron salt (NaFeCl(OH) 12 N2O8) 2.5ml / L, trace elements (manganese sulfate tetrahydrate, zinc sulfate heptahydrate, boric acid, copper sulfate pentahydrate, ammonium molybdenum tetrahydrate) 5ml / L, pH=6.

[0107] H1 nutrient solution formula: that is, the complete formula of Hoagland's solution. In the above H0 formula, 607 mg of potassium nitrate is added to each liter of H0 nutrient solution.

[0108] Plant treatment method: Tom cherry tomato seeds were soaked in warm water and then grown in seedling trays (50 seed trays, commercial seedling medium treatment). When the seedlings had two leaves and one heart, they were transplanted into nutrient pots (10cm×10cm) for substrate cultivation (the volume ratio of peat to vermiculite was 2:1). The first treatment began 10 days after transplanting: different concentrations of bacterial solution (10mL per plant) and Hogland nutrient solution with different potassium levels (40mL per plant) were irrigated around the roots, and then irrigated once every 10 days, for a total of 3 times. Conventional management was used for the rest of the time, and each treatment remained consistent.

[0109] Plant growth index determination

[0110] Fresh weight and dry weight of the above-ground and underground parts of Tom cherry tomatoes: Cut the plant from the root neck, use a 1% electronic balance to weigh the above-ground and underground parts of the plant respectively, and determine the fresh weight and dry weight.

[0111] Leaf area: measured by leaf area meter.

[0112] Each index was measured 30 days after the plants were treated with bacterial solution, and the same applies below.

[0113] Plant physiological index determination

[0114] Root activity: triphenyltetrazolium chloride method (TTC method);

[0115] Chlorophyll a, chlorophyll b and carotenoid content: Chlorophyll was extracted by 80% acetone immersion method and the content was determined by spectrophotometer.

[0116] Determination of physical and chemical properties of matrix

[0117] Rhizosphere soil collection method: dig out the plant roots and gently shake the plant roots to shake off the attached matrix. Wear sterile gloves to collect the fallen matrix as rhizosphere soil in a sterile sample bag. Randomly select 5 plants for each treatment for measurement. Each index was measured 30 days after the plants were treated with the bacterial solution.

[0118] Total nitrogen content: Kjeldahl method

[0119] Total phosphorus: sodium hydroxide fusion-molybdenum antimony colorimetric method;

[0120] Total Potassium: Flame Photometry

[0121] Alkaline Hydrolysis Nitrogen: Alkaline Hydrolysis Diffusion Method

[0122] Quick-acting potassium: ammonium acetate extraction-flame photometry;

[0123] Available phosphorus: NaHCO3 extraction-molybdenum blue colorimetry;

[0124] Urease activity: sodium phenol-sodium hypochlorite colorimetric method;

[0125] Catalase activity: potassium permanganate titration method;

[0126] Sucrase activity: 3,5-dinitrosalicylic acid colorimetric method;

[0127] Alkaline phosphatase activity: disodium phenyl phosphate colorimetric method.

[0128] 2.1 Effects of potassium-solubilizing bacteria on tomato growth indicators

[0129] The two-way ANOVA results showed that the K factor (potassium-dissolving bacteria) and the H factor (different concentrations of potassium element Hoagland solution) had a significant effect on tomato plant height, stem thickness and leaf area, and these two factors had a significant interaction effect on plant height.

[0130] Among the four treatments, compared with K0H0, the stem diameter, plant height, and leaf area of ​​K1H1 were 1.39 times, 1.46 times, and 5.18 times of those of K0H0, respectively; compared with K0H1, the plant height and leaf area of ​​K1H1 were 1.19 times and 1.16 times of those of K0H1, respectively. (See Table 4)

[0131] Table 4 Effects of potassium-solubilizing bacteria on plant height, stem thickness and leaf area of ​​tomatoes

[0132]

[0133] Note: 1) K indicates the effect of applying potassium-dissolving bacteria on plant growth indicators; H indicates the effect of applying Hoagland nutrition without potassium on plant growth indicators; K&H indicates whether there is an interaction between K and H to affect plant growth indicators; "*" indicates that the degree of influence reaches a significant level of P < 0.05; "**" indicates that the degree of influence reaches an extremely significant level of P < 0.01; "N" indicates that the degree of influence is not significant. 2) The same below.

[0134] The two-way ANOVA results showed that the K factor (potassium-solubilizing bacteria) and the H factor (Hoagland's solution of potassium at different concentrations) had significant effects on the fresh and dry weight of the above- and underground parts of tomatoes and the seedling index, and these two factors had an interactive effect on the seedling index.

[0135] Among the four treatments, compared with K0H0, the aboveground fresh weight and underground fresh weight of K1H1 were 13.81 times, 13.22 times, 4.63 times, and 5.33 times of K0H0, respectively; compared with K0H1, the aboveground fresh weight and underground fresh weight of K1H1 were 2.89 times, 1.53 times, 1.09 times, and 1.07 times of K0H1, respectively. Among the four treatments, the seedling index after K1H1 was the highest, 5.89 times that of K0H0 and 1.08 times that of K0H1. (Table 5)

[0136] Table 5 Effects of potassium-solubilizing bacteria on above-ground and underground dry and fresh weights of tomatoes

[0137]

[0138] Note: Seedling index: Seedling index = (stem diameter / plant height + underground dry mass / aboveground dry mass) × whole plant dry mass.

[0139] Conclusion: Watering the cancer-causing Enterobacter strain K4 of the present invention can increase plant growth, change plant nutrient distribution, promote the improvement of plant seedling index, and promote plant growth.

[0140] 2.2 Effects of potassium-solubilizing bacteria on physiological parameters of tomato plants

[0141] Two-way ANOVA results showed that K factor (potassium-solubilizing bacteria) had a significant effect on tomato chlorophyll a, carotenoids and root activity, H factor (different concentrations of potassium element Hoagland solution) had a significant effect on chlorophyll and root activity, and these two factors had an interactive effect on root activity.

[0142] Among the four treatments, compared with K0H0, the chlorophyll a, chlorophyll b, carotenoids, and root activity of K1H1 were 2.10 times, 2.42 times, 1.73 times, and 6.06 times higher than those of K0H0, respectively; compared with K0H1, the chlorophyll a, chlorophyll b, carotenoids, and root activity of K1H1 were 1.05 times, 1.02 times, 1.06 times, and 4.21 times higher than those of K0H1, respectively. (Table 6)

[0143] Table 6 Effect of potassium-solubilizing bacteria on chlorophyll content of tomatoes

[0144]

[0145]

[0146] Conclusion: Watering the cancer-causing Enterobacter K4 strain of the present invention can promote the enhancement of tomato root activity and the accumulation of photosynthetic pigment synthesis, improve the absorption of nutrients by tomato roots and the ability of leaves to absorb, transmit and convert light energy, and promote plant growth.

[0147] 2.3 Effects of potassium-solubilizing bacteria on the physical and chemical properties of rhizosphere soil of tomato plants

[0148] 2.3.1 Effects of potassium-solubilizing bacteria on tomato soil nutrients

[0149] The two-way ANOVA results showed that K factor (potassium-dissolving bacteria) had a significant effect on the total potassium and available potassium content of tomato cultivation medium, and there was an interaction effect between this factor and H factor (different concentrations of potassium element Hoagland solution). Among the four treatments, compared with K0H0, the total potassium and available potassium after K1H1 treatment were 1.16 times and 1.57 times of K0H0, respectively; compared with K0H1, the total potassium and available potassium after K1H1 treatment were 1.12 times and 1.09 times of K0H1, respectively.

[0150] The two-way variance analysis also showed that other indicators of the physical and chemical properties of tomato rhizosphere soil under different treatments also changed, among which the total nitrogen, total phosphorus and available phosphorus content increased most significantly after K1H1 treatment. Among the four treatments, compared with K0H0, the total nitrogen, total phosphorus and available phosphorus after K1H1 treatment were 1.60 times, 1.36 times and 1.90 times of K0H0 respectively; compared with K0H1, the total nitrogen, total phosphorus and available phosphorus after K1H1 treatment were 1.26 times, 1.32 times and 1.28 times of K0H1 respectively. (Table 7)

[0151] Table 7 Effects of potassium-solubilizing bacteria on nitrogen, phosphorus and potassium content in tomato soil

[0152]

[0153] 2.3.2 Effects of potassium-solubilizing bacteria on enzyme activities in tomato soil

[0154] Two-way ANOVA results showed that although K factor (potassium-dissolving bacteria) and H factor (different concentrations of potassium element Hoagland solution) had no significant effect on tomato soil sucrase, there was an interactive effect between the two factors on catalase, alkaline phosphatase, sucrase and urease.

[0155] The enzyme activities of tomato rhizosphere soil changed under each treatment. Among the four treatments, compared with K0H0, the contents of catalase, alkaline phosphatase, sucrase, and urease in K1H1 treatment were 1.38 times, 1.06 times, 2.52 times, and 2.59 times of those in K0H0, respectively; compared with K0H1, the contents of catalase, alkaline phosphatase, sucrase, and urease in K1H1 treatment were 1.47 times, 1.29 times, 1.33 times, and 1.83 times of those in K0H1, respectively.

[0156] (Table 8)

[0157] Table 8 Effects of potassium-solubilizing bacteria on enzyme activities in tomato soil

[0158]

[0159] Conclusion: Watering the cancer-causing Enterobacter K4 strain of the present invention increases the content of soluble potassium in the soil, thereby increasing the potassium level in the rhizosphere soil of tomatoes, and the content of total potassium and available potassium are significantly increased; at the same time, other nutrient indicators in the soil are also increased, such as total nitrogen, total phosphorus, available phosphorus and soil enzyme activity. The cancer-causing Enterobacter K4 strain of the present invention can activate the insoluble potassium in the soil, thereby increasing the level of available potassium in the soil, optimizing the supply of soil nutrients, and promoting plant growth.

[0160] Example 4 Growth-promoting effect of the fermentation broth of the cancer-producing Enterobacter K4 strain CGMCC No. 30972 of the present invention

[0161] The strain is fermented to prepare a potassium-solubilizing microbial agent, which is used in vegetable production, including leafy vegetables coriander and lettuce, and perennial vegetable Lanzhou lily (the edible organ is the underground bulb).

[0162] Formula of fermentation liquid: 2g bean cake powder, 2mg vitamin B2, 1g potassium dihydrogen phosphate, 0.4g humic acid and 2g soluble starch are prepared into 100ml formula liquid, and NaOH solution and HCl solution are used to adjust pH7, and sterilized in a high temperature sterilizer at 121℃ for 20min. When the solution is cooled to below 20℃, 0.15g catalase is weighed on the clean bench and added to the formula liquid. Use an inoculation loop (diameter 3.5mm) to pick 4 inoculation loops of activated cultured K4 strains to inoculate the above formula liquid, seal with sealing film and place on a shaker for culture. The culture conditions are 27℃, 2 days, 180r / min, pH=7, and light-shielded culture. The number of viable potassium-dissolving bacteria in the fermentation liquid is 315 million / ml, which meets the national agricultural microbial agent standard GB20287-2006 (the standard requires that the number of viable bacteria of liquid microbial agents must reach 2×10 8 / ml).

[0163] Case 1: Verify the growth-promoting effect of K4 strain fermentation liquid on coriander by watering

[0164] Coriander was selected as the experimental material. Two treatments were set up, namely CK (no potassium-dissolving bacteria, watering with sterile water) and T1 (watering with K4 strain fermentation liquid), with 35 plants in each treatment and 3 replicates.

[0165] After the coriander seeds are soaked in warm water, seedlings are grown in plug trays (72 plug trays, the substrate is a commercial organic seedling nutrient substrate - Zhuang Miao No. 1, the pH value is 5.5-6.5, and the moisture content is ≤30%). When the seedlings grow to two leaves and one heart, they are transplanted into nutrient pots (10cm×10cm×7.6cm) filled with substrate for cultivation. The first treatment began 10 days after transplanting: watering with 10-fold dilution of K4 strain fermentation liquid (50 ml per plant), CK watering with sterile water (50 ml per plant), and then watering once every 7 days, for a total of 4 times, and measuring plant growth indicators. Observe and record morphological indicators such as plant root length, plant height, aboveground weight and underground weight. The test results are shown in Tables 9 and Figure 8 .

[0166] Table 9 Effect of K4 strain fermentation broth on coriander growth

[0167]

[0168] The fermentation liquid formula has a good effect on promoting the growth of coriander. Compared with the control group CK, the coriander treated with T1 group has taller plants, more leaves, and a larger leaf area ( Figure 8 ). The results of one-way ANOVA showed that the differences among the treatments were significant, and the fermentation liquid had a good overall effect on the growth promotion of coriander. Compared with CK, the aboveground fresh weight, underground fresh weight, plant height, and root length of T1 treatment were 1.46 times, 2.68 times, 1.17 times, and 1.30 times that of CK (Table 9).

[0169] Conclusion: The fermentation liquid of Enterobacter K4 strain has a significant growth-promoting effect on coriander, which can increase the growth of coriander and promote the growth of plants.

[0170] Case 2: Verify the growth-promoting effect of K4 strain fermentation liquid on lettuce by watering

[0171] The experiment selected lettuce as the test material. Two treatments were set up, namely CK (no potassium-dissolving bacteria, watering with sterile water) and T2 (watering with K4 strain fermentation liquid), with 35 plants in each treatment and 3 replicates.

[0172] After soaking the lettuce seeds in warm water, seedlings were grown in plug trays (72 plug trays, the substrate was a commercial substrate organic seedling nutrient substrate - Zhuang Miao No. 1, pH value was 5.5-6.5, water ≤ 30%). When the seedlings grew to two leaves and one heart, they were transplanted into nutrient pots (10cm×10cm×7.6cm) filled with substrate. The first treatment began 10 days after transplanting: watering with 10-fold dilution of K4 strain fermentation liquid (50ml per plant), CK watering with sterile water (50ml per plant), and then watering once every 7 days, for a total of 5 times, and measuring plant growth indicators.

[0173] Table 10 Effect of K4 strain fermentation liquid on lettuce growth

[0174]

[0175] Compared with the control group CK, the lettuce treated in the T2 group had stretched leaves and good plant growth ( Fig. 9 ). The results of one-way ANOVA showed that the differences among the treatments reached a significant level (P < 0.05), and the fermented liquid had a good overall growth-promoting effect on lettuce. Compared with CK, the above-ground fresh weight, underground fresh weight, plant height, and root length of T2 treatment were 2.44 times, 1.02 times, 1.37 times, and 1.07 times that of CK (Table 10).

[0176] Conclusion: The fermentation liquid of Enterobacter K4 strain has a significant growth-promoting effect on lettuce, which can increase plant growth, promote plant growth, increase the fresh weight of the aboveground part, and improve yield.

[0177] Case 3: Watering the fermentation liquid of K4 strain to verify its growth-promoting effect on Lanzhou lily in field production

[0178] This case was designed to verify the growth-promoting effect of K4 strain fermentation liquid on vegetable plants under field soil cultivation conditions (to distinguish it from Case 1 and Case 2, which were substrate cultivation and potted conditions).

[0179] The experiment selected Lanzhou lily as the experimental material and was carried out at the lily bulb production base of Gansu Kangyuan Modern Agriculture Co., Ltd. The experimental field was one-year-old Lanzhou lily. Two treatments were set up, namely CK (no potassium-dissolving bacteria, watering with sterile water) and T3 (watering with K4 strain fermentation liquid). The experiment was a randomized block experiment design with 3 replications; the plot area was 0.5m*0.7m, and the planting density was 100 plants / m 2 (Equally spaced rows are planted on flat ridges, with a row spacing of 10cm*10cm). The first treatment began on September 28, 2024: each plot was irrigated with a 10-fold dilution of the K4 strain fermentation liquid (2000ml per treatment), and CK was irrigated with an equal amount of sterile water. Thereafter, it was irrigated once every 15 days, for a total of 3 times. The rest of the field management was routine. Sampling and measurement were carried out on November 4, 2024, and 30 plants were randomly selected from each group of plots to measure growth indicators. Observe and record the horizontal and vertical diameters (mm) of the Lanzhou lily: the maximum horizontal and vertical diameters of the bulb, measured with a vernier caliper; sphericity index: vertical diameter / horizontal diameter value; underground fresh weight (g) and bulb mass (g), recorded on a balance.

[0180] Field observation results showed that the bulb weight of Lanzhou lily treated with K4 fermentation liquid increased significantly, the root system became stronger, the lily leaves became darker, the withering time of the aboveground parts in autumn was delayed, and the growth period was extended ( Fig.10 , Fig.11). The results of one-way ANOVA showed that the differences among the treatments reached a significant level (P < 0.05), and the fermentation liquid had a good overall growth-promoting effect on Lanzhou lily: after the fermentation liquid treatment, the root system of the lily was well developed and the bulb weight increased; compared with CK, the underground fresh weight and the weight of a single bulb increased significantly by 33.33% and 39.33%, respectively; at the same time, the shape of the bulb was changed, and its spherical index increased significantly by 5.34%, which improved the commercial quality (Table 11).

[0181] Table 11 Effect of K4 strain fermentation liquid on the growth of Lanzhou lily bulbs

[0182]

[0183] Note: Bulb spherical index = bulb longitudinal diameter / bulb transverse diameter

[0184] Conclusion: The microbial fertilizer prepared by the cancer-causing Enterobacter K4 strain has a stable effect in field soil cultivation and has a significant growth-promoting effect on Lanzhou lily. It can promote the growth of lily roots, accelerate the growth of bulbs, increase the sphericity index, and improve yield and commercial quality.

Claims

1. A cancer-causing Enterobacterium with potassium-dissolving and growth-promoting effects, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration with a deposit number of CGMCC No.30972.

2. A cancer-causing enterobacterium bacterial liquid, characterized in that: The method is obtained by expanding and culturing the cancer-causing Enterobacter CGMCC No. 30972 strain described in claim 1.

3. The cancer-causing Enterobacterium bacterial liquid according to claim 2, characterized in that: The expanded culture specifically includes: inoculating the cancer-causing Enterobacter CGMCC No.30972 strain into a culture medium, and culturing it at 28±1° C. on a shaking table.

4. The cancer-causing Enterobacterium bacterial liquid according to claim 3, characterized in that: The culture medium is LB liquid culture medium.

5. Use of the cancer-causing Enterobacter according to claim 1 or the cancer-causing Enterobacter bacterial liquid according to claim 2, 3 or 4 in the preparation of a potassium-solubilizing product.

6. Use of the cancer-causing Enterobacter according to claim 1 or the cancer-causing Enterobacter bacterial solution according to claim 2, 3 or 4 in the preparation of products for improving soil physical and chemical properties and promoting crop growth.

7. The use according to claim 6, characterized in that: The crops include at least one of leafy vegetables and nightshade vegetables; The leafy vegetables include baby cabbage; The nightshade vegetables include tomatoes.

8. Use of the cancer-causing Enterobacter strain of claim 1 to prepare a microbial agent by fermentation in vegetable production, wherein the vegetables include leafy vegetables such as coriander and lettuce, and perennial vegetable Lanzhou lily.

9. The use according to claim 8, characterized in that: The fermentation is specifically as follows: the cancer-causing Enterobacter CGMCC No. 30972 strain is inoculated into the fermentation formula liquid, sealed with a sealing film and placed on a shaker for culture, the culture conditions are 27° C., 2 days, 180 r / min, pH=7, and light-shielded culture.

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