Endophytic Pantoea CBPS-16 and its application

Abstract

The present invention relates to an endophytic Pantoea ( Pantoea sp. )CBPS‑16, with the deposit number of CGMCC No.30593. This strain can efficiently release silicon, dissolve phosphorus and potassium, has good IAA production, iron carrier and nitrogen fixation functions, and is also acid- and alkali-resistant, salt-tolerant, heavy metal-tolerant, drought-tolerant and can prevent and control plant pathogenic fungi. This strain can be used in the production of bio-organic fertilizers, bio-pesticides, soil conditioners and / or artificial substrates, and can significantly promote plant growth and improve soil quality.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms, and particularly relates to an endophytic Pantoea CBPS-16 and an application thereof. Background Art

[0002] Most of the silicon in the soil exists in the form of various silicates. The effective silicon that can be absorbed and utilized by plant roots only accounts for 0.1% to 0.2% of the total silicon in the soil. Finding multiple ways to improve soil silicon nutrition and plant silicon absorption has always been a focus of research at home and abroad.

[0003] Silicon-releasing microorganisms can decompose silicate minerals in the soil, accelerate the mineral decomposition process in a variety of ways, and release soluble silicon for plants to absorb and utilize. They play an important role in increasing crop yield and quality, enhancing crop disease resistance and stress resistance, improving soil quality, and increasing fertilizer utilization. At present, the silicon-releasing microorganisms isolated and screened mainly include bacteria, fungi, and actinomycetes, such as Bacillus, Pseudomonas, Rhizobium, Aspergillus niger, Streptomyces, etc.

[0004] There are a large number of silicon-releasing functional microorganisms in the environment, which can effectively release silicon, phosphorus, potassium and other elements in the soil. When applied to the soil, they can not only significantly increase soil nutrients and promote plant growth, but also avoid a series of ecological and environmental problems caused by the large-scale application of chemical fertilizers, such as soil compaction, decreased organic matter content, groundwater and surface pollution. Therefore, screening and exploring new and efficient silicon-releasing functional microorganisms, developing low-cost and environmentally friendly microbial agents, promoting plant growth and disease resistance, and thus improving soil quality are of great significance to the development of green ecological agriculture. Summary of the invention

[0005] The purpose of the present invention is to provide an endophytic Pantoea CBPS-16 which can efficiently release silicon, dissolve phosphorus and potassium, has good IAA production, iron carrier and nitrogen fixation functions, and is also acid- and alkali-resistant, salt-resistant, heavy metal-resistant, drought-resistant and disease-resistant. The strain can be used in the production of biological organic fertilizers and artificial substrates, and can significantly promote plant growth and improve soil quality.

[0006] The present invention adopts the following technical solution:

[0007] An endophytic Pantoea ( Pantoea sp. )CBPS-16, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, Beijing, China, with the deposit number CGMCC No. 30593 and the deposit date May 10, 2024.

[0008] Furthermore, the endophytic Pantoea CBPS-16 grows well on PB medium, the surface of a single colony is moist and smooth, yellow, opaque, sticky, with neat edges, the bacteria are short rod-shaped, Gram-negative, and spore-free. It is negative in the indole test, positive in the urease test, and cannot hydrolyze gelatin, starch, and cellulose, and can use mannitol, maltose, sucrose, glucose, galactose, and fructose as carbon sources for fermentation and acid production.

[0009] Furthermore, the endophytic Pantoea CBPS-16 has the ability to release silicon, dissolve phosphorus, dissolve potassium, and fix nitrogen.

[0010] Furthermore, endophytic Pantoea CBPS-16 can produce IAA and siderophores.

[0011] Furthermore, the endophytic Pantoea CBPS-16 can grow well at pH 5-10, NaCl concentration 0-6%, and temperature 15-45°C, and has good acid and alkali resistance, salt tolerance, drought resistance and wide temperature adaptability.

[0012] Furthermore, endophytic Pantoea CBPS-16 can tolerate heavy metal ions.

[0013] In particular, the endophytic Pantoea CBPS-16 can grow normally in a culture medium containing 2 mM copper ions, 5 mM lead ions, and 1 mM hexavalent chromium ions.

[0014] Furthermore, the endophytic Pantoea CBPS-16 can antagonize Fusarium graminearum, Verticillium dahliae of cotton and / or Gray leaf spot of tomato.

[0015] A use of the endophytic Pantoea CBPS-16 in promoting plant growth and / or improving soil quality.

[0016] A use of the endophytic Pantoea CBPS-16 in the preparation of bio-organic fertilizer, bio-pesticide, soil conditioner and / or artificial matrix.

[0017] A biological organic fertilizer comprising the above-mentioned endophytic Pantoea CBPS-16, the number of viable bacteria of which is not less than 10 8 Pieces / g.

[0018] A soil conditioner comprising the above-mentioned endophytic Pantoea CBPS-16, wherein the number of viable bacteria is not less than 10 8 Pieces / g.

[0019] Specifically, the soil conditioner is a heavy metal contaminated soil remediation agent.

[0020] Specifically, the soil conditioner is an acidic or alkaline soil repair agent.

[0021] A biological pesticide comprising the endophytic Pantoea CBPS-16.

[0022] Specifically, the biological pesticide is used to control Fusarium graminearum, Verticillium dahliae of cotton and / or gray leaf spot of tomato.

[0023] An artificial matrix comprising the endophytic Pantoea CBPS-16.

[0024] The beneficial effects of the present invention are as follows: First, the endophytic Pantoea CBPS-16 of the present invention not only has the effect of releasing silicon, but also can release phosphorus, potassium, fix nitrogen, produce siderophore and IAA, provide nutrients for plant growth, promote plant growth, etc. Secondly, the endophytic Pantoea CBPS-16 of the present invention has an antagonistic effect on Pseudomonas graminearum, Verticillium wilt of cotton and gray leaf spot of tomato, and can be used in the development of biocontrol agents. Thirdly, the endophytic Pantoea CBPS-16 of the present invention is not only salt-alkali and drought-resistant, but also heavy metal-resistant, and can be used for the improvement and quality improvement of saline-alkali soil, heavy metal-contaminated soil and tailings area soil, and can also provide excellent strain resources for the development of new biological organic fertilizers, soil conditioners, artificial substrates, etc., and has practical economic and social benefits and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the phylogenetic tree of strain CBPS-16.

[0026] Figure 2 The effect of temperature on the growth of strain CBPS-16.

[0027] Figure 3 The effect of pH concentration on the growth of strain CBPS-16.

[0028] Figure 4 This is the effect of drought stress on the growth of strain CBPS-16.

[0029] Figure 5 This is the effect of NaCl on the growth of strain CBPS-16. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the embodiments and drawings. The protection scope of the present invention is not limited to the embodiments, and any changes made by those skilled in the art within the scope defined by the claims also fall within the protection scope of the present invention.

[0031] Example 1 Isolation, identification and preservation of strain CBPS-16

[0032] The iron tailings sand samples were obtained from the rhizosphere soil of plants around a tailings pond in Chengde City, Hebei Province. The soil samples were diluted to 10 -5 ~10 -7After concentration, evenly spread it on the silicon-release medium, invert it in a constant temperature incubator at 30℃ and culture it for 2 days, select a single colony with a transparent circle and repeatedly streak it for purification, and transfer the purified bacteria to LB slant medium and store it in a refrigerator at 4℃ for later use.

[0033] The single colonies obtained from the initial screening were screened for silicon release ability using iron tailings as substrate and the increase in effective silicon content in the fermentation broth as the standard. During the rescreening process, the seed liquid was fermented from PB medium to the logarithmic growth phase, and then inoculated into the leaching and desiliconization medium at 8% volume, and cultured in a shaking incubator at 30°C and 180 r / min. Samples were taken on the 5th day, and the fermentation broth was centrifuged to obtain the supernatant, and the effective silicon content was determined by the molybdenum blue colorimetric method. As a result, a strain CBPS-16 with good silicon release efficiency was screened.

[0034] The above culture medium formulas are:

[0035] (1) Silicon-releasing culture medium: sucrose 5.0 g, Na2HPO4 2.0 g, MgSO4·7H2O 0.5 g, FeCl3 0.005 g, CaCO3 0.1 g, potassium feldspar 1.0 g, agar 15-20 g, water 1000 mL, pH 7.0-7.4.

[0036] (2) Mineral leaching and desiliconization culture medium: glucose 10 g, KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaCl2·2H2O 0.2 g, iron tailings 1.0 g, CaCO3 5 g, water 1000 mL, pH 7.0-7.2.

[0037] (3) PB medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl. For solid culture medium, add 15-20 g agar and 1000 mL water. pH 7.2-7.5.

[0038] The strain CBPS-16 grew on LB medium. The colonies were round, yellow, opaque, smooth, moist, sticky, and had neat edges. Under the microscope, the CBPS-16 bacteria were short rod-shaped, Gram-negative, without spores or capsules. Table 1 shows the strain CBPS-16 and endophytic Pantoea Pantoea endophytica Physiological and biochemical characteristics of the model strain showed that strain CBPS-16 could not hydrolyze gelatin, was negative in the indole test, and was positive in the urease test. It could use mannitol, maltose, sucrose, glucose, galactose, and fructose as carbon sources for fermentation and acid production, but could not use starch and cellobiose as carbon sources for fermentation and acid production.

[0039] Table 1 Physiological and biochemical characteristics of strain CBPS-16

[0040] .

[0041] The total DNA of CBPS-16 strain was extracted using a bacterial genomic DNA extraction kit, and it was used as a template. The universal 16SrDNA primers: 27 F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492 R (5'-GGTTACCTTGTTACGACTT-3') were used for amplification. A band of about 1500 bp was obtained by agarose electrophoresis. The PCR amplification product was sent to Genewise Biotechnology Co., Ltd. for sequencing.

[0042] The sequencing results were submitted to NCBI for homology comparison analysis with the existing 16S rDNA sequences in the database, and the 16S rDNA gene sequences of closely related strains were selected from Genebank, and the phylogenetic tree was constructed using MEGA7.0. Figure 1 As shown, Pantoea endophytica strain 596 (NR_178843.1) was the closest relative, with a similarity of 99.72%.

[0043] Based on the morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, strain CBPS-16 was identified as Pantoea ( Pantoea sp. ) in the endophytic Pantoea ( Pantoea endophytica ). The strain was deposited at the General Microbiology Center of China Microbiological Culture Collection Administration on May 10, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 30593.

[0044] Example 2 Stress resistance of strain CBPS-16

[0045] PB liquid medium was used as the basic medium, and the growth rate was OD 600 The value was used as an indicator to investigate the effects of different initial pH, temperature, NaCl and PEG6000 (simulated drought) concentrations on the growth of strain CBPS-16. The initial pH of the culture medium was set to 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, the culture temperature was 15℃, 20℃, 25℃, 30℃, 35℃, 40℃ and 45℃, the NaCl concentration in the culture medium was 0, 0.5%, 1%, 2%, 4%, 6%, 8%, 10%, 12% and 15%, and the PEG6000 concentration in the culture medium was 0, 10%, 20%, 30%. The CBPS-16 seed solution was inoculated with 2% (v / v) in a 30 mL PB liquid shake flask and cultured at 30℃ and 180 r / min for 24 h. The fermentation liquid OD was measured. 600 The stress resistance of strain CBPS-16 was determined by

[0046] The results showed that (see Figures 2~5 ), strain CBPS-16 showed a certain drought tolerance and could grow when the drought degree PEG6000 content was 20%. Strain CPBS-16 had a good wide temperature adaptability and could grow normally at 15℃~45℃, and the best growth temperature was 35℃. 600 The value reached 1.847. The strain CBPS-16 showed good acid and alkali resistance, and it could grow normally at pH 5-10, and it grew best at pH 6, indicating that it had strong acid and alkali resistance and environmental adaptability, and could be used in acidic or alkaline soils, which had obvious advantages in the development of soil conditioners. The strain CBPS-16 also showed good salt tolerance, and it grew normally at 0-6% NaCl, and it grew best at 2% NaCl concentration, indicating that it had good salt tolerance, and it could also ensure that it was not inhibited when conducting saline-alkali soil remediation, which had great advantages.

[0047] Example 3 Silicon release characteristics of strain CBPS-16

[0048] Prepare the seed solution of strain CBPS-16 and adjust the concentration of the solution to 1×10 9 CFU / mL, the seed solution was inoculated into the leaching and desiliconization medium (mineral sand particle size 0.15 mm, mineral sand concentration 2.5%) with an inoculum size of 2% (v / v), and 2% (v / v) blank PB culture solution was inoculated into the leaching and desiliconization medium as a control, with a liquid volume of 50 mL (100 mL in a shake flask), and cultured on a shaker at 30°C and 120 r / min for 7 days, with 3 replicates, and fermentation broth samples were taken at 1 d, 3 d, 5 d, and 7 d, and the fermentation broth samples were rotated at 8000 r·min -1 After centrifugation for 10 min, the supernatant was filtered with a 0.22 μm membrane and the effective silicon content in the fermentation broth after culture was determined by the NYT1121.15-2006 molybdenum blue colorimetric method.

[0049] The results showed that strain CBPS-16 had the best silicon release effect at 5 days of culture, with an effective silicon content of 29.7 mg / L, which was 2.3 times that of the control group.

[0050] Example 4 Determination of Phosphorus Solubilization, Potassium Solubilization and Nitrogen Fixation Capacity of Strain CBPS-16

[0051] NBRIP phosphate solubilization medium: glucose 10 g, Ca3(PO4) 25 g, (NH4)2SO4 0.5 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, MgCl2·6H2O 5 g, agar 15 g, 0.4% bromophenol blue 6 mL, ultrapure water 1 L, pH 7.0~7.2.

[0052] Potassium-solubilizing medium: sucrose 10 g, Na2HPO4 1 g, (NH4)2SO4 0.5 g, MgSO4·7H2O 1 g, yeast powder 0.2 g, NaCl 0.1 g, CaCO3 0.1 g, FeCl3 0.005 g, potassium feldspar 5 g, water 1000 mL, pH 7.0~7.2.

[0053] Nitrogen fixation activity detection medium (Ashby nitrogen-free medium): mannitol 10 g, KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaSO4·2H2O 0.2 g, CaCO3 5 g, pH 7.2.

[0054] The seed solution of strain CBPS-16 was prepared and inoculated on NBRIP phosphate-dissolving medium and potassium-dissolving medium, respectively. After culturing in a constant temperature incubator at 30°C for 2 days, its growth and whether there was a dissolution zone were observed.

[0055] The calculation method is: dissolution capacity = dissolution zone diameter D / colony diameter d.

[0056] The seed solution concentration of strain CBPS-16 was adjusted to 1×10 9 CFU / mL, inoculated into nitrogen fixation activity detection medium at a volume ratio of 2%, cultured at 30℃ for 2 days, and observed its growth. After three transfers, the strain was still able to grow, indicating that strain CBPS-16 has the function of self-generated nitrogen fixation.

[0057] The results showed that in NBRIP phosphate-dissolving medium and potassium-dissolving medium, there were obvious transparent circles around the CBPS-16 colonies, and the D / d values ​​were greater than 2, indicating that it had good phosphate-dissolving and potassium-dissolving abilities. The strain CBPS-16 could grow after three consecutive transfers in nitrogen-fixing medium, indicating that the strain had nitrogen-fixing ability.

[0058] Example 5 Siderophore and IAA production capacity of strain CBPS-16

[0059] Prepare the seed solution of strain CBPS-16, inoculate it on CAS test medium, and culture it at 30℃ for 2 days. If a yellow-green halo appears, it means that the siderophore is produced. Adjust the concentration of the bacterial solution to 1×10 9CFU / mL, inoculated into LB liquid culture medium (containing 100 mg / L L-tryptophan) at a volume ratio of 2%, placed on a shaker at 30℃ and 180 r / min for 1-2 d, took 50 μL of the supernatant after centrifugation at 8000 r / min, added 50 μL of Salkowski colorimetric solution, dropped it on a white porcelain plate and protected from light for 30 minutes. If pink appears, it is positive, indicating that the strain can secrete IAA. The darker the color, the greater the intensity of secretion. No color change is negative, indicating that the strain cannot secrete IAA.

[0060] The results showed that when CBPS-16 was inoculated on the CAS detection medium, a clear yellow-green halo was produced around the colony, indicating that it has the ability to produce siderophores; the color reaction was positive, indicating that it has the ability to produce IAA, which indirectly indicates that it has the ability to promote plant growth.

[0061] Example 6 Antagonism of strain CBPS-16 against plant pathogens

[0062] The plate confrontation method was used to determine whether strain CBPS-16 had an antagonistic effect on plant pathogens: the pathogenic fungus block was inoculated in the center of the PDA plate, and the strain CBPS-16 was inoculated at equal distances on both sides of the block, and cultured at 25°C for 48 h, repeated 3 times. The inability of pathogens to grow normally near strain CBPS-16 indicated that there was an antagonistic effect. The results showed (see Table 2) that strain CBPS-16 had a significant antagonistic effect on pseudograminearum Fusarium, cotton wilt, and tomato gray leaf spot.

[0063] Table 2 Antagonism of strain CBPS-16 against some pathogenic fungi

[0064] .

[0065] The antibacterial activity of CBPS-16 against three pathogenic fungi was determined by the growth rate method. That is, CBPS-16 was cultured in PB liquid medium for 24 h, centrifuged, and the supernatant was sterilized by 0.22μm sterile filter membrane. A certain amount of sterile filtrate was added to PDA medium at 40-45℃, mixed and poured into a 9 cm culture dish, and no sterile filtrate was added as a blank control. After the culture medium solidified, the three pathogenic bacteria were inoculated in the center of each plate, repeated 3 times, and cultured at 28℃. The colony diameter was measured by the cross method, and the inhibition rate was calculated. The results showed that the inhibition rate of CBPS-16 against the three pathogens was more than 65%.

[0066] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial block diameter) × 100.

[0067] Example 7 Tolerance of strain CBPS-16 to copper, lead and chromium

[0068] Use ultrapure water to mix lead acetate (Pb 2+ ), copper sulfate (Cu 2+ ), potassium dichromate (Cr 6+ ), cadmium chloride (Cd 2+ ) were prepared into metal ion solutions with a mass concentration of about 100 mM as stock solutions. In subsequent experiments, each metal ion stock solution was added to the PB culture medium in a certain amount to make the culture medium contain 0.5 mM, 1.0 mM, 2.0 mM, and 5.0 mM Cu, respectively. 2+ , Pb 2+ Cr 6+ and Cd 2+ , sterilized at 121°C, poured into plates, strain CBPS-16 was inoculated on different culture media, and its tolerance to heavy metal ions was determined based on the growth of the strain.

[0069] The results are shown in Table 3, indicating that strain CBPS-16 can grow normally in the medium containing 2 mM copper ions, 5 mM lead ions, and 1 mM hexavalent chromium ions, but cannot grow normally in the medium containing 0.5~1.0 mM cadmium ions. 2+ , Pb 2+ Cr 6+ It has a certain tolerance, which indirectly indicates that strain CBPS-16 can be used for the remediation of soil contaminated by these three metal ions.

[0070] Table 3 Tolerance of strain CBPS-16 to heavy metal ions

[0071] .

[0072] Example 8 Effects of strain CBPS-16 on corn plant growth and soil nutrients

[0073] Prepare the seed solution of strain CBPS-16 and adjust the concentration of the solution to 1×10 8 CFU / mL, based on 100 mL·kg -1 Mix in the matrix, which is a composite of 40% garden soil, 20% iron tailings sand and 40% vermiculite. For the control, mix the same volume of PB culture medium into the matrix and conduct a corn pot test. In a 25℃ greenhouse, keep the three best-growing seedlings in each pot after seed germination, and measure the aboveground indicators of the soil and plants and the root growth status after 28 days. Each group of experiments was treated in parallel with 3 groups.

[0074] Table 4 Effects of strain CBPS-16 on corn plant growth

[0075] .

[0076] The growth of corn is shown in Table 4. The strain CBPS-16 significantly promoted the growth of corn. Compared with the CK treatment group, the plant height, stem diameter, fresh weight and dry weight of corn treated with bacterial solution increased by 24.12%, 28.21%, 41.45% and 37.93%, respectively. The chlorophyll content of the treatment group was significantly higher than that of the CK group, increasing by 11.25%.

[0077] Table 5 Effect of strain CBPS-16 on soil fertility (unit: mg / kg)

[0078] .

[0079] As shown in Table 5, the application of CBPS-16 bacterial solution in the corn pot experiment can significantly increase the content of nitrate nitrogen, available silicon, available phosphorus, available potassium and organic matter in the soil, which increased by 10.28%, 22.81%, 44.47%, 15.88% and 11.31% respectively compared with the control. This further shows that the application of CBPS-16 bacterial solution can effectively increase the nutrient content of soil and improve soil nutrients.

[0080] Example 9 Effect of strain CBPS-16 on corn plant growth under saline-alkali stress

[0081] Mix Na2SO4, NaCl, NaHCO3 and Na2CO3 in a ratio of 4:1:4:1 (molar ratio) to prepare a saline-alkali stock solution. Preparation of saline-alkali soil: Mix garden soil and vermiculite in a volume ratio of 2:3, and use the prepared saline-alkali solution to soak the soil as the test soil. Prepare the fermentation liquid of strain CBPS-16 and adjust the concentration of the bacterial liquid to 1×10 9 CFU / mL, four treatments were set up, and appropriate amount of bacterial solution was added according to the soil quality in the pot. The bacterial content in treatment one (T1) was 1×10 6 CFU / kg, the bacterial content in treatment 2 (T2) was 1×10 7 CFU / kg, the bacterial content in treatment three (T3) was 1×10 8 CFU / kg, with no bacterial solution as the control CK, and 3 groups of parallel treatments for each treatment, a corn pot test was conducted. In a 25℃ greenhouse, after the seeds germinated, the three best-growing seedlings were retained in each pot, and the aboveground indicators and root growth conditions of the plants were measured 28 days later.

[0082] Table 6 Effects of strain CBPS-16 on corn plant growth under saline-alkali stress

[0083] .

[0084] Under saline-alkali stress, applying the bacterial agent CBPS-16 to the soil can effectively promote the growth of corn plants. The specific results are shown in Table 6. After adding the bacterial solution, the growth of the plants in the treatment group was better than that in the control group. When the bacterial solution application amount was 1×10 7 When the concentration of CFU / kg was 1.577%, the growth rate was the best, and the stem diameter, plant height, root length, fresh weight and dry weight increased by 13.77%, 16.17%, 58.13%, 35.29% and 40.91% respectively compared with the control. This further shows that the strain CBPS-16 can be used for ecological restoration of saline-alkali soil.

[0085] The present invention has been described in detail according to the above-mentioned embodiments. It should be noted that the above embodiments are only for the purpose of illustrating the invention. Without departing from the spirit and essence of the present invention, those skilled in the art can design various alternatives and improvements of the present invention, which should all be understood to be within the scope of protection of the present invention.

Claims

1. An endophytic Pantoea Pantoea sp. CBPS-16, characterized in that The deposit number is CGMCC No.30593.

2. A use of the CBPS-16 as claimed in claim 1 in promoting plant growth or improving soil quality.

3. Use of the CBPS-16 as claimed in claim 1 in the preparation of bio-organic fertilizer, bio-pesticide, soil conditioner or artificial matrix.

4. A use of the CBPS-16 as claimed in claim 1 in the remediation of heavy metal contaminated soil, wherein the heavy metal is divalent copper ion, divalent lead ion or hexavalent chromium ion.

5. A saline-alkali soil remediation agent, characterized in that: It comprises the CBPS-16 as claimed in claim 1.

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