Endophytic bacteria for promoting tea seedling growth and increasing tea polyphenol content and application thereof

Abstract

The present invention provides an endophytic bacterium that promotes the growth of tea seedlings and improves the content of tea polyphenols, and an application thereof in the preparation of a microbial agent. The endophytic bacterium is strain SJ1, which is classified and named as Pantoea agglomerans, and is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No. 29420. The present invention also provides a microbial agent and an application thereof. The strain SJ1 of the present invention has the characteristics of solubilizing phosphorus and producing indoleacetic acid, and can significantly promote the growth of tea seedlings, increase the content of tea polyphenols in tea leaves, and increase the phosphorus content in the rhizosphere soil, roots and leaves of tea seedlings, and plays an important role in improving the utilization of phosphorus nutrients by tea seedlings, promoting the growth of tea seedlings, and improving the quality of tea leaves.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microorganisms, and in particular to endophytic bacteria for promoting the growth of tea seedlings and increasing the content of tea polyphenols and applications thereof. Background Art

[0002] Tea is one of the three major beverages in the world and an important economic crop in my country. Tea contains a large number of secondary metabolites that are beneficial to human health, including tea polyphenols, caffeine and free amino acids. Among them, tea polyphenols are the main component that determines the color, aroma, taste and efficacy of tea. They also have multiple physiological activities such as anti-oxidation, radiation protection, anti-aging, lowering blood lipids, lowering blood sugar, and antibacterial.

[0003] In order to increase tea yield and tea polyphenol content, fertilizers are applied in traditional ways. At present, tea gardens mainly apply single-element fertilizers and compound fertilizers. If such fertilizers are applied for a long time, problems such as soil compaction, changes in soil microecological environment, and reduced tea yield and quality will occur. Therefore, the use of microbial fertilizers or bacterial agents to produce organic, green, pollution-free tea is of great significance for improving tea yield and quality and protecting the ecological environment.

[0004] Endophytic bacteria exist in plants for a long time and usually form a stable mutually beneficial relationship with plants, which can affect the growth and development of plants and the synthesis of secondary metabolites. Endophytic bacteria can mainly promote the growth and development of plants by producing indoleacetic acid (IAA), fixing nitrogen, solubilizing phosphorus, and enhancing the absorption of soil nutrients by plants. At present, there are literature reports on the isolation and diversity of endophytic bacteria in tea trees, and the main endophytic bacteria in tea trees are publicly known to be Spirillum, Microbacterium, Acinetobacter, Bacillus, Burkholderia, etc., but there are not many studies on microbial strains promoting tea seedling growth and tea quality.

[0005] Screening endophytic bacteria of tea trees with growth-promoting effects and developing corresponding microbial agents will help reduce dependence on chemical fertilizers in tea garden management, improve tea tree growth, and promote the utilization of nutrients. At the same time, it can give full play to the regulatory potential of endophytes on plant secondary metabolism, effectively promote the synthesis of secondary metabolites in tea trees, and provide innovative agricultural management strategies for the sustainable development of the tea industry. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an endophytic bacterium for promoting the growth of tea seedlings and increasing the content of tea polyphenols and its application.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] An endophytic bacterium that promotes the growth of tea seedlings and increases the content of tea polyphenols. The endophytic bacterium is strain SJ1, classified and named Pantoea agglomerans, and was deposited and shown to be alive at the General Microbiological Center of China National Microbiological Culture Collection on December 27, 2023. The address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 29420.

[0009] The bacterial strain SJ1 has round, yellow colonies on LB medium, with smooth and opaque surfaces and neat edges; the bacterial strain is Gram-negative, amylase-negative, catalase-positive, can utilize mannitol, starch, sucrose, maltose, and glucose, tolerates 7% sodium chloride, and grows well at 20-35°C.

[0010] As one of the preferred embodiments of the present invention, the 16S rDNA sequence of the strain SJ1 is shown as SEQ ID NO.1.

[0011] As one of the preferred embodiments of the present invention, the strain SJ1 is used to dissolve poorly soluble inorganic phosphorus and organic phosphorus, and to produce indoleacetic acid.

[0012] As one of the preferred embodiments of the present invention, the poorly soluble inorganic phosphorus and organic phosphorus include tricalcium phosphate and calcium phytate.

[0013] As one of the preferred embodiments of the present invention, the strain SJ1 is used to promote the growth of tea seedlings and increase the tea polyphenol content of tea leaves.

[0014] The invention discloses an application of the endophytic bacteria for promoting the growth of tea seedlings and increasing the content of tea polyphenols in the preparation of microbial fertilizers.

[0015] A bacterial agent is prepared by using the above-mentioned endophytic bacteria which can promote the growth of tea seedlings and increase the content of tea polyphenols.

[0016] As one of the preferred embodiments of the present invention, the bacterial agent is a biological liquid bacterial agent, and the effective live bacteria count of the strain SJ1 is greater than 200 million / ml.

[0017] An application of the above-mentioned bacterial agent is used to promote the growth of tea seedlings and / or increase the content of tea polyphenols in tea leaves.

[0018] An application of the above-mentioned bacterial agent is used to increase the phosphorus content in the soil and promote the absorption and utilization of phosphorus nutrients by tea seedlings.

[0019] The advantages of the present invention compared to the prior art are:

[0020] (1) The present invention screened out a bacterial strain that can dissolve poorly soluble inorganic phosphorus and organic phosphorus. The strain SJ1 has the ability to dissolve tricalcium phosphate up to 229.29 mg / L and calcium phytate up to 272.10 mg / L, and can produce the plant growth hormone - indoleacetic acid.

[0021] (2) Compared with the uninoculated control, the strain SJ1 of the present invention can significantly improve the growth of tea seedlings, and the plant height, aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight are increased by 10.94%, 67.09%, 35.40%, 76.47% and 35.29%, respectively.

[0022] (3) The strain SJ1 of the present invention can significantly increase the tea polyphenol content of tea leaves by 18.89%, and can improve the quality of tea leaves while promoting the growth of tea seedlings.

[0023] (4) Inoculation of the strain SJ1 of the present invention can increase the effective phosphorus content in the rhizosphere soil of tea seedlings, increase soil fertility, and improve the soil ecological environment.

[0024] (5) Inoculation of the strain SJ1 of the present invention can increase the total phosphorus content in the leaves and roots of tea seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a normal growth colony image of strain SJ1 in Example 2;

[0026] Figure 2 is a staining microscopic image of strain SJ1 in Example 2;

[0027] Figure 3 is the phylogenetic tree of strain SJ1 in Example 2;

[0028] Figure 4 It is a growth state diagram of potted plants of treatment group SJ1 and control group CK in Example 5;

[0029] Figure 5 The effect of strain SJ1 on the tea polyphenol content of tea leaves in Example 5 (in the figure, "*" indicates significant difference between treatments, p<0.05);

[0030] Figure 6 The effect of strain SJ1 on soil available phosphorus content in Example 6 (in the figure, "**" indicates significant difference between treatments, p < 0.01);

[0031] Figure 7 The effect of strain SJ1 in Example 6 on the phosphorus content of tea seedling leaves (in the figure, "*" indicates a significant difference between treatments, p < 0.05);

[0032] Figure 8This is the effect of strain SJ1 in Example 6 on the phosphorus content in the roots of tea seedlings (in the figure, "**" indicates a significant difference between treatments, p<0.01). DETAILED DESCRIPTION

[0033] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0034] Example 1: Isolation of strains

[0035] The Pantoea agglomerans strain SJ1 isolated and preserved in the present invention is isolated and purified from the root of tea tree, and the specific isolation steps are as follows:

[0036] The collected tea tree roots were first rinsed with tap water, the surface was rinsed with sterile water, and cut into small pieces of 2 to 3 cm in length with scissors. Then, the surface of the material was disinfected on a clean bench, and it was soaked in 70% ethanol solution for 1 min, soaked in 3.5% sodium hypochlorite for 3 to 4 min, and rinsed with sterile water for 3 to 4 times. 100 μL of the sterile water from the last rinse was applied to the LB plate as a blank control. After incubation, no bacterial colonies grew on the surface of the plate, indicating that the root surface was thoroughly disinfected.

[0037] Put the sterilized in vitro material into a sterile mortar, add a little sterile quartz sand for grinding, and add sterile water to dilute it by 10 -1 , 10 -2 , 10 -3 times; take 100 μL of each gradient and spread it on the LB plate, and invert it in a 30℃ incubator for culture. When colonies appear on the plate, select single colonies with vigorous growth, different sizes and shapes, streak them on a new LB solid medium plate, and culture them at 30℃ for 48h. Repeat the transfer operation 3 times. After confirming that there are no foreign bacteria, pick a single colony and inoculate it into LB liquid culture medium, place it in a shaker, and culture it at 28℃ and 180r / min for 28 to 36h; finally, use 40% glycerol suspension to preserve the strain at -80℃ for later use.

[0038] Example 2: Identification of strain SJ1

[0039] The physiological and biochemical characteristics of strain SJ1 were tested according to the Manual of Identification of Common Bacterial Systems.

[0040] When the strain SJ1 was grown in LB medium, Figure 1 As shown, the colonies are yellow, with neat edges, smooth and moist surface, and opaque. The bacteria are rod-shaped under a microscope and have no spores ( Figure 2). Physiological and biochemical characteristics of the bacteria: Gram staining is negative, amylase is negative, catalase is positive, and urease is negative. It can utilize mannitol, starch, sucrose, maltose, and glucose. It tolerates 7% sodium chloride and grows well at 20-35°C.

[0041] The genomic DNA of the strain SJ1 was extracted, and then the DNA was amplified by PCR using the universal primers 27F and 1492R of the bacterial 16S rRNA gene. The obtained PCR amplification products were sequenced. The BLAST analysis was performed based on the strain 16S rDNA sequence using the NCBI nucleic acid database. The analysis results showed that the strain SJ1 belonged to the genus Pantoea sp., and the homology with the 16S rDNA sequence of Pantoea agglomerans was as high as 99.1% to 99.7%.

[0042] See also Figure 3 Phylogenetic analysis showed that strain SJ1 and Pantoea agglomerans clustered into a large branch. Further combined with Gram staining and other physiological and biochemical test results, it was shown that strain SJ1 was a Pantoea agglomerans. The strain was named Pantoea agglomerans SJ1 and deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration with a deposit number of CGMCC No. 29420. The 16S rDNA sequence of strain Pantoea agglomerans SJ1 is shown in SEQ ID NO.1.

[0043] Example 3: Determination of phosphate solubilization ability and indoleacetic acid (IAA) production of strain SJ1

[0044] 1. Phosphorus-solubilizing ability of strain SJ1

[0045] The strain SJ1 screened by the present invention is inoculated into a test tube, and after shaking culture on a shaker for 24 to 30 hours, it is inoculated into 100 mL of an inorganic phosphorus culture medium containing tricalcium phosphate (10.0 g of glucose, 0.1 g of ammonium sulfate, 0.2 g of potassium chloride, 0.25 g of magnesium sulfate heptahydrate, 5.0 g of magnesium chloride hexahydrate, 5.0 g of tricalcium phosphate, 1000 mL of distilled water, pH 7.0, 115° C., and sterilized for 30 minutes) and 100 mL of an organic phosphorus culture medium containing calcium phytate (10.0 g of glucose, 0.1 g of ammonium sulfate, 0.2 g of potassium chloride, 0.25 g of magnesium sulfate heptahydrate, 5.0 g of magnesium chloride hexahydrate, 5.0 g of calcium phytate, 1000 mL of distilled water, pH 7.0, 115° C., and sterilized for 30 minutes) at a shaking temperature of 28 to 30° C. and 160 r / min for 3 days. The culture solution was centrifuged at 8000r / min, 20μL of supernatant was added with 1mL of molybdenum antimony anti-colorimetric agent, and the volume was adjusted to 10mL with distilled water. The color was developed for 15-20min, and the OD value at 700nm was measured. The 5mg / L phosphorus standard solution was gradiently diluted to phosphorus contents of 0.00, 0.25, 0.50, 1.00, 1.25, and 1.50mg / L to prepare a standard curve. Molybdenum antimony anti-colorimetric agent was added at room temperature, the color was developed for 15-20min, the absorbance was measured, and a standard curve was prepared. The standard curve was y=0.5191x+0.0218, and the correlation coefficient R 2 =0.9976.

[0046] According to the standard curve, the phosphorus content in the culture fluid of strain SJ1 containing tricalcium phosphate was 229.29 mg / L, and the phosphorus content in the culture fluid of strain SJ1 containing calcium phytate was 272.10 mg / L. This shows that the strain SJ1 has a strong ability to dissolve calcium phosphate and calcium phytate, and can convert insoluble phosphorus into soluble phosphorus.

[0047] 2. Determination of IAA secretion of strain SJ1

[0048] The standard curve was prepared using analytically pure IAA. The 0.2 mg / mL IAA standard solution was diluted to 0, 5, 10, 15, 20, 25, and 30 mg / L concentrations. The absorbance (OD 530 ). The standard curve was obtained as y = 0.0275x + 0.0131, and the correlation coefficient R 2 =0.9993.

[0049] The nitrogen culture medium (sucrose 10.0g, ammonium sulfate 1.0g, potassium hydrogen phosphate 2.0g, magnesium sulfate heptahydrate 0.5g, sodium chloride 0.1g, yeast extract 0.5g, pH 7.2, distilled water 1000mL) was dispensed into test tubes, 4mL per tube, and 1mL of filtered sterilized tryptophan was added after sterilization to make the final tryptophan concentration in the culture medium 0.5mg / mL. The target strain was inoculated into the culture medium, cultured on a shaker at 28℃ 150r / min for 2d, the culture solution was centrifuged at 6000r / min for 10min, 1mL of the supernatant was taken, 50μL 10mM / L orthophosphoric acid was added, and 2mL Sackowski's colorimetric agent was added, mixed thoroughly, and color developed at 25℃ in the dark for 30min, and the absorbance at 530nm was measured.

[0050] According to the standard curve, the IAA content in the culture medium of strain SJ1 was 27.46 mg / L.

[0051] Example 4: Activation of strain SJ1 and preparation of bacterial suspension

[0052] Use an inoculation loop to pick a loop from the SJ1 glycerol tube, streak it on LB solid medium aseptically, and culture it at 28℃ for 72h. Then select a single colony of the SJ1 strain with vigorous growth and streak it on three zones of a new LB solid medium plate. After culture, pick a single SJ1 colony and inoculate it into LB liquid medium, and culture it at 28℃ and 150-180rpm on a shaker for 20-30h.

[0053] Take 100 mL of the culture medium containing strain SJ1, centrifuge at 8000 r / min to collect the cells, wash the cells with sterile water, and resuspend the cells in 80-100 mL of sterile water to make the cell count reach 2×10 8 CFU / mL or above, the liquid bacterial agent of strain SJ1 is obtained.

[0054] Example 5: Strain SJ1 promotes tea seedling growth and increases tea polyphenol content

[0055] 1. Pot experiment and biomass

[0056] One-year-old cutting tea seedlings (Zhongcha 108, about 11 cm high) with the same growth size were selected and planted in pots (15 cm high and 25 cm in diameter). The nutrient soil used for tea seedling growth was mainly Fuyu brand plant culture medium, which was mixed evenly with vermiculite and perlite at a mass ratio of 2:1:1 and then divided into pots, with 3 kg in each pot. 2 mL of the above-mentioned bacterial suspension was applied near the root of each seedling as the treatment group (SJ1), while the control group (CK) was applied with an equal volume of sterile water. Normal growth under natural conditions, regular watering, planting for 2 and a half months (about 75 days), the tea seedlings were harvested to measure plant height, aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight.

[0057] Table 1 Effect of strain SJ1 on tea seedling biomass

[0058]

[0059]

[0060] Note: * indicates p<0.05, there is a significant difference between the treatment levels

[0061] The growth status of potted plants in the control group (CK) and treatment group (SJ1) is as follows Figure 4 The final biomass measurement results are shown in Table 1.

[0062] As can be seen from Table 1: after inoculation with the strain, the growth of tea seedlings was significantly enhanced. Compared with the control, plant height, aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight were significantly increased by 10.94%, 67.09%, 35.40%, 76.47% and 35.29%, all reaching a significant difference level.

[0063] 2. Determination of tea polyphenols content

[0064] Collect one bud and two leaves of tea seedlings, kill them at 105℃ and dry them at 80℃ to constant weight. Grind the dried sample in a mortar, weigh the sample and put it in a centrifuge tube, add it to a 70% methanol aqueous solution preheated at 70℃, stir it thoroughly with a glass rod to make it evenly wet, immediately transfer it to a 70℃ water bath, extract it for 10 minutes, cool it to room temperature after extraction, transfer it to a centrifuge and centrifuge it at 8000rpm for 10 minutes, transfer the supernatant to a 10mL volumetric flask, and extract the residue once with 5mL of 70% methanol aqueous solution, repeat the above operation, and combine the extracts to make up to 10mL. After diluting the extract 100 times, transfer 1.0mL of the dilution to a graduated test tube, add 5.0mL of Folin phenol reagent, and shake well. React for 3 to 8 minutes, add 4.0mL of 7.5% sodium carbonate solution, add pure water to make up to the scale, and shake well. Leave it at room temperature for 60 minutes. Measure the absorbance at 765nm.

[0065] The standard curve was prepared using gallic acid. The 1000 μg / mL gallic acid standard solution was diluted to 10, 20, 30, 40, and 50 μg / mL concentrations. The absorbance (OD 765 ), the standard curve is y = 0.0044x + 0.0041, and the correlation coefficient R 2 =0.997.

[0066] Substitute the absorbance value of the sample at 765nm into the regression equation and calculate according to the dilution multiple and sample mass to obtain Figure 5 .Depend on Figure 5It can be seen that after inoculation with strain SJ1, the content of tea polyphenols was significantly increased by 18.89% (p<0.05) compared with the uninoculated control group (CK).

[0067] Example 6: Strain SJ1 increases available phosphorus in soil and phosphorus content in tea seedlings

[0068] 1. Soil potting

[0069] One-year-old tea seedlings (Zhongcha 108, about 11 cm in height) with the same growth size were selected for the experiment and planted in pots (15 cm in height and 25 cm in diameter). The test soil was collected from the tea garden in Xiafuqiao Town, Huoshan County, Lu'an City, Anhui Province, and 3 kg was packed in each pot. After the tea seedlings were transplanted, 2 mL of the above bacterial suspension (2×10 8 ~9×10 8 CFU / mL) as the treatment group (SJ1), while the control group (CK) was given an equal volume of sterile water. The tea seedlings were grown outdoors and watered regularly. After more than 2 and a half months (about 80-85 days), the rhizosphere soil and tea seedlings were collected to determine the available phosphorus in the soil and the phosphorus content in the roots and leaves of the tea seedlings.

[0070] 2. Determination of soil available phosphorus

[0071] Determination of soil available phosphorus by ammonium fluoride-hydrochloric acid extraction method: weigh the air-dried sample that has passed through a 2mm sieve hole and place it in a 200mL plastic bottle, add ammonium fluoride-hydrochloric acid extractant, shake at 180r / min for 30min at 25℃, filter with phosphorus-free filter paper, draw 10mL of the filtrate into a 50mL volumetric flask, add 10mL of boric acid solution, shake well, add water to about 30mL, then add 2 drops of dinitrophenol indicator, adjust the solution to slightly yellow with sulfuric acid solution and ammonia solution, add 5mL of molybdenum antimony anti-coloring agent, make up to volume with pure water, let stand for 30min at room temperature above 20℃, use a 1cm optical path colorimetric dish at a wavelength of 700nm, take the standard solution as the zero point, and perform colorimetric determination after zeroing. Substitute the measured value into the standard curve regression equation to calculate the phosphorus content in the sample.

[0072] The results are as follows Figure 6 As shown. Figure 6 It can be seen that compared with the uninoculated control group (CK), the available phosphorus content in the rhizosphere soil of tea seedlings was significantly increased by 9.25% (p<0.01) after the application of strain SJ1.

[0073] 3. Strain SJ1 increases phosphorus content in leaves and roots of tea seedlings

[0074] Weigh the mixed and ground plant dry samples (tea seedling leaf and root samples) accurately and place them in a digestion tube, add concentrated sulfuric acid, shake gently and let stand overnight, cover with a small curved neck funnel, and first digest at low temperature on a digestion furnace. After the white smoke disappears, remove the digestion tube and cool it slightly, add about 0.5mL of 30% H2O2 drop by drop; shake the digestion tube gently, continue to heat and boil for about 5 minutes, remove it and let it cool slightly, add a few drops of H2O2 repeatedly, and then digest again. Repeat this several times (about 2 to 3 times). The amount of H2O2 added each time should be reduced gradually. After digestion until the solution is colorless or clear, continue to digest for 5 to 15 minutes to remove the remaining H2O2. Remove the digestion tube, cool it, rinse the curved neck funnel with a small amount of water, and the washing liquid flows into the digestion tube. Transfer the digestion solution to a 100mL volumetric flask without any damage and make up to volume. After filtering into a triangular flask with quantitative filter paper, draw a certain amount of filtrate into a 50mL volumetric flask, add water to about 30mL, add 2 drops of dinitrophenol indicator, adjust the solution to just slightly yellow with sodium hydroxide solution or sulfuric acid solution, then add 5mL of molybdenum antimony anti-color developer, and make up to volume. Place it in an environment above 20℃ for 30 minutes to develop color, and measure and record the absorbance at 700nm.

[0075] The results are as follows Figure 7 , Figure 8 As shown. Figure 7 , Figure 8 It can be seen that compared with the uninoculated control group (CK), after the application of strain SJ1, the total phosphorus content in the leaves and roots of tea seedlings increased significantly by 5.10% and 18.60%, respectively, indicating that the application of strain SJ1 can promote the absorption and utilization of phosphorus nutrition by tea seedlings.

[0076] In summary, the strain SJ1 of the present invention has the characteristics of solubilizing phosphate and producing indoleacetic acid, can significantly promote the growth of tea seedlings, increase the content of tea polyphenols in tea leaves, and increase the phosphorus content in the rhizosphere soil, roots and leaves of tea seedlings, which plays an important role in improving the utilization of phosphorus nutrients by tea seedlings, promoting the growth of tea seedlings, and improving the quality of tea.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An endophytic bacterium for promoting the growth of tea seedlings and increasing the content of tea polyphenols, characterized in that: The endophytic bacteria is strain SJ1, which is classified and named Pantoea agglomerans and deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No.29420.

2. The endophytic bacteria for promoting the growth of tea seedlings and increasing the content of tea polyphenols according to claim 1, characterized in that: The 16S rDNA sequence of the strain SJ1 is shown in SEQ ID NO.

1.

3. Use of the endophytic bacteria for promoting the growth of tea seedlings and increasing the content of tea polyphenols as claimed in any one of claims 1 to 2 in the preparation of microbial fertilizers.

4. A bacterial agent, characterized in that The tea seedling tea is prepared by using the endophytic bacteria for promoting the growth of tea seedlings and increasing the content of tea polyphenols as claimed in any one of claims 1 to 2.

5. The bacterial agent according to claim 4, characterized in that The bacterial agent is a biological liquid bacterial agent, and contains an effective live bacterial count of strain SJ1 greater than 200 million per milliliter.

6. A use of the bacterial agent as claimed in claim 4 or 5, characterized in that: Used to promote the growth of tea seedlings and / or increase the content of tea polyphenols in tea leaves.

7. A use of the bacterial agent as claimed in claim 4 or 5, characterized in that: Used to increase soil phosphorus content and promote tea seedlings' absorption and utilization of phosphorus nutrients.

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