Bacillus velezensis as well as metabolite and application thereof

Abstract

The invention belongs to the field of microbial secondary metabolites, and particularly relates to bacillus velezensis as well as metabolites and application thereof. The bacillus velezensis is classified and named as Bacillus velezensis Z-1, and is preserved in the Guangdong Microbial Culture Collection Center, the preservation number is GDMCC 64975, and the preservation date is August 12, 2024. The bacillus velezensis Z-1 provided by the invention has a relatively strong inhibition effect on mango colletotrichum gloeosporioides in vitro, and the bacteriostasis rate of fermentation liquor can reach 73.33%. The prepared Z-1 bacterial liquid is used for soaking mango fruits, the bacterial liquid can effectively prolong the shelf life of the picked mangoes, the storage period can reach 24 days, in addition, the softening rate of the mangoes is obviously reduced, the fruit skin is full, the mangoes are fresh and turquoise, and the sense organ and the storage quality are maintained at a relatively high level. The method provides a certain new theoretical basis for disease control, storage and fresh-keeping of the harvested mangoes.

Description

Technical Field

[0001] The invention belongs to the field of microbial secondary metabolites, and specifically relates to Bacillus velezinus and its metabolites and uses. Background Art

[0002] Bacillus, a Gram-positive bacterium, is a typical biocontrol microorganism. It is widely used in the prevention and control of fruit diseases because of its safety, greenness and good effect. A large number of studies have found that probiotic Bacillus has a good preservation effect on litchi, figs and mutton. On the other hand, Bacillus can also effectively inhibit the growth and reproduction of fruit and vegetable pathogens, thereby resisting the infection of pathogenic microorganisms on fruits. Therefore, Bacillus has a good application prospect in the prevention and control of fruit and vegetable diseases as an environmentally friendly biological control agent.

[0003] Mango, as the "king of tropical fruits", is deeply favored by consumers for its unique flavor and high nutritional value. However, mango is extremely susceptible to infection by pathogenic microorganisms during growth and storage. Among them, Colletotrichum gloeosporioides can lurk in fruits and leaves. As the spores multiply in large numbers, black spots appear on the surface of the fruit, seriously reducing its commercial value. Colletotrichum gloeosporioides, as a major pathogen of mango, seriously affects the growth and development of mango fruit. The prevention and control of Colletotrichum gloeosporioides has gradually become a major research hotspot. However, physical control requires auxiliary instruments, which brings a lot of inconvenience to the prevention and control of mango diseases. Chemical fungicides have high residues and are harmful to human health, and are gradually eliminated by the public. Therefore, continuing to find safe, low-residue, and environmentally friendly biological control has a very broad application prospect in the prevention and control of fruit diseases. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a Bacillus velezensis and its metabolites and uses. The purpose of the present invention is achieved by the following technical scheme: A Bacillus velezensis, classified and named: Bacillus velezensis Z-1, deposited in Guangdong Microbiological Culture Collection Center, with a deposit number of GDMCC 64975 and a deposit date of August 12, 2024.

[0005] A method for preparing a metabolite of Bacillus velez, comprising the following steps:

[0006] S1. Preparing seed solution: inoculating the Bacillus Velezii described in claim 1 into LB liquid culture medium, and performing shaking flask culture at 35-40° C. and 160-200 rpm / min for 22-26 hours to obtain seed solution;

[0007] S2. Fermentation: The seed solution obtained in step S2 is inoculated into LB liquid culture medium for fermentation. The fermentation temperature is 35-40°C, the fermentation speed is 160-200 rpm / min, and the fermentation time is 68-74h.

[0008] Furthermore, the inoculation amount of the seed solution in step S2 is 2%.

[0009] Application of the Bacillus Velez metabolites prepared by the method in preventing and controlling mango anthracnose.

[0010] Furthermore, the metabolites of Bacillus Velezii have an inhibitory effect on Colletotrichum gloeosporioides.

[0011] Application of the Bacillus Velez metabolites prepared by the method in delaying the yellowing of mangoes after picking.

[0012] Furthermore, the harvested mangoes were soaked in a solution of Bacillus Velez metabolites for 8 to 15 minutes, taken out after soaking, washed with sterile water and air-dried, wherein the effective concentration of bacteria in the solution of Bacillus Velez metabolites was 0.8 to 1.5×10 8 CFU / mL.

[0013] Furthermore, the mango is 70% mature. After being picked on the same day, 0.8-1.2 cm of the fruit stem is retained, the white liquid on the fruit stem is washed off, and the washed fruit is placed at room temperature.

[0014] The present invention has the following advantages: The present invention provides a Bacillus velezensis, which is classified and named as Bacillus velezensis Z-1, and is deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC 64975 and a deposit date of August 12, 2024. The Bacillus velezensis Z-1 provided by the present invention has a strong inhibitory effect on Colletotrichum gloeosporioides in vitro, and the fermentation liquid has an antibacterial rate of up to 73.33%. The prepared Z-1 bacterial solution was soaked in mango fruits and it was found that the bacterial solution can effectively prolong the shelf life of post-harvest mangoes, and the storage period can reach 24 days. In addition, the softening rate of mangoes is significantly reduced, the fruit skin is plump and presents a fresh green color, and the sensory and storage quality is maintained at a high level. The present invention provides a certain new theoretical basis for the prevention and control of post-harvest mango diseases and storage and preservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a picture of the confrontation between Bacillus Velez Z-1 plates at different times.

[0016] Figure 2These are stability diagrams of the inhibition rate of Bacillus Velez fermentation broth against Colletotrichum gloeosporioides after treatment in different ways, where Figure A: different pH values, Figure B: different treatment temperatures, and Figure C: different UV irradiation times. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with the accompanying drawings and embodiments, and the protection scope of the present invention is not limited to the following: Example 1: Isolation, purification and identification of biocontrol bacteria

[0018] The soil of the mango orchard of Hainan University was sampled using the five-point method. About 15 g of soil was taken from each group and placed in a 55°C constant temperature incubator to dry the water for later use. The dried soil was added with 250 mL of sterile water and placed in a shaker for shaking overnight at a speed of 100 rpm / min for 12 hours. The culture solution was centrifuged at room temperature for 10 minutes at 5000 rpm / min, and the supernatant was taken as the original concentration for gradient dilution (10 1 -10 5 ) were spread on LB solid plates, cultured upright for 40 minutes, and then cultured inverted overnight. Pick out the single colony on the plate, streak five plates at 37°C for purification and culture. Streak the purified strain on the PDA plate inoculated with anthrax (the strain streaking position is about 2.5 cm away from the plate) to observe its inhibition on anthrax gloeosporioides. The single colony with the most obvious antibacterial effect was sequenced by PCR, and the obtained sequence was further uploaded to NCBI and compared online by BLAST.

[0019] Bacillus velezensis Z-1 strain is a broad-spectrum biocontrol bacterium isolated from mango orchard soil in 2023. The bacterium is a Gram-positive bacterium with white wrinkled colonies. It was identified as Bacillus velezensis Z-1 based on physiological characteristics and molecular identification. It is classified and named as Bacillus velezensis Z-1 and is deposited in Guangdong Microbial Culture Collection Center with the deposit number GDMCC 64975 and the deposit date August 12, 2024. The deposit address is: 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0020] The detailed sequence information of Bacillus velezensis Z-1 is as follows:

[0021]

[0022] The results showed that the Z-1 genome sequence was 100% similar to the genome of Bacillus velez (Gene bank: MT611666.1), and was named Z-1. Figure 1 As shown, the growth and reproduction rate of Colletotrichum gloeosporioides can be significantly inhibited.

[0023] Example 2: Experiment on inhibiting the growth and reproduction of Colletotrichum gloeosporioides

[0024] Take the purified Bacillus Velezii magnetic bead strain stored at -80℃, streak it on the LB solid plate, and activate it by culturing it at 37℃ overnight. Pick out a ring of the activated Z-1 strain, inoculate it into the liquid LB culture medium, and culture it in a shake flask at 37℃ and 180rpm / min for 24h to obtain the seed solution. Inoculate 2% of the seed solution and ferment it in the LB liquid culture medium. Culture it at 37℃ and 180rpm / min for 72h, and measure the absorbance of the Z-1 culture solution at 600nm using a UV spectrophotometer. Add different amounts of sterile water to adjust the bacterial solution concentration to 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 CFU / mL for later use. Cultivate the gloeosporium anthracnose in a 28°C constant temperature incubator for about 7 days (the gloeosporium anthracnose plate with orange spores in the colony), take the cultured bacterial cake with a 1mL sterile pipette tip, and inoculate it on a solid PDA medium plate at the same time. Draw a cross in the center of the PDA plate, stick a filter paper at 2.5cm from the center, inoculate 10μL of sterile water and fermentation liquid of different concentrations on the filter paper, measure the mycelial radius of the gloeosporium anthracnose in the sterile water group and the groups treated with different concentrations of bacterial liquid, compare the antibacterial rates of different concentrations of bacterial liquid, and screen the optimal treatment concentration.

[0025] Antibacterial rate (%) = (R 无菌水 -R 处理 ) / R 无菌水 ×100

[0026] R 无菌水 : Mycelial radius of sterile water treatment group

[0027] R 处理 :Mycelial radius of the group treated with fermentation liquid of Bacillus Velez Z-1

[0028] The results are shown in Table 1. The antibacterial rate of Velez subtilis Z-1 against anthrax is as high as 73.33%. 4 -109 CFU / mL) showed an inhibitory effect on mango anthracnose, which first increased and then decreased. 8 The inhibition rate of Bacillus Velezii liquid with CFU / mL against Colletotrichum mangoeum was the highest at 73.33%.

[0029] Table 1 Effect of different concentrations of Bacillus Velez fermentation broth on the antibacterial rate of Colletotrichum gloeosporioides

[0030]

[0031] Example 3: Experiment on delaying yellowing of mango peel

[0032] Take the purified Bacillus Velezii magnetic bead strain stored at -80℃, streak it on the LB solid plate, and activate it by culturing it at 37℃ overnight. Pick out a ring of the activated Z-1 strain, inoculate it into the liquid LB culture medium, and culture it in a shake flask at 37℃ and 180rpm / min for 24h to obtain the seed solution. Inoculate 2% of the seed solution and ferment it in the LB liquid culture medium. Culture it at 37℃ and 180rpm / min for 72h, and measure the absorbance of the Z-1 culture solution at 600nm using a UV spectrophotometer. Add different amounts of sterile water to adjust the bacterial solution concentration to 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 CFU / mL is reserved.

[0033] Experimental group: Wash the freshly picked mangoes to remove the mud and sand, cut off the fruit stalk (about 1 cm away from the fruit stem), rinse with running water, remove the fruits with mechanical damage, pests and spots, and select and grade them according to their size for later use. 4-9 CFU / mL) of Bacillus Velezii liquid was added, and the fruit was taken out after 10 minutes, washed with sterile water, air-dried, and the flesh color index was detected using a colorimeter, the a* value (the level of transition from green to red) was recorded, and the yellowing rate of the fruit was observed.

[0034] Comparative Example 1: Wash the freshly picked mangoes to remove the mud and sand, cut off the fruit stalk (about 1 cm from the fruit stem), rinse with running water, remove the fruits with mechanical damage, pests and spots, and select and grade them according to their size for later use. Soak the pretreated fruits in sterile water, take them out and dry them after 10 minutes, and measure the color difference change at the peel.

[0035] The method for determining the color difference value (a* value) involved in the above experiment is as follows:

[0036] Different treatment groups (104 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 The fruits of different storage periods (0d, 12d, 24d) were placed in a colorimeter (CR-400, KONICA MINOLTA, Osaka, Japan) and sterile water, and the color changes of the peel were detected. Three equidistant points on the equator of the fruit were selected as measuring points, and the a* value (the level of transition from green to red) was recorded.

[0037] The experimental results are shown in Table 2. 8 The yellowing rate of mangoes treated with Bacillus Velezii liquid at 10 CFU / mL was significantly reduced. 4 and 10 9 CFU / mL bacterial solution treatment can accelerate the yellowing rate of mangoes. Compared with the sterile water treatment group, there is no significant difference in a* value, and the fruit turns yellow in flakes. 8 After treatment with bacterial solution of 0.1 CFU / mL, the a* value on the fruit surface was the lowest until the fruit peel surface turned yellow in blocks after 24 days of storage.

[0038] Table 2 Effects of different concentrations of Bacillus Velez fermentation broth on a* value of mango fruit

[0039]

[0040] Example 4: Experiment on reducing the incidence of mango anthracnose

[0041] Take the purified Bacillus Velezii magnetic bead strain stored at -80℃, streak it on the LB solid plate, and activate it by culturing it at 37℃ overnight. Pick out a ring of the activated Z-1 strain, inoculate it into the liquid LB culture medium, and culture it in a shake flask at 37℃ and 180rpm / min for 24h to obtain the seed solution. Inoculate 2% of the seed solution and ferment it in the LB liquid culture medium. Culture it at 37℃ and 180rpm / min for 72h, and measure the absorbance of the Z-1 culture solution at 600nm using a UV spectrophotometer. Add different amounts of sterile water to adjust the bacterial solution concentration to 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 CFU / mL is reserved.

[0042] Experimental group: Wash the freshly picked mangoes to remove the mud and sand, cut off the fruit stalk (about 1 cm away from the fruit stem), rinse with running water, remove the fruits with mechanical damage, pests and spots, and select and grade them according to their size for later use. 4-9 CFU / mL) of Bacillus Velezii liquid was added, and the mixture was removed after 10 minutes, washed with sterile water, and air-dried. The lesion index on the surface of fruits in different groups was measured to observe the disease degree of the fruits.

[0043] Comparative Example 2: Wash the freshly picked mangoes to remove the mud and sand, cut off the fruit stalks (about 1 cm from the fruit stem), rinse with running water, remove the fruits with mechanical damage, pests and spots, and select and grade them according to their size for later use. Soak the pretreated fruits in sterile water, fish out and dry them after 10 minutes, measure the surface spot index of fruits in different groups, and observe the disease degree of the fruits.

[0044] The determination method of the lesion index involved in the above experiment is as follows:

[0045] Different treatment groups (10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 The disease degree on the fruit surface was calculated for fruits stored at different periods (0d, 12d, 24d) with 100 CFU / mL and sterile water. The lesion grading standard was based on the ratio of lesion area to fruit surface: grade 0, no lesions; grade 1, lesion area <1 / 10; grade 2, 1 / 10≤lesion area <1 / 5; grade 3, 1 / 5≤lesion area <1 / 2; grade 4, lesion area ≥1 / 2.

[0046] The disease index calculation formula is as follows:

[0047] Disease index = (number of fruits at each level × number of lesion levels) / (total number of fruits × number of lesion levels)

[0048] The experimental results are shown in Table 3. 8 The disease index on the surface of mango fruit treated with Bacillus Velez bacteria solution with CFU / mL was significantly reduced. 4 and 10 5 CFU / mL concentration of bacterial solution treatment mango fruit disease index did not significantly reduce, 10 9 CFU / mL of bacterial solution can effectively inhibit the disease of fruits, but if the concentration of bacterial solution is too high, it will accelerate the increase of the stomata diameter on the surface of the fruit, the stomata on the surface of the fruit will appear red, and black spots will be deposited, thereby reducing the sensory quality of the fruit.

[0049] Table 3 Effects of different concentrations of Bacillus Velez fermentation liquid on mango disease index

[0050]

[0051]

[0052] Example 5: Effect of different pH values ​​on the stability of the antibacterial rate of Bacillus velezinsis liquid

[0053] Take the purified Bacillus Velezii magnetic bead strain stored at -80℃, streak it on the LB solid plate, and activate it by culturing it at 37℃ overnight. Pick out a ring of the activated Z-1 strain, inoculate it into the liquid LB medium, and culture it in a shake flask at 37℃ and 180rpm / min for 24h to obtain the seed liquid. Inoculate 2% of the seed liquid and ferment it in the LB liquid medium. Culture it at 37℃ and 180rpm / min for 72h. Measure the absorbance of the Z-1 culture liquid at 600nm under a UV spectrophotometer and adjust the bacterial liquid concentration to 10 8 CFU / mL for later use. Use 10% NaOH and 10% HCL solution to adjust the pH of the prepared bacterial solution to 2.0, 7.0 and 9.0 respectively for later use. Cultivate the gloeosporium anthracnose bacteria in a 28°C constant temperature incubator for about 7 days (the gloeosporium anthracnose bacteria plate with orange spores in the colony), take the cultured bacterial cake with a 1mL sterile pipette tip, and inoculate it on a solid PDA culture medium plate at the same time. Draw a cross in the center of the PDA plate, stick a filter paper at 2.5cm from the center, inoculate 10μL of Bacillus Velez fermentation liquid with different pH values ​​on the filter paper, measure the mycelial radius of the gloeosporium anthracnose bacteria in the Bacillus Velez fermentation liquid treatment groups with different pH values, compare the antibacterial rates of different concentrations of bacterial liquid, and measure the stability of Bacillus Velez fermentation liquid under different pH conditions.

[0054] Antibacterial rate (%) = (R 无菌水 -R 处理 ) / R 无菌水 ×100

[0055] R 无菌水 : Mycelial radius of sterile water treatment group

[0056] R 处理 :Mycelial radius of the group treated with fermentation liquid of Bacillus Velez Z-1

[0057] The experimental results are as follows Figure 2 As shown in the figure, the acid-base stability of the sterile fermentation broth was tested and it was found that under acidic conditions, as the pH decreased, the Bacillus Velez fermentation broth produced a large amount of white precipitate, and the antibacterial activity decreased significantly. However, under neutral or alkaline conditions, the antibacterial activity of the fermentation broth remained basically unchanged, and the fermentation broth had better stability under neutral or alkaline conditions.

[0058] Example 6: Effect of different treatment temperatures on the stability of the antibacterial rate of Bacillus velezinsis liquid

[0059] Take the purified Bacillus Velezii magnetic bead strain stored at -80℃, streak it on the LB solid plate, and activate it by culturing it at 37℃ overnight. Pick out a ring of the activated Z-1 strain, inoculate it into the liquid LB medium, and culture it in a shake flask at 37℃ and 180rpm / min for 24h to obtain the seed liquid. Inoculate 2% of the seed liquid and ferment it in the LB liquid medium. Culture it at 37℃ and 180rpm / min for 72h. Measure the absorbance of the Z-1 culture liquid at 600nm under a UV spectrophotometer and adjust the bacterial liquid concentration to 10 8 CFU / mL for later use. Use 10% NaOH and 10% HCL solution respectively, place the prepared bacterial solution in a refrigerator (4℃), room temperature (25℃), and high temperature (heated to 75℃ in a water bath), and treat for 60 minutes respectively before use. Cultivate the gloeosporium anthracnose bacteria in a 28℃ constant temperature incubator for about 7 days (the gloeosporium anthracnose bacteria plate with orange spores in the colony), take the cultured bacterial cake with a 1mL sterile pipette tip, and inoculate it on a solid PDA medium plate at the same time. Draw a cross in the center of the PDA plate, stick a filter paper at 2.5cm from the center, and inoculate 10μL of the Bacillus Velez fermentation liquid treated at different temperatures on the filter paper, and measure the mycelial radius of the Bacillus Velez fermentation liquid treatment groups with different pH values, compare the antibacterial rates of different concentrations of bacterial liquid, and determine the stability of the Bacillus Velez fermentation liquid at different temperatures.

[0060] Antibacterial rate (%) = (R 无菌水 -R 处理 ) / R 无菌水 ×100

[0061] R 无菌水 : Mycelial radius of sterile water treatment group

[0062] R 处理 :Mycelial radius of the group treated with fermentation liquid of Bacillus Velez Z-1

[0063] The experimental results are as follows Figure 2 As shown, the stability of the fermentation broth at different temperatures was tested and it was found that there was no significant difference in the stability of the Bacillus Velez fermentation broth under low temperature (4°C), room temperature (25°C) and high temperature (65°C) conditions.

[0064] Example 7: Effect of UV irradiation on the stability of the antibacterial rate of Bacillus velezinsis liquid

[0065] Take the purified Bacillus Velezii magnetic bead strain stored at -80℃, streak it on the LB solid plate, and activate it by culturing it at 37℃ overnight. Pick out a ring of the activated Z-1 strain, inoculate it into the liquid LB medium, and culture it in a shake flask at 37℃ and 180rpm / min for 24h to obtain the seed liquid. Inoculate 2% of the seed liquid and ferment it in the LB liquid medium. Culture it at 37℃ and 180rpm / min for 72h. Measure the absorbance of the Z-1 culture liquid at 600nm under a UV spectrophotometer and adjust the bacterial liquid concentration to 10 8 CFU / mL for later use. Use 10% NaOH and 10% HCL solution respectively, place the prepared bacterial solution on a UV clean bench, and treat for 0min, 60min, and 120min respectively for later use. Cultivate the gloeosporium anthracnose bacteria in a 28℃ constant temperature incubator for about 7 days (gloeosporium anthracnose bacteria plate with orange spores in the colony), take the cultured bacterial cake with a 1mL sterile pipette tip, and inoculate it on a solid PDA medium plate at the same time. Draw a cross in the center of the PDA plate, stick a filter paper at 2.5cm from the center, and inoculate 10μL of the Bacillus Velez fermentation liquid treated at different temperatures on the filter paper. Determine the mycelial radius of the Bacillus Velez fermentation liquid treatment group with different pH values, compare the antibacterial rate of different concentrations of bacterial liquid, and determine the stability of the Bacillus Velez fermentation liquid at different temperatures.

[0066] Antibacterial rate (%) = (R 无菌水 -R 处理 ) / R 无菌水 ×100

[0067] R 无菌水 : Mycelial radius of sterile water treatment group

[0068] R 处理 :Mycelial radius of the group treated with fermentation liquid of Bacillus Velez Z-1

[0069] The experimental results are as follows Figure 2 As shown in the figure, the fermentation broth of Bacillus Velez has good stability to UV treatment. After different treatment times (0min, 60min and 120min), the antibacterial rate of Bacillus Velez fermentation broth against Colletotrichum gloeosporioides decreased, but there was no significant difference. Therefore, the fermentation broth of Bacillus Velez has high antibacterial stability against Colletotrichum gloeosporioides and can be used well under different conditions.

Claims

1. A Bacillus Velez, characterized in that The classification name is: Bacillus velezensis Z-1, deposited in Guangdong Provincial Microbiological Culture Collection Center, the deposit number is: GDMCC 64975, and the deposit date is: August 12, 2024.

2. A method for preparing a metabolite of Bacillus Velez, characterized in that: It includes the following steps: S1. Preparing seed solution: inoculating the Bacillus Velezii described in claim 1 into LB liquid culture medium, and performing shaking flask culture at 35-40° C. and 160-200 rpm / min for 22-26 hours to obtain seed solution; S2. Fermentation: The seed solution obtained in step S2 is inoculated into LB liquid culture medium for fermentation. The fermentation temperature is 35-40°C, the fermentation speed is 160-200 rpm / min, and the fermentation time is 68-74h.

3. The method for preparing a metabolite of Bacillus Velezii according to claim 2, characterized in that: The inoculation amount of the seed solution in step S2 is 2%.

4. Use of the Bacillus Velez metabolites prepared by the method according to claim 2 in preventing and treating mango anthracnose.

5. The use according to claim 4, characterized in that: The metabolites of Bacillus Velez have an inhibitory effect on Colletotrichum gloeosporioides.

6. Use of the Bacillus Velez metabolites prepared by the method according to claim 2 in delaying the yellowing of mangoes after picking.

7. The use according to claim 4 or 6, characterized in that: Soak the harvested mangoes in the solution of Bacillus Velez metabolites for 8 to 15 minutes, remove them, wash them with sterile water and air-dry them. The effective concentration of the bacteria in the solution of Bacillus Velez metabolites is 0.8 to 1.5 × 10 8 CFU / mL.

8. The use according to claim 7, characterized in that: The mangoes are 70% mature and are picked on the same day, with 0.8-1.2 cm of the fruit stems retained, white liquid on the fruit stems washed off, and the washed fruits placed at room temperature.

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