Bacillus velezensis GBW-J5 as well as fungicide and application thereof

Abstract

The invention provides bacillus velezensis GBW-J5 as well as a fungicide and application thereof, and belongs to the technical field of microorganisms. The preservation number of the bacillus velezensis GBW-J5 is CGMCC (China General Microbiological Culture Collection Center) No.33830, and the bacillus velezensis GBW-J5 is separated from a penaeus vannamei shed culture water body, can be metabolized to generate abundant enzyme systems such as protease, amylase, cellulase and xylanase, and particularly has outstanding cellulase production capacity. The bacillus velezensis GBW-J5 is subjected to seed solution preparation, immersion fermentation and spray drying to obtain bacillus velezensis GBW-J5 bacterial powder, and the bacillus velezensis GBW-J5 bacterial agent can effectively decompose aged and dead algae in an aquaculture pond in the application of aquaculture water, so that the algal phase of the water is stabilized, and the water quality is improved. Stress reaction of animals is reduced, generation of algal toxins is prevented, poison to bred animals is reduced, and the breeding success rate is increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a strain of Bacillus velezensis GBW-J5, a bacterial agent and an application thereof. Background Art

[0002] Bacillus Velez is a Gram-positive bacterium belonging to the Fungi kingdom, Firmicutes phylum, Bacillaceae family, genus Bacillus, and species Bacillus Velez. It has attracted widespread attention for its rapid growth, strong stress resistance, rich metabolites, harmlessness to humans and animals, and environmental friendliness. Currently, Bacillus Velez is widely used in agricultural cultivation. Its metabolites, such as lipopeptides, can be used to inhibit plant pathogens and produce growth hormones to promote plant growth.

[0003] Microalgae play a vital role in maintaining the ecological balance of aquatic bodies. They provide natural food for aquatic animals, are the primary source of oxygen in the water, and purify water quality. However, in the middle and later stages of aquaculture, due to factors such as feeding, fertilization, and the excretion of aquatic animals, organic matter accumulates continuously, leading to excessive levels of nutrients such as nitrogen and phosphorus in the water, and the algae population increases dramatically. Sudden changes in algae populations can cause environmental deterioration. Algal respiration consumes large amounts of oxygen, leading to hypoxia in the water. Furthermore, the mass death of algae can lead to the proliferation of microorganisms, further depleting dissolved oxygen in the water and worsening water quality, impacting the health, production, and success rate of aquaculture animals. To address this issue, there are currently a wide variety of products on the market for controlling algae, and most of them are physical and chemical means, which only treat the symptoms but not the root cause. Existing biological products are not very effective. Currently, there is relatively little research and application of Bacillus Velez in eliminating aging and dead algae in aquaculture ponds. Therefore, the research and development of Bacillus Velez for eliminating and controlling algae is of great significance for developing new functions of Bacillus Velez, stabilizing the algae phase in water bodies, preventing the production of algal toxins, and reducing harm to farmed animals. Summary of the Invention

[0004] The present invention provides a strain of Bacillus Velez GBW-J5, a bacterial agent and an application thereof. The Bacillus Velez GBW-J5 can produce abundant enzymes and has an outstanding ability to produce cellulase, which can decompose cellulose, pectin and the like in algae cell walls. The bacterial powder obtained after fermentation and spray drying is applied to aquaculture to decompose aged and dead algae in ponds and prevent excessive algae growth.

[0005] In order to achieve the above-mentioned purpose, the present invention is implemented through the following technical solutions: The present invention provides a strain of Bacillus velez GBW-J5, which is classified as Bacillus velez Bacillus velezensis, The deposit number is CGMCC No.33830.

[0006] Furthermore, the colonies of the Bacillus velezensis GBW-J5 are milky white, with a rough surface and slightly irregular edges. The bacteria are rod-shaped and aggregated into short chains or bead-like arrangements. The spores are oval and located near the center or near the end of the non-expanded sac.

[0007] Furthermore, the 16S rRNA sequence of the Bacillus velezensis GBW-J5 is shown as SEQ ID No. 1.

[0008] Furthermore, the suitable growth temperature of the Bacillus velezensis GBW-J5 is 25-40° C., the suitable pH is 4.5-9.0, and the tolerable NaCl concentration range is 0-25 g / L.

[0009] Furthermore, the optimum temperature of the Bacillus velezensis GBW-J5 is 35° C., the optimum pH value is 7.5, and the maximum concentration of NaCl that can be tolerated is 25 g / L.

[0010] Furthermore, the Bacillus Velez GBW-J5 has the ability to produce enzymes, and the enzymes include at least one of cellulase, protease, amylase, lipase and xylanase.

[0011] Furthermore, the Bacillus Velezii GBW-J5 has a high cellulase production capacity.

[0012] The present invention also provides a bacterial agent obtained by fermenting Bacillus Velezii GBW-J5.

[0013] Furthermore, the bacterial agent includes Bacillus Velez powder, and the bacterial content of the Bacillus Velez powder is 1.0-1.25×10 11 CFU / g.

[0014] Furthermore, the Bacillus Velez powder is prepared by the following preparation method: (1) Seed liquid preparation: using the Bacillus velezii GBW-J5 as the strain, picking a single colony and culturing it in a seed culture medium to obtain a Bacillus velezii seed liquid; (2) Submerged fermentation: inoculating the Bacillus velezensis seed solution into a submerged fermentation medium and culturing the solution to obtain a Bacillus velezensis fermentation solution; (3) Spray drying: The Bacillus velezii fermentation broth is dried using a carrier by spray drying to obtain Bacillus velezii powder.

[0015] Furthermore, the components of the seed culture medium in step (1) include 3g / L~6g / L glucose, 4g / L~6g / L sucrose, 8g / L~10g / L tryptone, 4g / L~6g / L yeast extract powder, and 6g / L~10g / L NaCl. The culture conditions are a temperature of 30°C~40°C, a rotation speed of 160rpm~230rpm, a pH of 7~8, and a culture time of 22-26h.

[0016] Furthermore, the components of the seed culture medium are preferably 4 g / L glucose, 5 g / L sucrose, 9 g / L tryptone, 5 g / L yeast extract powder, and 8 g / L NaCl. The preferred culture conditions are temperature 37° C., pH 7.5, and rotation speed 210 rpm for 24 hours.

[0017] Furthermore, the components of the immersion fermentation medium in step (2) include 6g / L~10g / L corn starch, 10g / L~14g / L sucrose, 4g / L~6g / L yeast extract powder, 8g / L~10g / L tryptone, 0.2g / L~0.6g / L MgSO4, 0.1g / L~0.4g / L MnSO4, and 6g / L~10g / L NaCl. The culture conditions are a temperature of 30°C~37°C, a rotation speed of 160rpm~230rpm, a pH of 7~8, and a culture time of 24~48h.

[0018] Furthermore, the inoculation amount of the Bacillus velezensis seed liquid in step (2) is 5% to 15% based on the volume of the submerged fermentation medium; Furthermore, the optimal components of the immersion fermentation medium in step (2) are 8 g / L corn starch, 12 g / L sucrose, 5 g / L yeast extract powder, 9 g / L tryptone, 0.4 g / L MgSO4, 0.2 g / L MnSO4, and 8 g / L NaCl; and the optimal inoculation amount of the Bacillus velezensis seed liquid is 10%.

[0019] Furthermore, the carrier in step (3) is light calcium carbonate, and the addition ratio of the light calcium carbonate is 8-12% based on the volume of the Bacillus velezensis fermentation broth.

[0020] Furthermore, the spray drying conditions in step (3) are: air inlet temperature 170°C~200°C, air outlet temperature 72~85°C, and drying time 8-13 seconds.

[0021] Furthermore, the optimal conditions for spray drying are an inlet air temperature of 195°C, an outlet air temperature of 75°C, and a drying time of 10 seconds; and the optimal addition ratio of the light calcium carbonate is 10%.

[0022] The present invention also provides the use of the Bacillus Velez bacteria agent in decomposing dead and aged algae in aquaculture ponds.

[0023] Furthermore, the bacterial agent is Bacillus Velez powder, the dosage of the Bacillus Velez powder ranges from 500 to 1000 g / mu·m, and the usage days are 1 to 4 days.

[0024] Furthermore, the pond farming includes whiteleg shrimp pond farming.

[0025] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: The present invention screened a new strain of Bacillus Velez GBW-J5 from the water body of a small-scale aquaculture of whiteleg shrimp in vannamei. The strain can metabolize and produce a rich enzyme system including protease, amylase, cellulase and xylanase, especially with outstanding cellulase production capacity. The Bacillus Velez GBW-J5 of the present invention was fermented by immersion and spray-dried to obtain bacterial powder, which also contained a high content of viable bacteria (1.2×10 11 CFU / g) and a large number of metabolites, the Bacillus Velez powder is dissolved in pond water at a dosage of 500-1000g / mu·m at the breeding site and then sprinkled on the key areas of dead algae. It can be used for 1-4 days. It can quickly decompose and eliminate aging and dead algae, quickly eliminate water quality problems such as "boiling water" and "panda eyes" caused by excessive algae reproduction, maintain a good algae phase in the water body, restore the water body to be fertile, tender and refreshing, and indirectly improve the success rate of aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 To compare the cellulase production ability of different strains; Figure 2 The enzyme production capacity of Bacillus velez GBW-J5; Figure 3 This is the colony image of Bacillus velez GBW-J5 on NA medium; Figure 4 This is a crystal violet-stained microscopic examination of the vegetative body of Bacillus velezinis GBW-J5; Figure 5 This is a crystal violet-stained microscopic examination of the spores of Bacillus velezinis GBW-J5; Figure 6 The effect of culture temperature on the growth of Bacillus velez GBW-J5; Figure 7 The effect of culture medium pH on the growth of Bacillus velez GBW-J5; Figure 8 is the effect of NaCl concentration in the culture medium on the growth of Bacillus velez GBW-J5; Figure 9 Cellulase digestion circle on the plate by Bacillus velezensis GBW-J5; Figure 10 The in vitro effect of Bacillus Velez GBW-J5 on decomposing dead algae; Figure 11 This is the effect of using Bacillus Velez GBW-J5 in small sheds for breeding whiteleg shrimp. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0028] In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0029] Nutrient agar (NA) medium: Add 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride, and 15 g of agar per liter of distilled water and adjust the final pH to 7.3 ± 0.2.

[0030] Cellulose culture medium: 20g sodium carboxymethyl cellulose, 0.5g yeast extract, 2.5g disodium hydrogen phosphate, 1.5g potassium dihydrogen phosphate, 2.5g / L peptone, 20g agar powder, 1L water, pH 7.2. The color development solution is Congo red staining solution: 1g Congo red, 1L water; the eluent (1 mol / L NaCl solution) is: 58.5g NaCl, 1L water. After adding Congo red to the cellulose culture medium, rinse with 1 mol / L NaCl solution to ensure a clearer enzymatic zone.

[0031] Casein culture medium: 5 g casein, 1 g yeast extract, 20 g agar, pH 7.0, dilute to 1 L with distilled water, and sterilize at 121°C for 30 min. The color developing solution is an acidic mercuric reagent: 15 g HgCl₂, 20 mL concentrated HCl, and distilled water to 100 mL. If casein is difficult to dissolve, add some NaOH to make it alkaline. Stir under magnetic heating until completely dissolved, then adjust the pH to 7.0 with dilute hydrochloric acid.

[0032] Starch medium: 2g soluble starch, 10g peptone, 5g beef extract, 5g sodium chloride, 20g agar, pH 7.2, dilute to 1L with distilled water, and sterilize at 121°C for 30 min. The color developing solution is Lugol's iodine solution: 1g iodine tablets, 2g potassium iodide, and 300ml distilled water. To prepare, first dissolve the potassium iodide in a small amount of water, then dissolve the iodine tablets in the potassium iodide. Once the iodine is completely dissolved, add enough water. Soluble starch can be prepared by first using a small amount of cold water, stirring, and then adding it to boiling water for better dissolution. The color of the color developer disappears quickly during development, so measurements should be taken as soon as possible.

[0033] Lipid culture medium: 10 g peptone, 5 g yeast extract, 10 g NaCl, 2 mL tributyrin, 20 g agar powder, 1 L water, pH 7.5. Lipase hydrolysis zones can be directly observed.

[0034] Xylan culture medium: xylan 10g, peptone 5g, NH4NO3 5g, MgSO4 0.3g, NaCl 5g, K2HPO4 2g, (NH4)2SO4 1g, yeast powder 0.3g, agar 20g, water 1L, pH 7.2; the color developer is DNS: refer to the preparation method of DNS reagent in "GB_T 23874-2009 Determination of xylanase activity in feed additives".

[0035] Example 1: Screening, Identification and Preservation of Bacillus velezensis GBW-J5 (1) Screening and cultivation of Bacillus velezensis GBW-J5 In the small shed area of ​​whiteleg shrimp in Yancheng, Jiangsu Province, ponds with large-scale algae death were selected. Aquaculture water containing dead algae was taken and ground evenly with a grinder in the laboratory. The algae were gradiently diluted with sterile water and spread on nutrient agar (NA) medium. After multiple separation and purification, multiple single colonies were obtained. The cultured multiple single colonies were numbered as GBW-K1, GBW-K2, GBW-K3, GBW-J1, and GBW-J5.

[0036] Cellulose culture medium was used to determine the cellulase production capacity of GBW-K1, GBW-K2, GBW-K3, GBW-J1, and GBW-J5 strains. GBW-J5 was found to have the strongest cellulase production capacity ( Figure 1 ).

[0037] At the same time, casein culture medium, starch culture medium, fat culture medium and xylan culture medium were used to test the ability of GBW-J5 to produce protease, amylase, lipase and xylanase. It was found that GBW-J5 can produce a rich enzyme system ( Figure 2 ).

[0038] (2) Strain identification Morphological identification: The colony morphology of strain GBW-J5 is as follows Figure 3 As shown, the colony is milky white, with a rough surface and slightly irregular edges. Figure 4 As shown, the bacteria are rod-shaped, aggregated into short chains or beads, and the spores ( Figure 5 ) is oval and located near the center or near the end in non-inflated sporangium.

[0039] Molecular identification: DNA of GBW-J5 was extracted as a template and amplified using 16S rRNA universal primers. The amplified fragment was sequenced and the 16S rDNA sequencing results of strain GBW-J5 (sequence as SEQ ID No. 1) were compared with the sequences in GenBank. The results showed that strain GBW-J5 was Bacillus velezensisThe homology was the highest, so the strain GBW-J5 was determined to be Bacillus velezinis.

[0040] (3) Strain preservation Bacillus velezis GBW-J5 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC); Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Deposit date: March 17, 2025; Bacillus velezis GBW-J5 Bacillus velezensis The deposit number is: CGMCC No.33830.

[0041] Example 2: Analysis of Growth Characteristics of Bacillus velezensis GBW-J5 Culture medium: Solid culture medium contains 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, and 20 g / L agar powder in distilled water and is sterilized by moist heat at 121°C for 20 min before use. Growth temperature analysis medium contains 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl, pH 7.0, in distilled water and is sterilized by moist heat at 121°C for 20 min before use. Growth pH analysis medium contains 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl, pH 4.0-9.5, in distilled water and is sterilized by moist heat at 121°C for 20 min before use. NaCl tolerance analysis medium contains 10 g / L tryptone, 5 g / L yeast extract, and 5-35 g / L NaCl, pH 7.0, in distilled water and is sterilized by moist heat at 121°C for 20 min before use.

[0042] Analytical method: Using Bacillus velezensis GBW-J5 as the strain, single colonies were picked and cultured in growth temperature analysis medium at 37°C, pH 7.0, and 200 rpm for 16 hours. The colonies were then inoculated into the above-mentioned culture medium at 2% of the total volume of the culture medium. The culture medium contained 10 g / L tryptone, 5 g / L yeast extract powder, 5-35 g / L NaCl, and a pH of 4.0-9.5. The medium was prepared with distilled water and sterilized with moist heat at 121°C for 20 minutes before use. After culturing at 200 rpm for 24 h, the OD value of the culture medium at 600 nm was measured. The culture temperatures of the growth temperature analysis group included 20°C, 25°C, 30°C, 35°C, 40°C, and 45°C; the culture temperature of the growth pH analysis group was 35°C, and the pH conditions included 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5; the culture temperature of the NaCl tolerance analysis group was 35°C, and the NaCl concentrations included 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, and 35 g / L.

[0043] Analysis results: Bacillus velezensis GBW-J5 can grow and reproduce rapidly in the temperature range of 25~40℃, but its optimal growth temperature is 35℃ ( Figure 6 ), can adapt to pH 4.5~9.0, and the optimal growth pH is 7.5 ( Figure 7 ), the bacteria can tolerate up to 25g / L NaCl ( Figure 8 ), the bacteria can secrete a large amount of cellulase after fermentation ( Figure 9 ).

[0044] Example 3: Preparation of Bacillus Velezii GBW-J5 powder 1. Culture medium: Seed liquid culture medium contains 4 g / L glucose, 5 g / L sucrose, 9 g / L tryptone, 5 g / L yeast extract, and 8 g / L NaCl. It is prepared with distilled water and sterilized by moist heat at 121°C for 20 min. Use after cooling. Immersion fermentation culture medium contains 8 g / L corn starch, 12 g / L sucrose, 5 g / L yeast extract, 9 g / L tryptone, 0.4 g / L MgSO4, 0.2 g / L MnSO4, and 8 g / L NaCl. It is prepared with distilled water and sterilized by moist heat at 121°C for 20 min. Use after cooling.

[0045] 2. Seed solution preparation: Using Bacillus velezensis GBW-J5 as the strain, pick a single colony and place it in a seed culture medium. Cultivate at 37°C, pH 7.5, and 200 rpm for 24 hours to obtain Bacillus velezensis GBW-J5 seed solution. 3. Submerged fermentation: Inoculate the submerged fermentation medium with 10% seed solution of Bacillus velezensis GBW-J5, based on the total volume of the submerged fermentation medium, and culture at 37°C, pH 7.5, and 200 rpm for 36 h to obtain a fermentation broth of Bacillus velezensis GBW-J5.

[0046] 4. Spray drying: Using light calcium carbonate as a carrier, the fermentation liquid of Bacillus velez GBW-J5 was spray dried under the following conditions: the inlet air temperature was 195°C, the outlet air temperature was 75°C, the carrier addition ratio was 10% based on the weight of the GBW-J5 fermentation liquid, the drying time was 10 seconds, and the Bacillus velez GBW-J5 powder was obtained. The live bacteria content of the powder was 1.2×10 11 CFU / g.

[0047] Example 4: In vitro test on decomposition of dead algae The Bacillus velezensis GBW-J5 powder described in this example was prepared in Example 3.

[0048] Pond water containing dead algae was collected from a small shrimp farm and placed in a white water scoop. A test group sprinkled 5g of Bacillus velezensis GBW-J5 powder on the dead algae. A control group without GBW-J5 was also set up. The changes in dead algae on the first and second days were observed. The results showed that the algae decomposition process in the test group with GBW-J5 was significantly faster than that in the control group ( Figure 10 ).

[0049] Example 5: Decomposition of aged dead algae pond test The Bacillus velezensis GBW-J5 powder described in this example was prepared in Example 3.

[0050] In Guangxi Hepu West Farm, a small shed for whiteleg shrimp farming was used. The shed was 40 meters long, 10 meters wide, and 1 meter deep. The stocking density was 60,000 shrimp per shed. After 75 days of farming, excessive algae appeared in the water, with old and dead algae floating on the water surface, seriously affecting the water quality and posing a potential threat to the success of whiteleg shrimp farming. To address this farming environment, Bacillus Velez GBW-J5 powder was used. 500g of it was dissolved in water and sprayed on each shed. This was applied on sunny mornings for two consecutive days. The effect was observed on the third day. It was found that the old and dead algae floating on the surface of the water in the shed were basically decomposed by Bacillus Velez GBW-J5, and the water surface was clean ( Figure 11 ), the water permeability is enhanced and the vitality of white shrimp is improved.

[0051] In summary, the Bacillus Velezii GBW-J5 provided by the present invention can produce a variety of enzymes, and has an outstanding ability to produce cellulase, which can decompose cellulose, pectin, etc. in the cell walls of algae, effectively eliminate aging and dead algae in aquaculture ponds, and has a significant effect on maintaining a good algal phase in the water body. It has broad application prospects in improving poor water quality caused by excessive algae reproduction in whiteleg shrimp aquaculture.

[0052] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A strain of Bacillus velezensis GBW-J5, characterized in that Bacillus velez Bacillus velezensis, The deposit number is CGMCC No.33830.

2. The Bacillus Velezii GBW-J5 according to claim 1, characterized in that The colony of the Bacillus velezensis GBW-J5 is milky white, with a rough surface and slightly irregular edges. The bacteria are rod-shaped and aggregated into short chains or beads. The spores are oval and located near the center or near the end in the non-expanded sporangium.

3. The Bacillus Velezii GBW-J5 according to claim 1 or 2, characterized in that The Bacillus velezensis GBW-J5 has the ability to produce enzymes, and the enzymes include at least one of cellulase, protease, amylase, lipase and xylanase.

4. A bacterial agent obtained by fermenting the Bacillus Velez GBW-J5 according to claim 1.

5. The microbial agent according to claim 4, characterized in that The bacterial agent includes Bacillus Velez powder, and the bacterial content of the Bacillus Velez powder is 1.0-1.25×10 11 CFU / g.

6. The microbial agent according to claim 5, characterized in that The Bacillus Velez powder is prepared by the following preparation method: (1) Seed liquid preparation: using the Bacillus velezii GBW-J5 as the strain, picking a single colony and culturing it in a seed culture medium to obtain a Bacillus velezii seed liquid; (2) Submerged fermentation: inoculating the Bacillus velezensis seed solution into a submerged fermentation medium and culturing the solution to obtain a Bacillus velezensis fermentation solution; (3) Spray drying: The Bacillus velezii fermentation broth is dried using a carrier by spray drying to obtain Bacillus velezii powder.

7. The microbial agent according to claim 6, characterized in that The components of the seed culture medium in step (1) include 3g / L~6g / L glucose, 4g / L~6g / L sucrose, 8g / L~10g / L tryptone, 4g / L~6g / L yeast extract powder, and 6g / L~10g / L NaCl. The culture conditions are a temperature of 30°C~40°C, a rotation speed of 160rpm~230rpm, a pH of 7~8, and a culture time of 22-26h.

8. The microbial agent according to claim 6, characterized in that The components of the immersion fermentation medium in step (2) include 6g / L~10g / L corn starch, 10g / L~14g / L sucrose, 4g / L~6g / L yeast extract powder, 8g / L~10g / L tryptone, 0.2g / L~0.6g / L MgSO4, 0.1g / L~0.4g / L MnSO4, and 6g / L~10g / L NaCl. The culture conditions are a temperature of 30°C~37°C, a rotation speed of 160rpm~230rpm, a pH of 7~8, and a culture time of 24~48h.

9. Use of the Bacillus Velez microbial agent according to any one of claims 4 to 8 in decomposing dead and aged algae in aquaculture ponds.

10. The use according to claim 8, characterized in that The bacterial agent is Bacillus Velez powder, the dosage of the Bacillus Velez powder is in the range of 500-1000 g / mu·m, and the usage days are 1-4 days.

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