AU2021105572A4 - Anti-ultraviolet microbial agent and application - Google Patents

Anti-ultraviolet microbial agent and application Download PDF

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AU2021105572A4
AU2021105572A4 AU2021105572A AU2021105572A AU2021105572A4 AU 2021105572 A4 AU2021105572 A4 AU 2021105572A4 AU 2021105572 A AU2021105572 A AU 2021105572A AU 2021105572 A AU2021105572 A AU 2021105572A AU 2021105572 A4 AU2021105572 A4 AU 2021105572A4
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bacillus subtilis
ultraviolet
microcapsules
marine
microbial agent
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Yunfei LIU
Jiajing Lv
Baohua Zhang
Chengsheng ZHANG
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Qingdao Agricultural University
Tobacco Research Institute of CAAS
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Institute Of Tobacco Res Of Caas
Qingdao Agricultural University
Tobacco Research Institute of CAAS
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Abstract

The present invention discloses an anti-ultraviolet microbial agent and an application, and relates to the technical field of microbes. The anti-ultraviolet microbial agent includes Bacillus subtilis microcapsules and marine Trichoderma asperellum HG1, wherein the Bacillus subtilis microcapsules include Bacillus subtilis having a collection number of CGMCC No.2843; and the marine Trichoderma asperellum HG1 has a collection number of CGMCC No.19276. In the present invention, the Bacillus subtilis bacterium agent is prepared through microencapsulation by utilizing silk fibroin and dopamine materials with excellent compatibility, thereby improving ultraviolet resistance. The Bacillus subtilis bacterium agent and the marine Trichoderma asperellum HG1 are further combined to prepare a biological bacterium agent, thereby enhancing an antibacterial effect on plant diseases. The prepared water dispersible granules have a control effect of 76.27% on Botrytis cinerea and a control effect of 70.06% on tobacco black shank.

Description

Description
ANTI-ULTRAVIOLET MICROBIAL AGENT AND APPLICATION
Technical Field
The present invention relates to the technical field of microbes, and particularly relates to an anti-ultraviolet microbial agent and an application.
Background
Although use of chemical pesticides plays a crucial role in ensuring agricultural production, environmental ecology is affected due to long-term use of the pesticides; and drug resistance of hazardous substances is further increased, thereby decreasing a control effect. Moreover, with the increasing requirements for food safety, pesticide residues also attract attention. Decreasing the use of the chemical pesticides and ensuring good development of agricultural production become problems that urgently need to be solved. Practice has proved that, application of a microbial agent that is environment-friendly and specific in control is an important way to control agricultural pests and weed damage. Bacillus subtilis may produce multiple active materials, has excellent antibacterial activity on multiple plant diseases, is high in growth rate, simple in nutritional requirement, non-pathogenic and wide in application, and is an ideal bio-control bacterium that may be directly applied to production. Although the Bacillus subtilis has wide applications, related preparation products in the current market have defects of short storage time, low activity, poor control effect and sensitivity to ultraviolet light. Improving stress resistance of the Bacillus subtilis, particularly ultraviolet resistance, is an important subject of preparation research. Marine Trichoderma asperellum HG1 serves as the most effective bio-control factor for controlling plant diseases, and has important functions of high stress resistance, high growth rate, long spore survival time, parasitism on pathogenic bacteria, and capacity of promoting plant growth and soil remediation. Previous
Description
studies have shown that, a combination of the Bacillus subtilis and the marine Trichoderma asperellum HG1 may achieve advantages of two different bacterium agents. By utilizing autophage activity substances of the Bacillus subtilis and dominant advantages of the marine Trichoderma asperellum HG1, the control effect is enhanced; and micro-ecology is improved. Silk fibroin serves as an important component of silk, is a low-density structural protein, and has irregular coil in a core domain to achieve tough strength. Dopamine (DA) is a biological neurotransmitter, structurally contains catechol base and amino, and may be subjected to auto-oxidative polymerization on the solid surface so as to form a polydopamine coating. The polydopamine coating has lots of phenolic hydroxyl groups and amino or imido, enhances adhesive property to surfaces of most of the materials (particularly smooth surfaces), and has excellent binding capacity and biocompatibility on cells and proteins. Therefore, the dopamine has wide applications in aspects of material surface modification, environmental control, drug delivery and biomedicine. Therefore, how to provide an anti-ultraviolet microbial agent is a problem that urgently needs to be solved by those skilled in the art.
Summary In view of this, the present invention provides an anti-ultraviolet microbial agent and an application. To achieve the above purpose, technical solutions of the present invention are as follows: The anti-ultraviolet microbial agent includes Bacillus subtilis microcapsules and marine Trichoderma asperellum HG1, wherein the Bacillus subtilis microcapsules include Bacillus subtilis having a collection number of CGMCC No. 2843; the marine Trichoderma asperellum HG1 has a collection number of
Description
CGMCC No. 19276; and a mass ratio of the Bacillus subtilis microcapsules to the marine Trichoderma asperellum HG1 is 4:1 to 9:1. Preferably, the mass ratio of the Bacillus subtilis microcapsules to the marine Trichoderma asperellum HG1 is 9:1. Preferably, a preparation method of the Bacillus subtilis microcapsules includes the following steps: 1) adding Bacillus subtilis and silk fibroin into a Tris/HCl buffer solution; and fully and uniformly mixing the materials to obtain bacterium suspension; 2) adding dopamine hydrochloride into the bacterium suspension obtained in the step (1); aerobically stirring in a dark place at 25-30°C for a certain time; and initiating cross-linking polymerization between the dopamine hydrochloride and the silk fibroin to obtain microcapsules; 3) centrifugally separating the microcapsules; washing the microcapsules with the Tris/HCl buffer solution and deionized water; and performing vacuum drying at 60-70°C for 24 h, thereby obtaining the Bacillus subtilis microcapsules. Preferably, in the step 1), content of the Bacillus subtilis in the bacterium suspension is 10-101 cfu/nL. Preferably, in the step 1), a concentration of the silk fibroin in the bacterium suspension is 10-20 g/L; and a pH value of the Tris/HCl buffer solution (the bacterium suspension) is 8.0-9.0. Preferably, in the step 2), a concentration of the dopamine hydrochloride in the bacterium suspension is 1-2 g/L; and stirring time is 10-12 h. Preferably, a concentration of the marine Trichoderma asperellum HG1 is 108-101cfu/g. Preferably, auxiliary materials are added to prepare water dispersible granules. The present invention further provides an application of the anti-ultraviolet microbial agent in control of crop diseases.
Description
Preferably, the control of crop diseases refers to control of Botrytis cinerea and tobacco black shank. Through the above technical solutions, compared with the prior art, the present invention discloses and provides the anti-ultraviolet microbial agent and the application. Compared with the prior art, the present invention has advantages as follows: 1. In the present invention, the silk fibroin and dopamine materials with excellent compatibility are selected; the Bacillus subtilis bacterium agent is prepared by microencapsulation; reaction conditions are mild; bacterium damage is slight; ultraviolet resistance is improved; a survival rate is up to 82.22% within 30 min after ultraviolet irradiation; the survival rate is up to 31.51% within 60 min after ultraviolet irradiation; and the survival rate is up to 0.64% only within 60 min in a control group. 2. In the present invention, the Bacillus subtilis bacterium agent and the marine Trichoderma asperellum HG1 are further combined to prepare the biological bacterium agent; the antibacterial effect on the plant diseases is increased by utilizing the active materials secreted by the Bacillus subtilis and an occupied effect of the marine Trichoderma asperellum HG1; an antibacterial rate on the Botrytis cinerea bacteria and tobacco black shank bacteria is up to more than 70% respectively; the antibacterial effect of the biological bacterium agent is better than that of individual bio-control bacteria; and moreover, the antibacterial rate of a composite bacterium agent subjected to ultraviolet irradiation treatment on the two pathogenic bacteria is still maintained at 60% or more and is far higher than that in the control group without microencapsulation treatment. 3. In the present invention, the control effect of the composite bacterium agent, that is, the water dispersible granules, on the Botrytis cinerea is up to 76.27%; the control effect on the tobacco black shank is up to 70.06%; and the control effect is respectively better than that of a single bio-control bacterium agent.
Description
Description of Drawings
To more clearly describe the technical solution in the embodiments of the present invention or in the prior art, the drawings required to be used in the description of the embodiments or the prior art will be simply presented below. Apparently, the drawings in the following description are merely the embodiments of the present invention, and for those ordinary skilled in the art, other drawings can also be obtained according to the provided drawings without contributing creative labor. Fig. 1 is a microphoto of a number of colonies on a coated plate of Bacillus subtilis provided by the present invention and a Bacillus subtilis bacterium agent prepared in embodiment 1 after ultraviolet irradiation treatment and a change chart of a survival rate, wherein CK is non-embedded Bacillus subtilis; and SP is a Bacillus subtilis microcapsule bacterium agent.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and fully described below in combination with the drawings in the embodiments of the present invention. Apparently, the described embodiments are merely part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those ordinary skilled in the art without contributing creative labor will belong to the protection scope of the present invention. Embodiments of the present invention disclose an anti-ultraviolet microbial agent and an application. A microbial material Bacillus subtilis in the embodiments has a collection number of CGMCC No.2843 and is the same as a microbe collected in a patent CN201110093415.4; and marine Trichoderma asperellum HG1is the same as a
Description
microbe collected in a patent CN202010349559.0. The microbial material is collected by the inventor. Silk fibroin is purchased from Xi'an Quanao Biotechnology Co., Ltd. Embodiment 1 Preparation of Bacillus subtilis microcapsules: Bacillus subtilis having a collection number of CGMCC No.2843 and a sterilized silk fibroin solution were added into a Tris/HCl buffer solution (pH=8.5); the materials were fully and uniformly mixed to obtain bacterium suspension, wherein a concentration of the Bacillus subtilis was up to 10" cfu/mL, and a concentration of the silk fibroin was g/L; dopamine hydrochloride was added into the above bacterium suspension; a concentration of the dopamine hydrochloride was maintained as 2 g/L; the bacterium suspension was aerobically stirred in a dark place at 25-30°C at a rate of 400 r/min for 12 h; cross-linking polymerization between the dopamine hydrochloride and the silk fibroin was initiated to obtain microcapsules; the microcapsules were separated and collected; and the microcapsules were washed with the Tris/HCl buffer solution and deionized water, thereby obtaining the Bacillus subtilis microcapsules. The prepared Bacillus subtilis microcapsules and the non-embedded Bacillus subtilis having the collection number of CGMCC No.2843 were respectively tiled in a sterile culture dish; the culture dish was irradiated under an ultraviolet lamp (30 W) by 20 cm for 30 min and 60 min; the irradiated test item was diluted and cultured on a PCA culture medium; the number of colonies was observed; and the survival rate was calculated. It can be seen that, the ultraviolet resistance of the microencapsulated Bacillus subtilis is well improved; after 60 min from irradiation treatment, the survival rate is still up to 31.51%; and the survival rate of the Bacillus subtilis in the control group is close to zero. Embodiment 2
Description
Antibacterial effects of different combinations of Bacillus subtilis microcapsules and marine Trichodermaasperellum HG1 Botrytis cinerea bacteria and tobacco black shank bacteria are test bacteria (all provided by Qingdao Agricultural University). On premise of ensuring total number of living bacteria, combinations of Bacillus subtilis having a collection number of CGMCC No.2843, Bacillus subtilis microcapsules and marine Trichoderma asperellum HG1 are respectively prepared (specific combinations are shown as Table 1). Bacteria count of the marine Trichoderma asperellum HG1 is up to 10-10"cfu/g; different combinations are respectively provided with normal treatment and ultraviolet treatment 30 min; and antibacterial activity of the combinations on the above test bacteria is determined. Table 1 Composite bacterium formula of different combinations Combination Bacillus subtilis Bacillus subtilis Marine Trichoderma (parts) microcapsules (parts) asperellum HG1 (parts) 1 0 9 1 2 0 8 2 3 9 0 1 4 8 0 2
The control group 1 only contains the Bacillus subtilis having the collection number of CGMCC No.2843. The control group 2 only contains the marine Trichoderma asperellum HG1. The number of living bacteria in the control groups 1 and 2 is the same as the total number of living bacteria in the combinations 1-4. With the adoption of a plate confrontation method, the antibacterial effects of the composite bacteria in different combinations and the individual bio-control bacteria in the control groups on the Botrytis cinerea bacteria and tobacco black shank bacteria are determined. Specifically, pathogenic bacteria for test were activated on a PDA plate; a bacterium cake was made in a colony edge area by a puncher after the colonies grew all over the plate; then the bacterium cake was transferred to the center of the PDA plate; then the composite bacteria in different combinations and the
Description
bio-control bacteria in the control groups subjected to normal treatment or ultraviolet treatment were inoculated in a place that had the same distance away from the bacterium cake of the pathogenic bacteria; four test bacteria were inoculated in each dish; meanwhile, blank control was set, i.e., a pathogenic bacteria PDA plate without any inoculated bio-control strain. Three repeats were set in each treatment. After the pathogenic bacteria were cultured under culture conditions of the pathogenic bacteria for a certain time, colony growth of the pathogenic bacteria was determined; and an antibacterial rate was calculated. A calculation formula is as follows: The antibacterial rate (%)=(a colony diameter of the blank control-a colony diameter of confrontation culture)/the colony diameter of the blank controlx100%. Specific results are shown as Table 2. Table 2 Inhibition effects of different composite bacterium combinations on the pathogenic bacteria Antibacterial rate(%)
Combination No ultraviolet irradiation treatment Ultraviolet irradiation treatment of 30min Botrytis cinerea Tobacco black shank Botrytis cinerea Tobacco black bacteria bacteria bacteria shank bacteria Combination 1 83.24 81.64 67.49 65.15 Combination 2 81.38 79.45 64.57 62.39 Combination 3 79.87 77.16 46.19 48.21 Combination 4 78.86 72.27 44.62 45.38 Control group 1 68.34 61.34 Control group 2 65.21 55.73
The results in Table 2 show that, the antibacterial effects of the composite bacteria in different combinations on the Botrytis cinerea bacteria are better than those of individual bio-control bacteria; an inhibition rate is up to 75% or more; the combination of the Bacillus subtilis subjected to microencapsulation treatment and the marine Trichoderma asperellum HG1 and the combination that is not subjected to microencapsulation treatment have non-significant inhibition rate differences on the Botrytis cinerea bacteria, which indicates that the microencapsulation treatment does not affect the antibacterial activity of the bio-control bacteria. When a mass
Description
ratio of the Bacillus subtilis bacterium agent subjected to microencapsulation treatment to the Trichoderma is 9:1, the antibacterial effect is the best; and the antibacterial rate is up to 83.24%. The antibacterial rates of all the combinations subjected to ultraviolet treatment are all decreased, but the antibacterial rate of the combination of the non-microencapsulated Bacillus subtilis and the marine Trichoderma asperellum HG1 is greatly decreased; the antibacterial rates are all % or less; and the antibacterial rate of the microencapsulated Bacillus subtilis and the marine Trichodermaasperellum HG1 is still maintained at 60% or more. Inhibition effects of different combinations on the tobacco black shank bacteria are also better than those of the individual bio-control bacteria. When a mass ratio of the Bacillus subtilis bacterium agent subjected to microencapsulation treatment to the marine Trichoderma asperellum HG1 is 9:1, the antibacterial effect is the best; and the antibacterial rate is up to 81.64%. The inhibition effects of all the combinations subjected to ultraviolet irradiation treatment are all decreased, but the antibacterial rates of the combinations of the microencapsulated Bacillus subtilis and the marine Trichoderma asperellum HG1 are still maintained at 60% or more; excellent inhibition effects are achieved; the antibacterial rate of the combination 1 is the highest and is up to 65.15%; and the antibacterial activity of the non-microencapsulated Bacillus subtilis and the Trichoderma is decreased to be 50% or less. Embodiment 3 Preparation of water dispersible granules from Bacillus subtilis having a collection number of CGMCC No.2843 and marine Trichoderma asperellum HG1 The microbial composite bacteria in the present invention may be further prepared into water dispersible granules suitable for agricultural use according to the above compound optimal formula combination 1 and control by using an existing method. The percent in the formula is a percentage by mass.
Description
Table 3 shows preparation embodiments and control of anti-ultraviolet combinations of the Bacillus subtilis bacterium agent and marine Trichoderma asperellum HG1. Table 3 Formulas of water dispersible granules of different bacterium agents Marine Bacillus subtilis Trichoderma Group microcapsules asperellum Formula Dosage form
HG1
5% of sodium carboxymethyl starch, 5% of saponin Water Embodiment 45% 5% powder, 0.7% of sodium carboxymethylcellulose, 1% of dispersible 3 sodium alginate and the balance of kaolin, totaling 100% granules
Marine Water dispersible Control Trichoderma granules Bacillussubtilis Formula group asperellum HG1
5% of sodium carboxymethyl starch, 5% of saponin Water Control dispersible 45% 5% powder, 0.7% of sodium carboxymethylcellulose, 1% of granules sodium alginate and the balance of kaolin, totaling 100%
Control 5% of sodium carboxymethyl starch, 5% of saponin Water 50% 0 powder, 0.7% of sodium carboxymethylcellulose, 1% of dispersible group 4 sodium alginate and the balance of kaolin, totaling 100% granules
Control 5% of sodium carboxymethyl starch, 5% of saponin Water 0 50% powder, 0.7% of sodium carboxymethylcellulose, 1% of dispersible group 5 sodium alginate and the balance of kaolin, totaling 100% granules
Notes: the total living bacteria counts in the used Bacillus subtilis, Bacillus subtilis microcapsules and marine Trichoderma asperellum HG1 are respectively 101°CFU/g. Control effects on Botrytis cinerea and tobacco black shank The above water dispersible granules were respectively applied to conducting pharmacological experiments for controlling the Botrytis cinerea and tobacco black shank in fields; drug delivery was performed once every 7 days in a spraying mode; drug delivery was performed twice totally; and results were inspected at an interval of 7 days for the last time. The results are shown as Tables 4 and 5. It can be seen from Table 4 that, the control effects of the composite water dispersible granules of the microencapsulated Bacillus subtilis and marine Trichoderma asperellum HG1on the Botrytis cinerea are better than the effects of
Description
combinations of non-microencapsulated Bacillus subtilis and marine Trichoderma asperellum HG1 and individual bio-control bacteria; and the highest control effect is up to 76.27%. The same results may be seen from Table 5. Table 4 Control effects of the water dispersible granules of different composite bacterium agents on the Botrytis cinerea (21 d) Treatment Dilution ratio Disease index Control effect(%)
CK - (88.42 ±1.98) a
Embodiment 3 500 (20.96 ±0.89)d 76.27
Control group 3 500 (27.68 ±1.34)c 68.69
Control group 4 500 (32.21 ±0.85)b 63.57
Control group 5 500 (33.15 ±1.21)b 62.50
Notes: different small letters behind the data of the same line represent significant level differences of P < 0.05. Table 5 Control effects of the water dispersible granules of different composite bacterium agents on the tobacco black shank (21 d)
Treatment Dilution ratio Disease index Control effect
CK - (84.65 ±2.31)a
Embodiment 3 400 (25.34 ±0.89)c 70.06
Control group 3 400 (31.84 ±0.91)c 62.39
Control group 4 400 (36.82 ±1.35)b 56.50
Control group 5 400 (34.65 ±1.13)b 59.06
Notes: different small letters behind the data of the same line represent significant level differences of P < 0.05. In the present invention, the material silk fibroin that has excellent compatibility with microbial bacteria is embedded with the Bacillus subtilis; and the Bacillus subtilis microcapsules are prepared by utilizing polymerization of the
Description
dopamine, so that ultraviolet stability is enhanced. Then, the optimum ratio is further determined by an optimized combination of the Bacillus subtilis and the marine Trichoderma asperellum HG1; and the environment-friendly preparations, that is, the water dispersible granules, are prepared. The field trials show that, the water dispersible granules of the combination of the anti-ultraviolet Bacillus subtilis and the marine Trichoderma asperellum HG1 have the control effects better than those of other control agents. Each embodiment in the description is described in a progressive way. The difference of each embodiment from each other is the focus of explanation. The same and similar parts among all of the embodiments can be referred to each other. The above description of the disclosed embodiments enables those skilled in the art to realize or use the present invention. Many modifications to these embodiments will be apparent to those skilled in the art. The general principle defined herein can be realized in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principle and novel features disclosed herein.

Claims (10)

Claims
1. An anti-ultraviolet microbial agent, comprising Bacillus subtilis microcapsules and marine Trichoderma asperellum HG1, wherein the Bacillus subtilis microcapsules comprises Bacillus subtilis having a collection number of CGMCC No. 2843; the marine Trichoderma asperellum HG1 has a collection number of CGMCC No. 19276; and a mass ratio of the Bacillus subtilis microcapsules to the marine Trichoderma asperellum HG1 is 4:1 to 9:1.
2. The anti-ultraviolet microbial agent according to claim 1, wherein the mass ratio of the Bacillus subtilis microcapsules to the marine Trichoderma asperellum HG1 is 9:1.
3. The anti-ultraviolet microbial agent according to claim 1, wherein a preparation method of the Bacillus subtilis microcapsules comprises the following steps: 1) adding Bacillus subtilis and silk fibroin into a Tris/HCl buffer solution; and fully and uniformly mixing the materials to obtain bacterium suspension; 2) adding dopamine hydrochloride into the bacterium suspension obtained in the step (1); aerobically stirring in a dark place at 25-30°C for a certain time; and initiating cross-linking polymerization between the dopamine hydrochloride and the silk fibroin to obtain microcapsules; 3) centrifugally separating the microcapsules; washing the microcapsules with the Tris/HCl buffer solution and deionized water; and performing vacuum drying at 60-70°C for 24 h, thereby obtaining the Bacillus subtilis microcapsules.
4. The anti-ultraviolet microbial agent according to claim 3, wherein in the step 1), content of the Bacillus subtilis in the bacterium suspension is 10-10" cfu/mL.
5. The anti-ultraviolet microbial agent according to claim 3, wherein in the step 1), a concentration of the silk fibroin in the bacterium suspension is 10-20 g/L; and a pH value of the Tris/HC buffer solution (the bacterium suspension) is 8.0-9.0.
Claims
6. The anti-ultraviolet microbial agent according to claim 3, wherein in the step 2), a concentration of the dopamine hydrochloride in the bacterium suspension is 1-2 g/L; and stirring time is 10-12 h.
7. The anti-ultraviolet microbial agent according to claim 1, wherein a concentration of the marine Trichoderma asperellum HG1 is 10'-10cefu/g.
8. The anti-ultraviolet microbial agent according to any one of claims 1-7, wherein auxiliary materials are added to prepare water dispersible granules.
9. An application of the anti-ultraviolet microbial agent of any one of claims 1-8 in control of crop diseases.
10. The application according to claim 9, wherein the control of crop diseases refers to control of Botrytis cinerea and tobacco black shank.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114258923A (en) * 2021-12-30 2022-04-01 南京工业大学 Application of trichoderma asperellum microbial agent in prevention and treatment of crop diseases
CN115007076A (en) * 2022-06-17 2022-09-06 重庆大学 Ultraviolet light and moisture dual-triggered polyurethane microcapsule, and preparation method and application thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114258923A (en) * 2021-12-30 2022-04-01 南京工业大学 Application of trichoderma asperellum microbial agent in prevention and treatment of crop diseases
CN114258923B (en) * 2021-12-30 2022-10-14 南京工业大学 Application of trichoderma asperellum microbial agent in prevention and treatment of crop diseases
CN115007076A (en) * 2022-06-17 2022-09-06 重庆大学 Ultraviolet light and moisture dual-triggered polyurethane microcapsule, and preparation method and application thereof

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