Chaetomium globosum NtCg1 and application of chaetomium globosum NtCg1 in embedding biocontrol bacterium microorganism microcapsule

Abstract

The invention belongs to the technical field of microbial agents, and relates to chaetomium globosum NtCg1 and application thereof in embedding biocontrol bacterium microbial microcapsules. The chaetomium globosum NtCg1 has an obvious antagonistic effect on various root rot pathogenic bacteria, the control effect on mung bean root rot reaches 62.16%, the control effect on tobacco root rot reaches 59.62%, and the root rot phenomenon caused by infection of the root rot is effectively restrained. According to the invention, the chaetomium globosum NtCg1 and the acremonium corioli NtSi1 are adopted to prepare the sodium alginate embedded biocontrol bacterium microorganism microcapsule, the control stability is improved through the synergistic effect of strains, and a better root rot control effect is achieved.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and relates to a type of Chaetomium globosum NtCg1 and its application in encapsulating biocontrol microbial microcapsules. Background Technology

[0002] The main pathogens of root rot include Fusarium, Pythium, and Phytophthora. These pathogens survive in the soil and are spread through rainwater or irrigation water. They infect the plant root system, causing the vascular tissue to rot, which in turn leads to wilting and death of the plant. In the early stages of the disease, only a few lateral roots and fibrous roots are infected, gradually spreading to the main root. This weakens the plant's ability to absorb water and nutrients, and the above-ground parts show symptoms such as yellowing and wilting due to insufficient nutrient supply. If not controlled in time, root rot can lead to reduced crop yield or even total crop failure.

[0003] Traditional control methods include selecting disease-resistant varieties, agricultural measures (such as crop rotation, raised bed cultivation, and application of phosphorus and potassium fertilizers), and chemical control. However, chemical control easily leads to drug resistance in pathogens and may pollute the soil and agricultural products. For example, long-term use of chemical agents such as methyl thiophanate and hymexazol has caused problems with environmental residues and pathogen resistance. In addition, traditional methods are unable to fundamentally restore the imbalance of soil microecology, leading to recurrent root rot. In recent years, microbial control technology has become a research hotspot in the field of root rot control due to its advantages such as environmental friendliness, sustainability, and the ability to induce plant resistance.

[0004] In recent years, synthetic microbial communities have gained increasing attention in the field of synthetic biology research. They possess characteristics and functions not found in individual populations, enabling the entire community to perform complex functions that a single strain cannot accomplish. For example, in agricultural production, the overuse of veterinary drugs and pesticides results in agricultural wastewater containing large amounts of antibiotics and organophosphate waste, causing significant environmental harm. Researchers can construct microbial communities (MSBCs) using two species to degrade antibiotics and organophosphates respectively, achieving effective degradation of harmful substances in wastewater. However, most attempts to co-cultivate these artificial microbial communities fail due to the winner-takes-all dynamic in nutrient competition. Therefore, there is an urgent need to provide a compound microbial agent that can prevent and control plant root rot and coexist in the same space. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a *Chaetomium globosum* strain and its applications. The *Chaetomium globosum* NtCg1 strain provided by this invention exhibits significant antagonistic effects against various root rot pathogens and can be used to prepare root rot control agents. Furthermore, this invention provides a method for preparing sodium alginate-encapsulated biocontrol microcapsules, using two biocontrol strains, *Acer synoviae* and *Chaetomium globosum*, which demonstrate excellent biocontrol effects against various pathogens, such as mung bean root rot and tobacco root rot.

[0006] This invention is achieved through the following technical solution: A type of Chaetomium, characterized in that the Chaetomium is Chaetomium globosum ( Chaetomium globosum The NtCg1 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 20, 2025, with accession number CGMCC NO.41759.

[0007] The present invention also provides the application of the above-mentioned Chaetomium globosum NtCg1 in the preparation of a fungal agent for the prevention and control of root rot.

[0008] Furthermore, the root rot is mung bean root rot or tobacco root rot.

[0009] Furthermore, the pathogen causing the root rot is *Fusarium oxysporum* (…). Fusarium oxysporum Fusarium solani () Fusarium solani Fusarium ( ) Fusarium falciforme Alternaria ( ) Alternaria sp ) and Curvularia ( Curvularia spicifera Any one or more of the following.

[0010] Furthermore, the root rot control agent is used in one or more of the following applications: Used to inhibit the growth of root rot pathogens; Used to promote plant growth after root rot infection; Used to prevent and control root rot caused by plant root rot pathogens; It is used to increase the content of antioxidant enzymes in plants.

[0011] Furthermore, the antioxidant enzyme includes one or more of peroxidase, polyphenol oxidase, and catalase.

[0012] This invention also provides a method for preparing sodium alginate-encapsulated biocontrol microbial microcapsules, which involves combining *Chaetoceros* NtCg1 with *Acer truncatum* (…). Sarocladium implicatum The bacterial culture of NtSi1 was mixed with activated carbon and then encapsulated with sodium alginate solution to obtain sodium alginate-encapsulated biocontrol microbial microcapsules. The *Acer synoviae* NtSi1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 20, 2025, with accession number CGMCC NO.41760.

[0013] Furthermore, after embedding with sodium alginate solution, the solution was slowly added dropwise to calcium chloride solution using a syringe and cured at 2-6°C for 1-3 hours.

[0014] The present invention also provides a sodium alginate-encapsulated biocontrol microbial microcapsule, which is prepared by the above-described preparation method.

[0015] The present invention also provides a method for preventing and controlling plant root rot by applying the above-mentioned Chaetomium globosum or the above-mentioned sodium alginate-encapsulated biocontrol microbial microcapsules to the plant root growth environment.

[0016] Beneficial technical effects of the present invention: The *Chaetomium globosum* NtCg1 provided by this invention has significant antagonistic effects against various root rot pathogens, achieving a control efficacy of 62.16% against mung bean root rot and 59.62% against tobacco root rot, effectively curbing root rot caused by root rot infection.

[0017] This invention also uses *Chaetomium globosum* NtCg1 and *Acer truncatum* NtSi1 to prepare sodium alginate-encapsulated biocontrol microcapsules. Through the synergistic effect of the strains, the control stability is improved, resulting in better root rot control. It has broad-spectrum resistance, is environmentally friendly, and is easy to industrialize. Attached Figure Description

[0018] Figure 1A Phylogenetic tree of Chaetomium NtCg1; Figure 1B Phylogenetic tree of *Acer synoviae* NtSi1; Figure 2 The plate control efficacy of two biocontrol fungi, Chaetomium globulus NtCg1 and Acerobiota intercalatum NtSi1, for root rot; Figure 3 Phenotypic images of mung bean plants, leaves, and roots after treatment with biocontrol bacteria; where A is a mung bean plant after treatment with biocontrol bacteria solution; B is a mung bean leaf after treatment with biocontrol bacteria solution; and C is a phenotypic image of mung bean roots after treatment with biocontrol bacteria solution. Figure 4 Phenotypic images of tobacco plants, leaves, and roots after treatment with biocontrol bacteria, biocontrol bacterial groups, and microcapsule biocontrol bacterial groups; A is a morphological image of tobacco plants; B is an image of tobacco leaves; C is an image of tobacco roots; D is an image of tobacco root sections. Figure 5A The plant height of mung beans after treatment with biocontrol bacteria, biocontrol bacterial groups, and microencapsulated biocontrol bacterial groups; Figure 5B Fresh weight of the above-ground parts of mung beans after treatment with biocontrol bacteria, biocontrol bacterial groups, and microencapsulated biocontrol bacterial groups; Figure 5C The root length of mung beans after treatment with biocontrol bacteria, biocontrol bacterial groups, and microencapsulated biocontrol bacterial groups; Figure 5D Fresh root weight of mung beans after treatment with biocontrol bacteria, biocontrol bacterial groups, and microencapsulated biocontrol bacterial groups; Figure 5E The chlorophyll content of mung beans after treatment with biocontrol bacteria, biocontrol bacterial groups, and microencapsulated biocontrol bacterial groups; Figure 6A The plant height of tobacco plants after treatment with biocontrol bacteria, biocontrol bacterial groups, and microcapsule biocontrol bacterial groups; Figure 6B Fresh weight of the aboveground parts of tobacco after treatment with biocontrol bacteria, biocontrol bacteria groups, and microcapsule biocontrol bacteria groups; Figure 6C The root length of tobacco after treatment with biocontrol bacteria, biocontrol bacterial groups, and microcapsule biocontrol bacterial groups; Figure 6D Fresh root weight of tobacco after treatment with biocontrol bacteria, biocontrol bacterial groups and microcapsule biocontrol bacterial groups; Figure 6E The chlorophyll content of tobacco treated with biocontrol bacteria, biocontrol bacterial groups, and microencapsulated biocontrol bacterial groups; Figure 7A Changes in peroxidase (POD) activity after treatment with biocontrol bacteria, biocontrol bacterial communities, and microencapsulated biocontrol bacterial communities; Figure 7B The changes in polyphenol oxidase (PPO) activity after treatment with biocontrol bacteria, biocontrol bacterial groups, and microencapsulated biocontrol bacterial groups; Figure 7C Changes in catalase (CAT) activity after treatment with biocontrol bacteria, biocontrol bacterial communities, and microcapsule biocontrol bacterial communities. Detailed Implementation

[0019] To better understand the present invention, it will be further described below with reference to the accompanying drawings and embodiments, but this is not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0020] Example 1: A type of Chaetomium globosum, wherein the Chaetomium globosum strain NtCg1 is deposited at the China General Microbiological Culture Collection Center on January 20, 2025, with accession number CGMCC NO.41759.

[0021] Example 2: Application of Chaetomium globosum NtCg1 described in Example 1 in the preparation of a fungal agent for the prevention and control of root rot.

[0022] In this embodiment, the root rot disease is mung bean root rot or tobacco root rot.

[0023] In this embodiment, the pathogen causing root rot is any one or more of Fusarium oxysporum, Fusarium solani, Fusarium spp., Alternaria alternata, and Curvularia spp.

[0024] In this embodiment, the root rot control agent is used in one or more of the following applications: Used to inhibit the growth of root rot pathogens; Used to promote plant growth after root rot infection; Used to prevent and control root rot caused by plant root rot pathogens; It is used to increase the content of antioxidant enzymes in plants.

[0025] In this embodiment, the antioxidant enzyme includes one or more of peroxidase, polyphenol oxidase, and catalase.

[0026] Example 3: A method for preparing sodium alginate-encapsulated biocontrol microbial microcapsules, wherein the bacterial solutions of Chaetomium globosum NtCg1 and Acer negundo NtSi1 described in Example 1 are respectively mixed with activated carbon, and then encapsulated with sodium alginate solution to obtain sodium alginate-encapsulated biocontrol microbial microcapsules. The *Acer synoviae* NtSi1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 20, 2025, with accession number CGMCC NO.41760.

[0027] In this embodiment, after embedding with sodium alginate solution, it was slowly added to calcium chloride solution using a syringe and cured at 2~6℃ for 1~3 h.

[0028] Example 4: A sodium alginate-encapsulated biocontrol microcapsule was prepared using the preparation method described in Example 3.

[0029] Example 5: A method for preventing and controlling plant root rot, comprising applying *Chaetomium globosum* as described in Example 1 or sodium alginate-encapsulated biocontrol microcapsules as described in Example 4 to the plant root growth environment. The screening and sequencing process of biocontrol strains with effects against multiple plant pathogenic fungi in this invention is as follows: The *Chaetomium globosum* described in this invention ( Chaetomium globosum NtCg1 was deposited on January 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.41759, located in Beijing, China.

[0030] The cross-branched apical mold described in this invention ( Sarocladium implicatum NtSi1 was deposited on January 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.41760, located in Beijing, China.

[0031] Sample collection: Tobacco rhizosphere soil was collected from diseased tobacco fields in Zhengzhou, Henan Province, and then packaged, labeled, and brought back to the laboratory for separation.

[0032] Isolation of Chaetomium globosum and Atoplastrum crossbredii: The dilution-spreading method and temperature screening method were used. Collected soil was air-dried, ground finely, and 10 g was weighed into an Erlenmeyer flask, with 90 mL of sterile water added. The above solution was prepared into dilutions of different concentrations. These dilutions, from low to high concentration, were sequentially and evenly spread onto dried LB and PDA plates and incubated at 28 ℃ for 2 days. Colonies with different morphological and color characteristics were selected for further purification on PDA plates. The purified strains were preserved in glycerol. Single hyphae were picked and cultured on PDA agar plates for three consecutive purification cycles to obtain pure strains. The purified strains were then preserved as slant agar for later use.

[0033] Molecular identification of *Chaetoceros* and *Atoporphyra*: Genomic DNA of the two isolated strains was extracted using the CTAB method. The ITS regions of eukaryotic organisms were amplified using Vazyme's 2×Taq Master Mix enzyme and primer pair ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') / ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The sequences were then sent to Qingke Biotechnology Co., Ltd. for sequencing to obtain nucleotide sequence information. The obtained sequences were submitted to the GenBank sequence database for analysis and alignment. Sequences with high homology were selected, and a phylogenetic tree was constructed using molecular evolutionary genetic analysis software (Mega 5.0). Therefore, it was confirmed that the strain isolated from the soil was indeed *Atoporphyra*. Figure 1A ), Chaetomium coccidioides ( Figure 1B ).

[0034] The following experiments demonstrate the effectiveness of the biocontrol strains and agents of the present invention, which are capable of controlling various plant pathogenic fungi, in controlling tobacco root rot.

[0035] Plate antagonism detection of Chaetomium globosum and Acer synergium: Using root rot pathogens as targets, the antagonistic activity of isolated rhizosphere antagonistic bacteria was detected. The specific method was as follows: A 6 mm diameter root rot pathogen mycelial disc was placed in the center of a 90 mm φ PDA plate. Biocontrol bacteria were inoculated around the pathogen, which had been cultured for 3 days, at a distance of 30 mm. A control without biocontrol bacteria was used. The plates were incubated at 28℃, and the degree of antagonism was determined based on the area of ​​inhibition. The experiment was repeated three times. Plate confrontation tests verified that *Chaetoceros globosa* and *Acer negundo* had antagonistic effects against four soil-borne pathogenic fungi. Figure 2 As shown. By Figure 2 The results showed that strains NtCg1 and NtSi1 both had significant antagonistic effects against the pathogen.

[0036] Experiment on the control of tobacco root rot by two biocontrol bacteria encapsulated in sodium alginate: (1) Preparation of pathogenic fungal spore suspension: Five root rot pathogenic fungi were cultured on PDA plates (200 g potato, 20 g sucrose, 20 g agar, natural pH, 1000 mL distilled water) for 7 days. The five root rot pathogenic fungi included Fusarium oxysporum (Fo), Fusarium falcatum (Ff), Fusarium solani (Fs), Alternaria alternata (As), and Curvularia spp. (Cs). The pathogenic fungi cultured on the PDA plates were gently scraped off with a pipette tip, rinsed with 10 mL of sterile water, centrifuged at 8000 rpm / min for 5 min to remove the supernatant precipitate, and resuspended in sterile water. The conidia were counted using a hemocytometer, and the concentration was adjusted to 5 × 10⁻⁶ with sterile water. 7 Approximately [number] spores / ml. Mix the conidial suspensions of five root rot pathogenic fungi.

[0037] (2) Preparation of biocontrol bacteria solution: After culturing the biocontrol bacteria in PDA (200 g potato, 20 g sucrose, natural pH, 1000 mL distilled water) liquid medium for 3 days, centrifuge at 30℃, 8000 rpm / min for 5 min to remove the supernatant and resuspend the bacterial solution in sterile water.

[0038] (3) Sodium alginate encapsulation of two biocontrol bacteria: The biocontrol bacteria cultured in PDA liquid medium (20 mL of biocontrol bacteria solution per pot, 100 mL for each treatment) were centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and sterile water was added to repeat the above steps 3 times. The spore precipitates of each bacteria were added to the same volume of 15 g / L sterile activated carbon and shaken at 200 rpm / min for 3 h for adsorption. The mixture of activated carbon and bacteria was added to the same volume of 15 g / L sterile sodium alginate solution for encapsulation. After mixing, it was slowly added dropwise to 2% sterile calcium chloride solution using a syringe and solidified at 4℃ for 2 h. The encapsulated microspheres were removed, the surface of the microspheres was rinsed with sterile water to remove ionic solutions, and stored at 4℃ for later use.

[0039] (4) Inoculation of mung bean seedlings with pathogenic fungal suspension and biocontrol agent: Seven days after mung bean seedlings germinated, a substrate of vermiculite:nutrient soil = 3:1 was added, and five treatments were set up: control group (CK) without biocontrol agent and pathogenic fungal suspension; 20 mL pathogenic fungal suspension (P); 20 mL pathogenic fungal suspension and 20 mL Chaetomium globosum biocontrol agent (P+NtCg1); 20 mL pathogenic fungal suspension and 20 mL Acer synergum biocontrol agent (P+NtSi1); 20 mL pathogenic fungal suspension and 20 mL Acer globosum and Acer synergum biocontrol agent (P+C). Each treatment was tested in 5 independent pots. Disease incidence was observed 45 days after inoculation, and the disease control effect was statistically analyzed.

[0040] (5) Inoculation of tobacco seedlings with pathogenic fungal suspension and biocontrol agents: Twenty days after tobacco seedling emergence, a substrate of vermiculite:nutrient soil = 3:1 was added, and six treatments were set up: control group (CK) without biocontrol agent and pathogenic fungal suspension; 20 mL pathogenic fungal suspension (P); 20 mL pathogenic fungal suspension and 20 mL Chaetomium globosum biocontrol agent (P+NtCg1); 20 mL pathogenic fungal suspension and 20 mL Acer synergum biocontrol agent (P+NtSi1); 20 mL pathogenic fungal suspension and 20 mL Chaetomium globosum and Acer synergum biocontrol agent (P+C); 20 mL pathogenic fungal suspension and 20 g Chaetomium globosum and Acer synergum microcapsule-encapsulated bacterial group (P+M). Each treatment was tested in 5 independent pots. Disease incidence was observed 15, 30, and 45 days after inoculation, and the disease control effect was statistically analyzed.

[0041] The results of the biocontrol efficacy test on mung bean potted plants showed that the control efficacy of inoculation with NtCg1 was 62.16%, that of inoculation with NtSi1 was 66.21%, and that of inoculation with both NtCg1 and NtSi1 was 71.62%. The incidence rate and disease index also decreased accordingly (Table 1). Without biocontrol, the roots turned black and rotted. Figure 3 (C). After treatment with single biocontrol bacteria and microbial communities, root rot was effectively controlled. Figure 3 (C). Plant samples were taken and relevant indicators were measured. Figure 5A Plant height, Figure 5B Fresh weight of aboveground parts Figure 5C Root length Figure 5D Fresh root weight and Figure 5E Chlorophyll analysis revealed that different biocontrol bacteria treatments all showed significant biocontrol effects after 45 days, with mixed inoculation of biocontrol bacteria yielding the best results.

[0042] The results of the tobacco pot control efficacy test showed that the control efficacy of inoculation with NtCg1 was 59.62%, that of inoculation with NtSi1 was 63.46%, that of inoculation with both NtCg1 and NtSi1 was 78.85%, and that of inoculation with embedded NtCg1 and NtSi1 was 86.54%. The incidence rate and disease index also decreased accordingly (Table 2). Uninoculated plants showed root drop, blackening, and root rot, as seen in the section images (…). Figure 4 As shown in Figure D, root cells are damaged. Treatment with a single biocontrol bacterium, a microbial community, and microcapsules effectively curbed root rot. Figure 4 (As shown in C). Plant samples were taken and relevant indicators were measured. Figure 6A Plant height, Figure 6B Fresh weight of aboveground parts Figure 6C Root length Figure 6D Fresh root weight and Figure 6EChlorophyll levels showed no significant differences among the treatment groups at 15 days. At 45 days, all biocontrol treatments exhibited significant biocontrol effects, with the embedded formulation showing the best effect. Single-inoculation and mixed-inoculation biocontrol strains were less effective than the embedded formulation.

[0043] The tobacco-related illness grading standards are as follows: Grade 0: The affected area is less than or equal to 1%; Grade 1: Infected area 1%-25%; Grade 2: Infected area 26%-50%; Grade 3: Infected area 51%-75%; Level 4: Infected area 76%-89%; Level 5: The affected area is greater than or equal to 90%.

[0044] The incidence rate, disease index, and biocontrol effect are calculated using the following formula.

[0045] Incidence rate (%) = Number of infected plants / Total number of plants × 100 Disease index (%) = ∑(Disease level × Number of plants at that level) / (Highest disease level × Total number of plants) × 100.

[0046] Biological control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0047] Table 1. Biocontrol effect of biocontrol bacteria on mung bean root rot ( p <0.05) Table 2. Biocontrol effects of biocontrol bacteria on tobacco root rot ( p <0.05) The biocontrol effects of biocontrol bacteria include inducing plants to produce various defensive enzymes, including antioxidant enzymes such as peroxidase (POD), polyphenol oxidase (POO), and catalase (CAT). A preliminary investigation was conducted on the induction of POD, PPO, and CAT enzyme activities by single-strain treatments NtCg1 and NtSi1, mixed-strain treatment C, and encapsulated-strain treatment M. All treatments significantly increased the activities of POD, PPO, and CAT enzymes, with the encapsulated biocontrol bacteria showing a greater improvement in disease resistance than the traditional biocontrol bacteria. Figures 7A-7C This indicates that biocontrol bacteria enhance tobacco disease resistance by inducing the activation of antioxidant enzymes in tobacco.

[0048] 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, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Chaetomium globosum, characterized in that, The *Chaetomium globulae* strain is *Chaetomium globulae* NtCg1, deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 20, 2025, with accession number CGMCC NO. 41759.

2. The application of the *Chaetomium globosum* NtCg1 as described in claim 1 in the preparation of a fungal agent for the prevention and control of root rot.

3. The application according to claim 2, characterized in that, The root rot mentioned is either mung bean root rot or tobacco root rot.

4. The application according to claim 2, characterized in that, The pathogen causing root rot is any one or more of the following: Fusarium oxysporum, Fusarium solani, Fusarium spp., Alternaria alternata, and Curvularia spp.

5. The application according to claim 2, characterized in that, The root rot control agent is used in one or more of the following applications: Used to inhibit the growth of root rot pathogens; Used to promote plant growth after root rot infection; Used to prevent and control root rot caused by plant root rot pathogens; It is used to increase the content of antioxidant enzymes in plants.

6. The application according to claim 5, characterized in that, The antioxidant enzymes include one or more of peroxidase, polyphenol oxidase, and catalase.

7. A method for preparing sodium alginate-encapsulated biocontrol microbial microcapsules, characterized in that, The bacterial cultures of Chaetomium globosum NtCg1 and Acer negundo NtSi1 as described in claim 1 were respectively mixed with activated carbon, and then encapsulated with sodium alginate solution to obtain sodium alginate-encapsulated biocontrol microcapsules. The *Acer synoviae* NtSi1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 20, 2025, with accession number CGMCC NO.41760.

8. The method for preparing sodium alginate-encapsulated biocontrol microbial microcapsules according to claim 7, characterized in that, After embedding with sodium alginate solution, the solution was slowly added dropwise to calcium chloride solution using a syringe and cured at 2-6℃ for 1-3 hours.

9. A sodium alginate-encapsulated biocontrol microcapsule, characterized in that, It is prepared using the preparation method described in claim 7 or 8.

10. A method for preventing and controlling plant root rot, characterized in that, Apply the *Chaetoceros globosum* of claim 1 or the sodium alginate-encapsulated biocontrol microcapsules of claim 9 to the plant root growth environment.

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