Streptomyces sp. strain and use thereof for producing 6-oxo-6,7-dihydro-1h- pyrazolo[3,4-d]pyrimidine-3,5-dicarboxylic acid

Abstract

The application discloses a strain of stenotrophomonas maltophilia capable of degrading pyridaben and an application thereof, wherein the stenotrophomonas maltophilia strain is preserved in the China General Microbiological Culture Collection Center on December 24, 2024, and the preservation number is CGMCC No. 33180. The application also specifically discloses an application of the stenotrophomonas maltophilia strain in degrading pyridaben. The growth characteristics and degradation characteristics of the stenotrophomonas maltophilia strain PEB1 are preliminarily studied, the screened stenotrophomonas maltophilia has a good degradation effect on pyridaben, and provides a strain resource for bioremediation of farmland contaminated by pyridaben.

Description

Technical Field

[0001] This invention belongs to the field of microbial degradation technology of pyrimethanil in soil, specifically involving a strain of Stenotrophomonas maltophilia that can degrade pyrimethanil and its application. Background Technology

[0002] Pyrimethanil, a typical representative of succinate dehydrogenase inhibitors, has been registered and commercially used in 35 major agricultural countries worldwide (covering Asia, North America, and the European Union). Its target is highly consistent with SDHI compounds; it specifically binds to the succinate dehydrogenase complex in the inner mitochondrial membrane of pathogens, interfering with the electron transport chain, thus hindering the tricarboxylic acid cycle, impeding energy metabolism, and ultimately leading to pathogen death. This compound possesses broad-spectrum and highly effective fungicidal properties and is widely used to control diseases in fruit trees, vegetables, and turfgrass, such as rust, gray mold, scab, and leaf mold.

[0003] Pyrimethanil has attracted widespread attention due to its high persistence in soil environments, with existing data showing a half-life of 65-356 days in different soil types. This persistent residue not only significantly increases its risk of bioaccumulation through the food chain but also poses a potential threat to ecosystem stability and human health. Studies on its ecotoxicological effects have shown that zebrafish exposed to pyrimethanil during the critical embryonic to larval developmental stages can experience dose-dependent acute toxicity, manifested as decreased hatching rates, body segment malformations, and developmental abnormalities such as impaired locomotor function. Further mechanistic studies reveal that this compound can interfere with the activity of key enzymes in the melanin synthesis pathway, leading to abnormal differentiation of larval pigment cells, specifically manifested as disordered melanin deposition patterns and abnormal synthesis pathways. Given the cumulative effect of pyrimethanil's persistence in environmental media and its significant developmental toxicity, developing efficient and environmentally friendly pollution remediation technologies has become an urgent research direction in the fields of environmental science and ecotoxicology.

[0004] Compared to traditional physicochemical remediation technologies, which suffer from high energy consumption and secondary pollution, microbial degradation offers advantages such as high degradation efficiency and no secondary pollution, making it a recognized environmentally friendly pesticide degradation measure. Currently, there are few reports on the microbial degradation of pyraclostrobin. Podbielska et al., in evaluating the effects of *Trichoderma* and *Bacillus subtilis* on pyraclostrobin concentrations, found that the degradation efficiency of *Bacillus subtilis* on pyraclostrobin was approximately 5% during a 14-day experiment. For pyraclostrobin treated with *Trichoderma*, the degradation efficiency was 34.2% on day 3 and reached 56.9% on day 14. In the mixed culture experiment, the degradation efficiency of pyraclostrobin was 23.7% on day 3 and 29.1% on day 14. Dou Li isolated six dominant pyrimethanil-degrading bacterial strains from contaminated soil, namely *Pseudomonas* sp., *Paenarthrobacternicotinovorans*, *Acinetobacters* sp., *Serratiamarcescens*, *Pandoraeasp.*, and *Burkholderia* sp. These six strains exhibited the ability to degrade pyrimethanil in inorganic salt media, with a degradation range of 60%–75%. Furthermore, it was found that pH, salinity, and temperature in the culture environment could influence the biodegradation by altering the growth of the degrading bacteria. In soil remediation experiments, the presence of all six strains promoted the degradation of pyrimethanil in the soil, shortening the half-life by 2.6–30.4 days. However, the above study has the drawback of low pyrimethanil degradation efficiency. Currently, no patent reports have been found regarding the microbial degradation of pyrimethanil residual pollution.

[0005] Stenotrophomonas maltophilia is a Gram-negative bacterium belonging to the phylum Proteobacteria, subclass Gamma-β, and genus Stenotrophomonas. It is widely distributed in the environment, having been found in soil, water, and on animal surfaces. It shows potential in pesticide degradation, biocontrol, and plant growth promotion. However, its degradation activity against pyraclostrobin has not yet been reported. This invention discloses a pyrimethanil-degrading bacterium and its applications. This invention is the first to discover the degradation effect of *Stenotrophomonas maltophilia* on pyrimethanil, and screened a *Stenotrophomonas maltophilia* strain, PEB1, that can efficiently degrade pyrimethanil. Based on this invention, *Stenotrophomonas maltophilia* exhibits significant biodegradation effects on pyrimethanil, effectively degrading it over a wide temperature range and tolerating high concentrations of pyrimethanil. This strain can be effectively applied to environmental remediation scenarios such as pesticide production wastewater treatment, bioremediation of contaminated soil, and in-situ treatment of surface water. This invention provides a novel and highly efficient microbial resource for the remediation of organic pesticide pollution. Its strong environmental adaptability and high operational feasibility open up new avenues for the development of green bioremediation technologies. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a maltophilic oligotrophomonas strain that can efficiently degrade pyrimethanil. This strain can also be used to prepare a bioremediation agent for agricultural land contaminated by the pesticide pyrimethanil, and has good application prospects in soil remediation.

[0007] The purpose of this invention is to provide the application of Stenotrophomonas maltophilia in the degradation and remediation of pyrimethanil contaminated natural environments.

[0008] Another objective of this invention is to provide a maltophilic oligotrophomonas PEB1 strain capable of efficiently degrading pyrimethanil.

[0009] Another object of the present invention is to provide the application of the Stenotrophomonas maltophilia strain PEB1 in the degradation and remediation of natural environments contaminated with pyrimethanil.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention is the first to discover the degradation effect of Stenotrophomonas maltophilia on pyrimethanil, and a highly efficient and rapid pyrimethanil-degrading Stenotrophomonas maltophilia strain, PEB1, was screened and deposited at the China General Microbiological Culture Collection Center on December 24, 2024, with the accession number CGMCC No. 33180.

[0012] This strain (PEB1) was isolated from farmland soil subjected to long-term application of pyraclostrobin and obtained through artificial enrichment culture and multiple generations of purification. Experiments showed that it possesses significant degradation characteristics: in an inorganic salt medium with pyraclostrobin as the sole carbon source, the degradation rate exceeded 90% after 7 days; it also exhibits wide temperature range adaptability and tolerance to high pesticide concentrations. Soil remediation experiments showed that after 60 days of adding the strain, the degradation rate of pyraclostrobin in sterilized soil reached 52.3% (an increase of 6.0% compared to the blank control group of 46.5%); while in a non-sterilized system with competition from indigenous microorganisms, the degradation efficiency further increased to 60.4% (an increase of 8.7% compared to the control group of 51.7%), confirming its ecological competitive advantage. This strain can efficiently degrade pyraclostrobin residues in the environment, demonstrating important application value in the field of pesticide pollution bioremediation.

[0013] Therefore, the following applications should all fall within the scope of protection of this invention:

[0014] Applications of Stenotrophomonas maltophilia in the degradation of pyrimethanil or in the preparation of degrading agents.

[0015] Application of Stenotrophomonas maltophilia in the remediation of natural environments contaminated with pyraclostrobin or in the preparation of remediation agents.

[0016] Application of the Stenotrophomonas maltophilia strain PEB1 in the degradation of pyrimethanil or in the preparation of degrading agents.

[0017] Application of the Stenotrophomonas maltophilia strain PEB1 in the remediation of natural environments contaminated with pyraclostrobin or in the preparation of remediation agents.

[0018] The natural environment includes water bodies or soil, etc.

[0019] Preferably, the maltophilic oligotrophozoites is maltophilic oligotrophozoites strain PEB1.

[0020] This invention discloses for the first time the degradation effect of Stenotrophomonas maltophilia on pyrimethanil, and screened out a highly efficient Stenotrophomonas maltophilia strain PEB1 that degrades pyrimethanil. This provides a new and efficient microbial resource for the treatment of organic pesticide pollution. Its strong environmental adaptability and high operational feasibility open up new avenues for the development of green bioremediation technology.

[0021] Moreover, this bacterium can effectively degrade pyraclostrobin over a wide temperature range and tolerate high concentrations of pyraclostrobin (250 mg / L), proving that strain PEB1 can be used as an excellent pesticide-degrading bacterium for the bioremediation of environments contaminated with pyraclostrobin. Attached Figure Description

[0022] Figure 1 This is a colony morphology diagram of Stenotrophomonas maltophilia strain PEB1 after 2 days of culture on LB solid medium.

[0023] Figure 2 These are scanning electron microscope images and Gram staining images of Stenotrophomonas maltophilia strain PEB1.

[0024] Figure 3 This is a phylogenetic tree of the 16S rDNA of Stenotrophomonas maltophilia strain PEB1.

[0025] Figure 4 This is the growth curve of Stenotrophomonas maltophilia strain PEB1.

[0026] Figure 5 This is the degradation curve of Stenotrophomonas maltophilia strain PEB1.

[0027] Figure 6 The degradation effect of maltophilic oligotrophomonas strain PEB1 on pyrimethanil under different temperature conditions is shown.

[0028] Figure 7 The degradation effect of Stenotrophomonas maltophilia strain PEB1 on pyrimethanil under different initial concentrations.

[0029] Figure 8 The degradation effect of Stenotrophomonas maltophilia strain PEB1 on pyrimethanil under different inoculation conditions.

[0030] Figure 9 The effect of Stenotrophomonas maltophilia strain PEB1 on the degradation of pyrimethanil in sterilized soil.

[0031] Figure 10 The effect of Stenotrophomonas maltophilia strain PEB1 on the degradation of pyrimethanil in unsterilized soil. Detailed Implementation

[0032] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0033] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0034] The culture medium formulations described in the following examples are as follows:

[0035] Inorganic salt medium (MSM): prepared with 1g ammonium nitrate, 0.2g magnesium sulfate heptahydrate, 1g sodium chloride, 1.5g potassium dihydrogen phosphate, 0.5g dipotassium hydrogen phosphate, and 1000mL distilled water, pH 7.2; Solid medium: add 18-20g agar powder to every 1L of liquid medium.

[0036] LB medium: prepared with 10g tryptone, 5g yeast extract, 10g sodium chloride, and 1000mL distilled water, pH 7.2; solid medium: add 18-20g agar powder to every 1L of liquid medium.

[0037] Example 1: Isolation and Identification of Strains

[0038] 1. Screening and isolation of pyrimethanil-degrading strains:

[0039] Soil samples long-term contaminated with pyrimethanil were collected, air-dried in a cool place, and sieved. 5g of the sieved sample was added to 50mL of sterile MSM medium with a pyrimethanil concentration of 50mg / L and cultured in a shaker (28℃, 160rpm). Every 7 days, the culture was transferred at a 5% inoculum to inorganic salt liquid medium with progressively increasing pyrimethanil concentrations: 50mg / L, 100mg / L, 200mg / L, 300mg / L, 400mg / L, and 500mg / L. The final culture was plated onto inorganic salt solid medium containing 100mg / L pyrimethanil and incubated at 28℃ for 4–5 days. Colonies were then streaked and purified three times. The purified strain was then plated onto inorganic salt solid medium plates with a pyrimethanil concentration of 100mg / L, and a highly efficient degrading bacterium, designated PEB1, was isolated.

[0040] 2. Identification of strain PEB1

[0041] (1) Morphological identification:

[0042] The degrading strain PEB1 was incubated on LB agar plates at 30°C for 24 hours, and the colony morphology was observed. On LB agar, strain PEB1 colonies were opaque, bright yellow, round, raised, with neat edges, moist surface, and a glossy appearance. Figure 1 Scanning electron microscopy revealed that PEB1 was non-spore-forming, with short rod-shaped cells and no other special structures. Figure 2 )

[0043] (2) Physiological and biochemical identification:

[0044] The physiological and biochemical assays of strain PEB1 showed that it was negative for methyl red reaction, starch hydrolysis, indole, and VP tests, but positive for nitrate reduction and citrate tests. The results of its physiological and biochemical identification are shown in Table 1.

[0045] Table 1 Physiological and biochemical characteristics of strain PEB1 Note: +: Positive; -: Negative.

[0046] (3) Molecular biological identification of 16S rDNA:

[0047] Genomic DNA was extracted from strain PEB1. Using the extracted genome as a template, PCR amplification was performed using universal 16S rDNA primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1429R: 5'-GGTTACCTTGTTACGACTT-3'). The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequenced strain gene sequences were BLAST aligned in the NCBI database, and a phylogenetic tree was constructed using MEGA 11.0.

[0048] Phylogenetic tree of 16S rDNA as follows Figure 3 As shown, strain PEB1 has the highest homology with *Stenotrophomonas maltophilia* (GU254017) and is also very close to it in taxonomic position. Its culture characteristics and scanning electron microscopy observation characteristics are also most similar to *Stenotrophomonas maltophilia*. Therefore, the degrading bacteria screened in this invention were identified as *Stenotrophomonas maltophilia*.

[0049] Based on the above identification results, the strain PEB1 of this invention was identified as Stenotrophomonas maltophilia and was deposited at the China General Microbiological Culture Collection Center on December 24, 2024, with the accession number CGMCC No. 33180.

[0050] Example 2: Experiment on the degradation effect of strain PEB1 on pyrimethanil

[0051] 1. Experimental Methods

[0052] (1) Preparation of bacterial suspension: The screened degrading strain PEB1 was inoculated into LB medium and cultured with shaking at 30℃ and 160 rpm for 18 h. The bacterial cells were collected by centrifugation at 5000 r / min for 5 min, washed twice with sterile physiological saline, and resuspended in inorganic salt liquid medium to prepare bacterial suspension (OD). 600 =1), and conduct subsequent experiments.

[0053] (2) Growth curve determination: PEB1 bacterial suspension was inoculated into sterile LB medium at an inoculum rate of 2%. Samples were taken before incubation as a control, and the growth rate of the strain at 0 h of incubation was recorded. The culture was incubated at 28℃ and 150 rpm for 96 h. Samples were taken at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h to determine the OD of the samples at each time point.600 value.

[0054] (3) Degradation performance test: The bacterial suspension of strain PEB1 was inoculated into 100 mL of pyrimethanil (100 mg / L) inorganic salt liquid culture medium. The inorganic salt liquid culture medium without bacterial inoculation but with drug was used as a blank control. Samples were taken at 0, 1, 2, 3, 4, 5, 6 and 7 days, and the degradation of pyrimethanil was determined by HPLC.

[0055] (4) Chromatographic conditions:

[0056] HPLC: Agilent 1260

[0057] Column: DIKMA Leapsil-C18 (4.6mm × 150mm × 5μm)

[0058] Column temperature: 30℃;

[0059] Mobile phase: Acetonitrile: Water = 70:30 (V / V)

[0060] Flow rate: 1 mL / min

[0061] Injection volume: 20 μL

[0062] Detection wavelength: 230nm

[0063] The degradation rate of pyrimethanil is calculated using the following formula: Degradation rate (%) = (C – C0) / C × 100.

[0064] Where C is the initial concentration of pyrimethanil (mg / L), and C0 is the residual concentration of pyrimethanil in the inorganic salt liquid culture medium after the culture is completed (mg / L).

[0065] Quality control: The external standard method is used to calibrate the standard reference material to prepare the standard curve.

[0066] 2. Experimental Results

[0067] The results are as follows Figure 4 and Figure 5 As shown, the overall growth trend of the bacteria is as follows: 0–4 h is the adaptation period, 4–24 h is the logarithmic growth phase, and the bacterial mass increases rapidly. From 24 h onwards, it enters the stationary phase, lasting until 72 h, after which the bacterial mass begins to decrease, entering the decline phase. Strain PEB1 can utilize pyrimethanil as its sole carbon source, and the degradation rate changes with culture time. The residual rate of pyrimethanil gradually decreases with increasing time, then tends to plateau. After 5 days of culture, the degradation rate reaches a maximum of over 80%, after which the degradation rate of the strain slows down. On day 7, the degradation rate of pyrimethanil by strain PEB1 reaches 90%.

[0068] The results indicate that strain PEB1 can use pyrimethanil as the sole carbon source for growth and reproduction. When the concentration of pyrimethanil is 100 mg / L, the degradation rate reaches 90% after 7 days of culture, indicating that the strain has the ability to efficiently and rapidly degrade pyrimethanil.

[0069] Example 3: Study on the degradation characteristics of pyraclostrobin by strain PEB1

[0070] 1. Experimental Methods

[0071] (1) Effect of temperature on the degradation of pyraclostrobin by PEB1:

[0072] The bacterial suspension was inoculated at a rate of 3% into 50 mL of inorganic salt liquid medium with an initial concentration of 100 mg / L pyrimethanil. The medium was then cultured with shaking at temperatures of 20℃, 25℃, 30℃, and 35℃, and a rotation speed of 160 rpm. Samples were taken after 7 days of culture to determine the residual concentration of pyrimethanil.

[0073] (2) Effect of initial concentration of pyraclostrobin on the degradation of pyraclostrobin by PEB1:

[0074] The bacterial suspension was inoculated at a rate of 3% into 50 mL of inorganic salt liquid culture medium containing pyraclostrobin at initial concentrations of 10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, and 250 mg / L, respectively. The medium was then cultured with shaking at 30°C and 160 rpm. Samples were taken after 7 days of culture to determine the residual concentration of pyraclostrobin.

[0075] (3) Effect of inoculum size on the degradation of pyraclostrobin by PEB1:

[0076] The bacterial suspension was inoculated into 50 mL of inorganic salt liquid medium with an initial concentration of 100 mg / L of pyrimethanil at inoculation rates of 1%, 3%, 5%, 10%, and 20%, respectively. The medium was then cultured with shaking at 30 °C and 160 rpm. Samples were taken after 7 days of culture to determine the residual concentration of pyrimethanil.

[0077] 2. Experimental results are as follows Figures 6-8 As shown.

[0078] Figure 6 The study showed the effect of temperature on the degradation of pyraclostrobin by strain PEB1. Within the temperature range of 20–35 °C, the degradation efficiency of pyraclostrobin by strain PEB1 exhibited a significant temperature-independent characteristic. Notably, the degradation rate remained consistently above 85% (p>0.05) throughout the entire test temperature range, indicating that strain PEB1 possesses excellent wide-temperature-range degradation characteristics.

[0079] Figure 7The study investigated the effect of initial pyraclostrobin concentration on the degradation performance of strain PEB1. When the initial concentration of pyraclostrobin was 10–250 mg / L, the degradation rate initially increased and then decreased with increasing concentration. Strain PEB1 could utilize pyraclostrobin as its sole carbon source and tolerated a concentration of 250 mg / L. At an initial concentration of 100 mg / L, strain PEB1 achieved the highest degradation rate of pyraclostrobin, reaching 93.84%.

[0080] Figure 8 The study showed the effect of inoculum size on the degradation of pyrimethanil by strain PEB1. As the inoculum size increased, the degradation rate of pyrimethanil by strain PEB1 also increased. The optimal degradation effect of strain PEB1 on pyrimethanil was observed at an inoculum size of 3%. When the inoculum size increased from 5% to 20%, the degradation rate not only did not increase but actually showed a decreasing trend (p<0.05), although it still maintained a relatively high degradation rate.

[0081] The results showed that the degradation of pyrimethanil by strain PEB1 was not significantly affected by the environment. Under existing conventional conditions, it could efficiently and rapidly degrade pyrimethanil. For example, it could rapidly degrade pyrimethanil within a wide temperature range (20-35℃) and could tolerate 250 mg / L of pyrimethanil, which ensured its application in complex environments.

[0082] Example 4: Study on the degradation effect of strain PEB1 on pyrimethanil in soil

[0083] 1. Soil sample for testing

[0084] The test soil was taken from the experimental field of the new East Campus of Henan University of Science and Technology. It is yellow soil and has not been treated with pyraclostrobin for more than 5 years. Before use, remove impurities and air dry in a cool and ventilated place. Remove impurities and pass through a 2.0 mm sieve for later use.

[0085] Treatment 1 (Sterilized Soil): Accurately weigh 300g of the treated soil sample and place it in a 500mL Erlenmeyer flask. Seal the flask with a breathable sealing film and sterilize it at high temperature and humidity for 30min. After drying at 30℃, add an inorganic salt liquid culture medium containing pyrimethanil (final concentration of pyrimethanil: 50mg / kg) to the soil in a clean bench to bring the soil moisture content to 60% of field capacity. Mix the soil thoroughly. For the experimental group, add a suspension of degrading bacteria and mix it thoroughly with the soil. A soil sample of the same concentration without degrading bacteria was set up as a blank control. Samples were taken at 0 (2h), 1, 3, 5, 7, 14, 28, 36, 45, and 60 days to determine the pyrimethanil residue and calculate the degradation rate. Throughout the experiment, water was continuously added using a weighing method to maintain 60% field capacity.

[0086] Treatment 2 (Non-sterilized soil): Accurately weigh 300g of the treated soil sample and place it in a 500mL Erlenmeyer flask. Seal the flask with a breathable sealing film. Under aseptic conditions, add an inorganic salt liquid culture medium containing pyrimethanil (final concentration of pyrimethanil: 50mg / kg) to bring the soil moisture content to 60% of field capacity. Mix the soil thoroughly. For the experimental group, add a suspension of degrading bacteria and mix thoroughly with the soil. A soil sample of the same concentration without degrading bacteria was set up as a blank control. The sampling time and detection method were the same as for Treatment 1. Throughout the experiment, water was continuously added using a weighing method to maintain 60% field capacity.

[0087] 2. Measurement results are as follows Figure 9 and Figure 10 The degradation effects of the degrading bacteria on pyrimethanil showed little difference, effectively promoting its degradation. Furthermore, the degradation process of strain PEB1 exhibited a two-stage pattern: a lag phase of 0-3 days followed by rapid degradation. After 60 days of cultivation, the degradation rate in sterilized soil inoculated with PEB1 was 52.3%, significantly higher than the sterilized control group (46.5%, P<0.05). Under non-sterilized conditions, the degradation efficiency of PEB1 (60.4%) was 8.7% higher than the non-sterilized control group (51.7%) (P<0.05), and the residual concentration in the soil was significantly reduced.

[0088] The results showed that strain PEB1 exhibited stable degradation performance after being directly applied to the soil, providing a scientific basis for the soil remediation of pyraclostrobin by strain PEB1.

[0089] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A strain of Stenotrophomonas maltophilia PEB1 that degrades pyrimethanil, characterized by: The maltophilic oligotrophozoites PEB1 was deposited at the China General Microbiological Culture Collection Center on December 24, 2024, with accession number CGMCC No. 33180.

2. The application of Stenotrophomonas maltophiliae PEB1 as described in claim 1 in the degradation of pyrimethanil.

3. The application of the maltophilic oligotrophic strain PEB1 according to claim 1 in the remediation of natural environments contaminated with the fungicide pyrimethanil or in the preparation of pyrimethanil-degrading bacterial agents.

4. The application according to claim 3, characterized in that, The natural environment is water or soil.

5. A highly efficient microbial agent for degrading pyraclostrobin, characterized in that, Contains the maltophilic oligotrophomonas PEB1 as described in claim 1.

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