Prevention and control method of bacillus velezensis on epimedium anthrax bacteria and application of bacillus velezensis

Abstract

The invention discloses a method for preventing and controlling epimedium colletotrichum gloeosporioides by bacillus velezensis and application. The method comprises the following steps: step 1, preparing a bacillus velezensis seed solution; step 2, preparing a bacillus velezensis sterile fermentation broth; step 3, preparing raw materials; 4, preparing a biopesticide mother solution; step 5, filtering and sub-packaging; step 6, applying a biopesticide; the biological pesticide is used for preventing and controlling the epimedium colletotrichum gloeosporioides, and the antibacterial extract is matched with the bacillus velezensis sterile fermentation liquor with extremely high inhibition on the epimedium colletotrichum gloeosporioides to serve as a core antibacterial component to synergistically inhibit growth of pathogenic bacteria and disease diffusion; according to the biopesticide, the stability of a pesticide system is improved through the stabilizing aid, the active ingredients are promoted to be uniformly dispersed through the dispersing aid, the spreadability of leaves is enhanced, the acting time of the active ingredients on the leaves is prolonged through the moisturizing aid, the raw materials are green and environment-friendly, pollution to plants and the ecological environment is avoided, and high prevention and control efficiency and environmental safety are both considered.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, specifically to a method and application of Bacillus belye in controlling Epimedium anthracnose. Background Technology

[0002] Epimedium is a medicinal plant widely used in traditional Chinese medicine, possessing both high medicinal and economic value. However, in the large-scale cultivation of Epimedium, disease problems have consistently been a core bottleneck restricting stable yield and quality improvement. Anthracnose, in particular, is a significant threat and has become a major obstacle to the development of the Epimedium industry. Anthracnose is caused by infection with *Anthracnose karstii*. In the early stages of infection, irregular yellow spots appear on the leaf surface without obvious halos. As the disease progresses, these spots rapidly enlarge and merge, forming yellowish-brown lesions surrounded by deep yellow halos. Later, the lesions spread to the entire leaf, causing chlorosis and wilting. Simultaneously, the pathogen can spread from the petiole to the stem, causing stem rot and ultimately leading to the collapse and death of the entire plant, resulting in severe economic losses for growers.

[0003] To address the control of anthracnose in Epimedium, current technologies generally employ chemical fungicides. However, long-term reliance on chemical control not only leads to excessive pesticide residues in Epimedium, affecting its medicinal quality and safety, but also promotes the development of drug resistance in pathogens, reducing the effectiveness of subsequent control measures. Furthermore, the extensive use of chemical agents causes irreparable pollution to the soil, water, and air, contradicting the principles of green agriculture and sustainable development. Some technologies utilize biopesticides, which employ live microbial agents such as Bacillus subtilis, Trichoderma harzianum, and Pseudomonas fluorescens to achieve antibacterial effects. While these biopesticides are environmentally friendly, they are less effective against karst anthracnose pathogens and cannot effectively prevent the spread of the disease. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application of Bacillus belye in the prevention and control of Epimedium anthrax bacteria, in order to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for controlling the anthrax pathogen of Epimedium using Bacillus belye, comprising the following steps: Step 1, preparing Bacillus belye seed liquid; Step 2, preparing Bacillus belye sterile fermentation broth; Step 3, preparing raw materials; Step 4, preparing biological pesticide mother liquor; Step 5, filtering and dispensing; Step 6, applying biological pesticide.

[0006] In step one above, *Bacillus belye* strain is inoculated onto LB solid medium and activated at 37°C for 12 hours to obtain single colonies. These single colonies are then inoculated onto LB liquid medium and cultured on a shaker at 37°C and 200 rpm for 24 hours until the bacterial concentration stabilizes at 10⁻⁶. 8 -10 9 CFU / mL was used to obtain the seed culture;

[0007] In step two above, the seed culture is transferred to LB liquid medium at a volume inoculation ratio of 5%, and after culturing for 72 hours, the bacterial concentration stabilizes at 10. 8 -10 9 CFU / mL was used to obtain Bacillus berberis fermentation broth. The Bacillus berberis fermentation broth was centrifuged and filtered to obtain sterile Bacillus berberis fermentation broth.

[0008] In step three above, with the sum of the mass percentage content of each component being 1, deionized water, stabilizing agent, dispersing agent, moisturizing agent, antibacterial extract and Bacillus vesicles sterile fermentation broth are prepared according to the biological pesticide formula.

[0009] In step four above, room temperature deionized water is added to a mixing tank, a stabilizing agent is added, and the mixture is stirred until completely dissolved. Then, a dispersing agent and a moisturizing agent are added in sequence, and the mixture is stirred at 300 rpm for 10-15 minutes. Subsequently, an antibacterial extract and Bacillus vesiculosus sterile fermentation broth are added, and the mixture is stirred at 200 rpm for 25-30 minutes to obtain a biological pesticide mother liquor.

[0010] In step five above, the biological pesticide mother liquor is filtered through a 0.45μm sterile filter membrane, dispensed into sterile bottles, and sealed for storage.

[0011] In step six above, the prepared biological pesticide mother liquor is diluted with deionized water at a volume ratio of 1:40-50, and the diluted biological pesticide is applied to Epimedium.

[0012] Preferably, in step two, the cultivation conditions are as follows: 0-24h, temperature is 37℃, stirring speed is 200rpm, and aeration rate is 1:1V / V·min; 24-72h, temperature is 25℃, stirring speed is 200rpm, and aeration rate is 1:1.2V / V·min. During the cultivation process, the pH of the fermentation broth is stabilized at 7.0 by adding 0.5mol / L NaOH and HCl.

[0013] Preferably, in step two, the centrifugal filtration process specifically involves: placing the Bacillus berberis fermentation broth in a centrifuge and centrifuging at 12000 r / min for 15 min, maintaining the temperature at 25°C during the centrifugation process; collecting the supernatant; and filtering it through a 0.22 μm sterile filter membrane to obtain the sterile Bacillus berberis fermentation broth.

[0014] Preferably, in step three, the mass percentage content of each component in the biopesticide formulation is as follows: 45-55% deionized water, 0.1-0.3% stabilizing adjuvant, 1.5-2.5% dispersing adjuvant, 2.3-3.3% moisturizing adjuvant, 2.5-3.5% antibacterial extract, and 37-47% Bacillus vesiculosus sterile fermentation broth.

[0015] Preferably, the stabilizing agent is one or more of xanthan gum, guar gum, gum arabic, carrageenan, locust bean gum, and konjac glucomannan.

[0016] Preferably, the dispersing agent is one or more of sucrose fatty acid ester, tea saponin, soybean lecithin, alkyl glycoside, castor oil polyoxyethylene ether, and Tween 80.

[0017] Preferably, the moisturizing agent is one or more of glycerin, propylene glycol, sorbitol, xylitol, 1,3-propanediol, and polyethylene glycol 400.

[0018] Preferably, the antibacterial extract is prepared by compounding a plant-derived antibacterial agent and Bacillus subtilis metabolic liquid in a mass ratio of 2-3:1. The plant-derived antibacterial agent is one or more of chlorogenic acid, baicalin, forsythoside, allicin, matrine, and eugenol.

[0019] Preferably, in step six, the application time of the biological pesticide is: 8:00-10:00 AM and 4:00-6:00 PM on a sunny and windless day; the application method is: spraying the Epimedium diseased plants and healthy plants within a 0-80cm radius around the diseased plants, with the application amount per Epimedium plant controlled at 15-20mL; the application frequency of the biological pesticide is: after the first application, repeat the application once every 48 hours, and three consecutive applications constitute one control cycle. If light rain occurs within 24 hours after application, a supplementary application is required within 12 hours after the rain. When supplementing the application, the dilution ratio of the biological pesticide remains unchanged, but the application amount is reduced to 80% of the normal amount.

[0020] Application of a method for controlling Epimedium anthrax bacteria using Bacillus belye, the method can be applied to the control of anthrax bacteria.

[0021] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention uses biological pesticides to control Epimedium anthracnose fungus. It uses antibacterial extracts combined with sterile fermentation broth of Bacillus vesiculosus, which has a strong inhibitory effect on Epimedium anthracnose fungus, as the core antibacterial component to synergistically inhibit the growth of pathogens and the spread of disease. The biological pesticide improves the stability of the pesticide system through stabilizing adjuvants, promotes the uniform dispersion of active ingredients and enhances leaf spreadability through dispersing adjuvants, and prolongs the action time of active ingredients on leaves through moisturizing adjuvants. Moreover, the raw materials are green and environmentally friendly and will not cause pollution to plants and the ecological environment, thus taking into account both high control efficiency and environmental safety. Attached Figure Description

[0022] Figure 1 This is a flowchart of the method of the present invention;

[0023] Figure 2 Figure A shows the experimental results of Experiment Example 1; A represents the inhibitory effect of different concentrations of CFS on the growth of karst anthrax bacteria; B represents the inhibition rate of different concentrations of CFS on karst anthrax bacteria. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Please see the appendix Figure 1 The present invention provides an embodiment of a method for controlling the anthrax pathogen of Epimedium using Bacillus belye, comprising the following steps: Step 1, preparing Bacillus belye seed liquid; Step 2, preparing Bacillus belye sterile fermentation broth; Step 3, preparing raw materials; Step 4, preparing biological pesticide mother liquor; Step 5, filtering and dispensing; Step 6, applying biological pesticide.

[0026] In step one above, *Bacillus belye* strain is inoculated onto LB solid medium and activated at 37°C for 12 hours to obtain single colonies. These single colonies are then inoculated onto LB liquid medium and cultured on a shaker at 37°C and 200 rpm for 24 hours until the bacterial concentration stabilizes at 10⁻⁶. 8 -10 9 CFU / mL was used to obtain the seed culture;

[0027] In step two above, the seed culture was transferred to LB liquid medium at a volume inoculation ratio of 5% and cultured under the following conditions: 0-24h, temperature 37℃, stirring speed 200rpm, aeration rate 1:1V / V·min; 24-72h, temperature 25℃, stirring speed 200rpm, aeration rate 1:1.2V / V·min. During the culture, the pH of the fermentation broth was stabilized at 7.0 by adding 0.5mol / L NaOH and HCl. After 72h of culture, the cell concentration stabilized at 10. 8 -10 9 CFU / mL was used to obtain the Bacillus berberis fermentation broth. The Bacillus berberis fermentation broth was placed in a centrifuge and centrifuged at 12000 r / min for 15 min. The centrifugation process was maintained at 25℃. The supernatant was collected and filtered through a 0.22 μm sterile filter membrane to obtain the sterile Bacillus berberis fermentation broth.

[0028] In step three above, taking the sum of the mass percentages of each component as 1, according to the biopesticide formula, deionized water, stabilizing agent, dispersing agent, moisturizing agent, antibacterial extract, and Bacillus vesiculosus sterile fermentation broth are prepared. The mass percentages of each component in the biopesticide formula are as follows: 50% deionized water, 0.2% stabilizing agent, 2% dispersing agent, 2.8% moisturizing agent, 3% antibacterial extract, and 42% Bacillus vesiculosus sterile fermentation broth. The stabilizing agent is xanthan gum, the dispersing agent is sucrose fatty acid ester, the moisturizing agent is glycerin, and the antibacterial extract is a compound of plant-derived antibacterial agent and Bacillus subtilis metabolite in a mass ratio of 2:1. The plant-derived antibacterial agent is chlorogenic acid.

[0029] In step four above, room temperature deionized water is added to a mixing tank, a stabilizing agent is added, and the mixture is stirred until completely dissolved. Then, a dispersing agent and a moisturizing agent are added in sequence, and the mixture is stirred at 300 rpm for 10-15 minutes. Subsequently, an antibacterial extract and Bacillus vesiculosus sterile fermentation broth are added, and the mixture is stirred at 200 rpm for 25-30 minutes to obtain a biological pesticide mother liquor.

[0030] In step five above, the biological pesticide mother liquor is filtered through a 0.45μm sterile filter membrane, dispensed into sterile bottles, and sealed for storage.

[0031] In step six above, the prepared biological pesticide stock solution is diluted evenly with deionized water at a volume ratio of 1:40-50. The diluted biological pesticide is then applied to Epimedium. The application time is 8:00-10:00 AM and 4:00-6:00 PM on sunny, windless days. The application method is to spray the Epimedium diseased plants and healthy plants within a 0-80cm radius around them. The amount applied to each Epimedium plant is controlled at 15-20mL. The frequency of application is as follows: after the first application, repeat the application once every 48 hours, and three consecutive applications constitute one control cycle. If light rain occurs within 24 hours after application, a supplementary application is required within 12 hours after the rain. When supplementing the application, the dilution ratio of the biological pesticide remains unchanged, but the amount applied is reduced to 80% of the normal amount.

[0032] Example 2:

[0033] Referring to the method of Example 1, only the mass percentage content of each component in the biological pesticide formulation in step three was changed to: 45% deionized water, 0.2% stabilizing agent, 2% dispersing agent, 3.3% moisturizing agent, 3.5% antibacterial extract and 46% Bacillus vesiculosus sterile fermentation broth.

[0034] Example 3:

[0035] Referring to the method of Example 1, only the mass percentage content of each component in the biopesticide formulation in step three was changed to: 55% deionized water, 0.3% stabilizing adjuvant, 1.5% dispersing adjuvant, 3.2% moisturizing adjuvant, 3% antibacterial extract, and 37% Bacillus vesiculosus sterile fermentation broth.

[0036] Example 4:

[0037] Referring to the method in Example 1, only the stabilizing adjuvant in the biological pesticide formulation in step three was replaced with guar gum, the dispersing adjuvant with soybean lecithin, the moisturizing adjuvant with propylene glycol, and the plant-derived antibacterial agent with eugenol.

[0038] Experimental Example 1:

[0039] To verify the antibacterial effect of the *Bacillus belyssiensis* sterile fermentation broth proposed in this invention on *Bacillus karstiensis*, the following experiment was conducted: The *Bacillus belyssiensis* sterile fermentation broth (CFS) prepared in Example 1 was mixed with potato dextrose agar at different volume ratios and poured into plates to prepare plates containing 1%, 5%, and 10% *Bacillus belyssiensis* sterile fermentation broth as three experimental groups. The control group used an equal volume of sterile LB liquid medium mixed with PDA. *Bacillus karstiensis* stipes with a diameter of 6 mm were inoculated in the center of the plates in both the experimental and control groups. Each treatment was repeated three times. After 5 days of dark incubation at 28°C, the mycelial diameter was measured using the cross-cross method, and the inhibition rate was calculated. The inhibition rate was calculated using the following formula: Inhibition rate = (Mycelial diameter of control group - Mycelial diameter of treatment group) / Mycelial diameter of control group × 100%, where the mycelial diameter of both the experimental and control groups was reduced by the stipe diameter of 6 mm. The experimental results are attached. Figure 2 As shown, 10% CFS inhibited the growth of anthrax bacteria by 77.65%, 5% CFS inhibited the growth of anthrax bacteria by 43.97%, and 1% CFS inhibited the growth of anthrax bacteria by 20.57%. This indicates that CFS contains highly effective antibacterial active substances that can directly inhibit the growth of anthrax bacteria mycelia.

[0040] Experimental Example 2:

[0041] To verify the control effect of the biological pesticide proposed in this invention on Epimedium anthracnose, the following experiment was conducted: Three treatment groups were set up, with each group having three replicates, and each replicate consisting of 10 Epimedium seedlings. The specific treatments were as follows: Blank control group: After the plant leaves were lightly pricked three times, 10 mL of sterile water was sprayed intensively, and the plants were incubated at 28℃ for 24 hours, followed by spraying with 15 mL of sterile water; Pathogen control group: After the plant leaves were lightly pricked three times, 10 mL of a bacterial concentration of 10 was sprayed intensively. 6 CFU / mL of *Anthracnose karstii* spores were incubated at 28°C for 24 hours, followed by spraying with 15mL of sterile water. In the biological pesticide treatment group, after lightly pricking the plant leaves three times, 10mL of a 10% bacterial concentration was sprayed intensively. 6CFU / mL of *Anthracnose karstii* spores were incubated at 28°C for 24 hours, followed by spraying with 15 mL of a diluted biological pesticide solution. This diluted solution was prepared by diluting the biological pesticide stock solution from Example 1 with deionized water at a volume ratio of 1:50. All treated plants were incubated in an incubator at 28°C, 80% relative humidity, and a 12-hour light cycle. After the initial application, the corresponding liquid was sprayed every 48 hours for a total of three treatments. Seven days after the treatment, the number of diseased leaves in each group was recorded according to the diseased leaf grading standard, and the incidence rate was calculated. Incidence rate (%) = (Number of diseased leaves / Total number of leaves) × 100%; Based on the incidence rates of the control group and the treatment group, the control rate was calculated as follows: Control rate (%) = [(Incidence rate of pathogen control group - Incidence rate of biological pesticide treatment group) / Incidence rate of pathogen control group] × 100%; The experimental results are shown in the table below. It can be seen that the incidence rate of the pathogen control group reached 81.25%, and some plants withered and collapsed. The incidence rate of the biological pesticide treatment group was only 13.43%, and the control rate reached 83.47%, proving that biological pesticides can significantly inhibit the occurrence and spread of Epimedium anthracnose.

[0042]

[0043] Based on the above, the advantages of this invention are as follows: When used, this invention achieves effective control of Epimedium anthracnose fungus through biological pesticides. The biological pesticides utilize antibacterial extracts and sterile fermentation broth of Bacillus vesiculosus as the main antibacterial components. Stabilizing agents increase the stability of the biological pesticide system, preventing stratification and precipitation. Dispersing agents promote uniform dispersion of active ingredients in the fermentation broth, improving the spreadability of the biological pesticide on leaves. Moisturizing agents enhance the adhesion and moisture retention of the biological pesticide on the surface of Epimedium leaves, prolonging the action time of the active ingredients. The antibacterial extract is a compound of plant-derived antibacterial agents and Bacillus subtilis metabolites. The plant-derived antibacterial agents are natural antibacterial components, while the Bacillus subtilis metabolites can assist in decomposing the cell walls of anthracnose fungus. The sterile fermentation broth of Bacillus vesiculosus has a strong inhibitory effect on Epimedium anthracnose fungus. The three components work synergistically to effectively inhibit karst anthracnose fungus. Furthermore, the raw materials used in the biological pesticides are green and environmentally friendly, and will not pollute the plants or the ecological environment.

[0044] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A method for controlling Epimedium anthrax pathogens with Bacillus belye, comprising the following steps: Step 1: Prepare Bacillus belyss seed culture; Step 2: Prepare Bacillus belyss aseptic fermentation broth; Step 3: Prepare raw materials; Step 4: Prepare biological pesticide mother liquor; Step 5: Filter and dispense; Step 6: Apply biological pesticide; The characteristic feature is: In step one above, *Bacillus belye* strain is inoculated onto LB solid medium and activated at 37°C for 12 hours to obtain single colonies. These single colonies are then inoculated onto LB liquid medium and cultured on a shaker at 37°C and 200 rpm for 24 hours until the bacterial concentration stabilizes at 10⁻⁶. 8 -10 9 CFU / mL was used to obtain the seed culture; In step two above, the seed culture is transferred to LB liquid medium at a volume inoculation ratio of 5%, and after culturing for 72 hours, the bacterial concentration stabilizes at 10. 8 -10 9 CFU / mL was used to obtain Bacillus berberis fermentation broth. The Bacillus berberis fermentation broth was centrifuged and filtered to obtain sterile Bacillus berberis fermentation broth. In step three above, with the sum of the mass percentage content of each component being 1, deionized water, stabilizing agent, dispersing agent, moisturizing agent, antibacterial extract and Bacillus vesicles sterile fermentation broth are prepared according to the biological pesticide formula. In step four above, room temperature deionized water is added to a mixing tank, a stabilizing agent is added, and the mixture is stirred until completely dissolved. Then, a dispersing agent and a moisturizing agent are added in sequence, and the mixture is stirred at 300 rpm for 10-15 minutes. Subsequently, an antibacterial extract and Bacillus vesiculosus sterile fermentation broth are added, and the mixture is stirred at 200 rpm for 25-30 minutes to obtain a biological pesticide mother liquor. In step five above, the biological pesticide mother liquor is filtered through a 0.45μm sterile filter membrane, dispensed into sterile bottles, and sealed for storage. In step six above, the prepared biological pesticide mother liquor is diluted with deionized water at a volume ratio of 1:40-50, and the diluted biological pesticide is applied to Epimedium.

2. The method for controlling Epimedium anthrax bacteria using Bacillus belye according to claim 1, characterized in that: In step two, the cultivation conditions are as follows: 0-24h, temperature is 37℃, stirring speed is 200rpm, and aeration rate is 1:1V / V·min; 24-72h, temperature is 25℃, stirring speed is 200rpm, and aeration rate is 1:1.2V / V·min. During the cultivation process, the pH of the fermentation broth is stabilized at 7.0 by adding 0.5mol / L NaOH and HCl.

3. The method for controlling Epimedium anthrax bacteria using Bacillus belye according to claim 1, characterized in that: In step two, the centrifugal filtration process is as follows: the Bacillus berberis fermentation broth is placed in a centrifuge and centrifuged at 12000 r / min for 15 min, while maintaining the temperature at 25°C during the centrifugation process. The supernatant is collected and filtered through a 0.22 μm sterile filter membrane to obtain the sterile Bacillus berberis fermentation broth.

4. The method for controlling Epimedium anthrax bacteria using Bacillus belye according to claim 1, characterized in that: In step three, the mass percentage content of each component in the biopesticide formulation is as follows: 45-55% deionized water, 0.1-0.3% stabilizing adjuvant, 1.5-2.5% dispersing adjuvant, 2.3-3.3% moisturizing adjuvant, 2.5-3.5% antibacterial extract, and 37-47% Bacillus vesiculosus sterile fermentation broth.

5. A method for controlling Epimedium anthrax bacteria using Bacillus belye according to claim 4, characterized in that: The stabilizing agent is one or more of xanthan gum, guar gum, gum arabic, carrageenan, locust bean gum, and konjac glucomannan.

6. A method for controlling Epimedium anthrax bacteria using Bacillus belye according to claim 4, characterized in that: The dispersing agent is one or more of the following: sucrose fatty acid ester, tea saponin, soybean lecithin, alkyl glycoside, castor oil polyoxyethylene ether, and Tween 80.

7. A method for controlling Epimedium anthrax bacteria using Bacillus belye according to claim 4, characterized in that: The moisturizing agent is one or more of glycerin, propylene glycol, sorbitol, xylitol, 1,3-propanediol, and polyethylene glycol 400.

8. A method for controlling Epimedium anthrax bacteria using Bacillus belye according to claim 4, characterized in that: The antibacterial extract is prepared by compounding plant-derived antibacterial agents and Bacillus subtilis metabolites in a mass ratio of 2-3:

1. The plant-derived antibacterial agents are one or more of chlorogenic acid, baicalin, forsythoside, allicin, matrine, and eugenol.

9. A method for controlling Epimedium anthrax bacteria using Bacillus belye according to claim 1, characterized in that: In step six, the application time for the biological pesticide is: 8:00-10:00 AM and 4:00-6:00 PM on a sunny, windless day; the application method is: spraying the Epimedium diseased plants and healthy plants within a 0-80cm radius around the diseased plants, with each Epimedium plant receiving 15-20mL of the pesticide; the frequency of application is: after the first application, repeat the application every 48 hours, for a total of 3 applications constituting one control cycle. If light rain occurs within 24 hours after application, a second application is required within 12 hours after the rain. When applying the second application, the dilution ratio of the biological pesticide remains unchanged, but the application amount is reduced to 80% of the normal amount.

10. The application of a method for controlling Epimedium anthrax bacteria using Bacillus belye, characterized in that: The method described can be applied to the prevention and control of anthrax bacteria.

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