Evaluation method of prevention and control effect of plant essential oil on sweet potato root rot and application of evaluation method

Abstract

The invention discloses a method for evaluating the prevention and control effect of plant essential oil on sweet potato root rot and application of the method. The method comprises the following steps: pouring a sterilized water agar culture medium into a culture dish, cooling, and inverting to prepare a moisturizing culture dish; sweet potato tuberous roots are cleaned, peeled, sterilized, sliced and wiped to be dry and then placed in a culture dish; coating the cinnamyl aldehyde solution on the surface of the water agar culture medium of a moisturizing culture dish, taking a proper amount of fusarium solani spore liquid, dropwise adding the fusarium solani spore liquid to the centers of sweet potato slices, sealing the culture dish, putting the culture dish in an incubator at 28 DEG C, culturing for 10 days, and observing the prevention and control effect of cinnamyl aldehyde on sweet potato rot. According to the method, experiments are designed according to the volatilization characteristics of cinnamyl aldehyde, nutritional ingredients on the surfaces of sweet potatoes and spores are removed by sterile water, the pathogenicity of pathogenic bacteria on fruits and vegetables such as sweet potatoes and the like can be accurately and efficiently evaluated by operations such as moisture preservation of a water agar culture medium and quantitative spore inoculation, and the prevention and control effect of essential oil such as cinnamyl aldehyde on postharvest diseases of sweet potatoes can be accurately and efficiently evaluated; and a method reference is provided for pathogenicity evaluation of food pathogenic bacteria and efficacy evaluation of volatile bacteriostatic agents.

Description

Technical Field

[0001] This invention belongs to the field of food preservation technology, specifically relating to the evaluation method and application of the effect of plant essential oils on the prevention and control of sweet potato root rot. Background Technology

[0002] Sweet potatoes are an important crop for food, feed, and industrial raw materials. However, their high sugar content, thin and brittle skin, and high water content make them prone to post-harvest rot. *Fusarium solani* is the main pathogenic fungus causing post-harvest sweet potato root rot, and also a major pathogenic fungus for potato dry rot and human keratitis (Document DOI: 10.1094 / PHYTO-01-16-0009-R, 10.1111 / mpp.12289). Chemically synthesized antibacterial agents have a recognized health risk. Unlike antibacterial agents such as potassium sorbate and carbendazim, plant essential oils such as cinnamaldehyde are volatile and can achieve vapor-phase preservation through non-contact methods, showing promising application prospects as green preservatives.

[0003] CN108849960A discloses a compound essential oil preparation for controlling *Heterophyllum oxysporum*, its preparation method, and its application in the prevention and control of sweet potato black rot. The method involves directly spraying the plant essential oil onto sweet potatoes for preservation experiments. However, direct spraying can lead to excessively high local drug concentrations, making it inaccurate in scientific experiments evaluating the antibacterial and preservative effects of the drug. CN117265060A discloses a method for identifying resistance to sweet potato soft rot, using a 5mm diameter punch to create holes and then injecting spore suspension for inoculation. However, the punching operation compresses the internal tissue of the sweet potato. After inoculation with pathogenic spores, the spores seep into the water-depleted flesh, interfering with the diffusion of exogenous volatile components and affecting the efficacy evaluation results of the volatile antibacterial agent. CN119775379A discloses the application of protein IbNFYA3 in regulating resistance to sweet potato soft rot, using discs with mycelia to inoculate the surface of sweet potato slices to evaluate the pathogenicity of the pathogen to sweet potatoes. However, mycelial discs are not conducive to controlling the biomass of inoculation, and are not accurate enough for evaluating the antibacterial effect of volatile components.

[0004] Therefore, in order to more accurately evaluate the antibacterial effect of plant essential oils such as cinnamaldehyde on Fusarium solani and their control effect on sweet potato root rot, a new evaluation method is urgently needed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for evaluating the effect of plant essential oils on the prevention and control of sweet potato root rot and its application, providing a methodological reference for evaluating the pathogenicity of food pathogens and the efficacy of volatile antibacterial agents.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for evaluating the control effect of plant essential oils on sweet potato root rot, comprising the following steps:

[0007] Step 1: Preparation of the moisturizing culture dish: Pour sterilized water agar medium into the culture dish in the biosafety cabinet. After the medium cools and solidifies, invert the culture dish for later use.

[0008] Step 2: Preparation of sweet potato slices: Wash the sweet potato tubers with water, peel them, and disinfect the surface of the peeled sweet potatoes. Then, in a biosafety cabinet, immerse the sweet potatoes in sodium hypochlorite for sterilization. After removing them, remove the sodium hypochlorite from the surface of the sweet potatoes, dry them with sterile air, and use a sterile knife to cut sweet potato slices with a thickness of 0.8-1.2 cm from the equatorial region. Then transfer them to the inside of the lid of a humidified culture dish for later use.

[0009] Step 3: Preparation of spore suspension: Pour physiological saline onto potato dextrose agar (PDA) culture plates containing Fusarium solani that have been growing for 7 days, scrape the spores from the surface of the colonies with a spreader, and filter the spores into sterile centrifuge tubes using sterile lens paper.

[0010] Step 4: Preparation of spore suspension for inoculation: Calculate the spore concentration of the washed suspension using a hemocytometer, and dilute the spore concentration to a suitable level with physiological saline for later use;

[0011] Step 5: Preparation of cinnamaldehyde solution: Cinnamaldehyde is mixed with a co-solvent to obtain an essential oil suspension, which is then vortexed and ultrasonically emulsified for 30 minutes for later use.

[0012] Step 6: Pathogenicity control experiment: A small amount of spore liquid was added to the center of a sweet potato slice. 100 μL of cinnamaldehyde solution was spread onto the surface of the water agar medium in a moist culture dish, allowing cinnamaldehyde to evaporate into the air inside the dish to reach a certain concentration range. The culture dish was sealed and placed in an incubator at 28℃ for 10 days. The control effect of cinnamaldehyde on sweet potato spoilage was then observed.

[0013] Furthermore, the evaluation method also includes the washing of sweet potato slices: the sweet potato slices are washed three times with sterile water, the surface moisture of the sweet potato slices is dried after removal, and then transferred to a humidified petri dish for later use.

[0014] Furthermore, the evaluation method also includes spore washing: the spore suspension is aspirated into a sterile centrifuge tube, the spores are collected by centrifugation, the spores are resuspended in physiological saline, and this operation is repeated to wash the spores three times.

[0015] Preferably, the centrifugation conditions for collecting spores are centrifugation at 5000×g for 3 minutes.

[0016] Preferably, in step one, the water agar medium is prepared by the following steps: 15 g / L of agar powder is heated and dissolved in reverse osmosis water (RO water), and then sterilized by high-pressure steam at 121°C for 20 min.

[0017] Preferably, in step two, the sweet potato is peeled to the extent that the outer skin and periderm are removed, while the flesh is retained; the sodium hypochlorite has a mass concentration of 1% and is soaked for 10 minutes.

[0018] Preferably, in step four, the solution for diluting the spores is 0.85% physiological saline, and the concentration of the spore solution used for inoculation is 1×10⁻⁶. 6 spores / mL

[0019] Preferably, in step five, the cosolvent used is 0.1% (v / v) Tween 80, and the proportion of cinnamaldehyde added to 0.1% Tween 80 is 0% to 12.8%.

[0020] Preferably, in step six, the inoculation volume of the spore solution is 1 μL, and the final concentration of cinnamaldehyde volatilized into the air inside the culture dish is 0.075 g / L to 0.3 g / L.

[0021] The present invention also provides the application of the above evaluation method in the evaluation of the pathogenicity of sweet potato and the effect of plant essential oil fumigation.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. This invention provides a simple device for evaluating the pathogenicity of sweet potato and the effectiveness of plant essential oil fumigation. The device comprises a tall petri dish, a fixed-volume water agar medium, thick sweet potato storage root slices representing the disease characteristics of sweet potato, accurate pathogen inoculation amounts, and a sealing film. Figure 1 Compared to existing technologies that use mycelium or perforation tests for pathogenicity evaluation, the culture device of this invention can ensure the accuracy of inoculation amount and the consistency of the wound surface in the sweet potato flesh, which is beneficial to ensuring the stability of the evaluation results of sweet potato pathogenicity or the effect of controlling sweet potato rot. Figure 2 and Figure 3 ).

[0024] 2. In the method for evaluating the preservative effect of this invention, sterile water is used to wash away the nutrients on the surface of the spores prepared from the bacterial culture plate and to wash away the nutrients on the cut surface of the sweet potato. Combined with this simple device for evaluating the pathogenicity of sweet potato and the effect of essential oil fumigation, it can eliminate the influence of nutrients on the PDA culture medium for culturing the bacterial culture dissolving into the spore suspension, and eliminate the influence of nutrients and plant immune components in the juice of different varieties of sweet potato on the pathogenicity of the pathogen. This allows for a more accurate analysis of the pathogenicity of the pathogen, the preservative effect of the antibacterial agent, and the influence of added nutrients on the pathogenicity and drug resistance of the pathogen. Figure 4 , Figure 5 and Figure 6 ).

[0025] Therefore, compared with existing technologies, the method for evaluating the control effect of cinnamaldehyde on sweet potato root rot according to the present invention can improve the stability and accuracy of the evaluation results of the pathogenicity or control effect on sweet potato rot by considering both the preparation of the culture device, the pathogen, and the preparation of the sweet potato. This invention is beneficial to the product development of novel non-contact slow-release food preservatives. Attached Figure Description

[0026] Figure 1 It is a device for evaluating the pathogenicity of Fusarium solani to sweet potatoes and the preservative effect of cinnamaldehyde fumigation;

[0027] Figure 2 This is an experimental flowchart for evaluating the pathogenicity of Fusarium solani to sweet potatoes and the preservative effect of cinnamaldehyde fumigation;

[0028] Figure 3 This demonstrates the control effect of cinnamaldehyde on sweet potato root rot caused by Fusarium solani;

[0029] Figure 4 This is an experimental flowchart for evaluating the pathogenicity of Fusarium rot on sweet potatoes and the preservative effect of cinnamaldehyde fumigation under conditions with and without surface sugars.

[0030] Figure 5 The effect of the lack of nutrients in the cut surface of sweet potato on the effect of cinnamaldehyde on the control of sweet potato root rot;

[0031] Figure 6 The study showed that the presence of sugar in the cut surface of sweet potatoes affects the effectiveness of cinnamaldehyde in controlling sweet potato root rot. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0033] Example 1

[0034] use Figure 1 The apparatus shown was used to evaluate the pathogenicity of Fusarium solani on sweet potatoes and the preservative effect of cinnamaldehyde fumigation. The experimental procedure is as follows: Figure 2 As shown, the specific steps are as follows:

[0035] Preparation of water agar medium: Dissolve 15 g / L agar powder in reverse osmosis water (RO water) by heating, and then sterilize by high pressure steam at 121℃ for 20 min.

[0036] Preparation of the moisturizing culture dish: Pour 15 mL of sterilized water agar medium into a 2 cm high culture dish. After the medium cools and solidifies, invert the culture dish for later use.

[0037] Preparation of sweet potato slices: Wash the sweet potato tubers thoroughly with tap water and peel off the skin with a peeler. Disinfect the surface of the sweet potatoes by spraying with 75% ethanol. In a biosafety cabinet, place the sweet potatoes in a large beaker and immerse them in 1% (m / v) sodium hypochlorite solution for 10 minutes to sterilize. Remove the sweet potatoes. Wipe off the sodium hypochlorite from the surface of the sweet potatoes with sterile dry gauze and air dry with sterile air for 5 minutes. Use a sterile knife to cut slices approximately 1 cm thick from the equatorial region. Place the prepared sweet potato slices into a humidified petri dish using sterile tweezers for later use.

[0038] Preparation of spore suspension: Pour 10 mL of 0.85% (m / v) physiological saline into a 7-day-old Fusarium solani culture plate. Spores are scraped from the surface of the colonies using a spreader. Filter the spores into a 10 mL sterile centrifuge tube using sterile triple-layer lens paper. Calculate the concentration of the filtered spore suspension using a hemocytometer. Dilute the spore concentration to 2 × 10⁻⁶ with 0.85% (m / v) physiological saline. 4 spores / mL, for later use.

[0039] Preparation of cinnamaldehyde solution: 0–160 μL of cinnamaldehyde was mixed with 0.1% (v / v) Tween 80 to obtain 1 mL of essential oil suspension. After vortex mixing, ultrasonic emulsification was performed for 30 min, and the solution was ready for use.

[0040] Pathogenicity control experiment procedure: 1 μL of spore suspension was dropped into the center of a sweet potato slice. 100 μL of cinnamaldehyde stock solution was spread onto the surface of water agar medium in a moistened culture dish, allowing cinnamaldehyde to evaporate into the air at final concentrations of 0.075 g / L, 0.15 g / L, and 0.3 g / L, respectively. The culture dishes were sealed with plastic wrap and incubated at 28℃ for 10 days. The control effect of cinnamaldehyde on sweet potato spoilage was then observed.

[0041] Experimental results showed that cinnamaldehyde effectively inhibited sweet potato rot caused by Fusarium rot. Compared with the control group without essential oil treatment, cinnamaldehyde treatment did not affect the surface morphology of sweet potatoes. Figure 3 (a)). The higher the concentration of the essential oil, the better the preservative effect of cinnamaldehyde. When the concentration of cinnamaldehyde reaches 0.3 g / L, it can completely inhibit the rot of sweet potatoes caused by Fusarium rot. Figure 3 This evaluation method can be used to evaluate the efficacy of cinnamaldehyde essential oil in controlling sweet potato rot.

[0042] Example 2

[0043] This embodiment provides a method for evaluating the control effect of cinnamaldehyde on sweet potato root rot, and the experimental procedure is as follows: Figure 4 As shown, the specific steps are as follows:

[0044] Preparation of water agar medium: Dissolve 15 g / L agar powder in reverse osmosis water (RO water) by heating, and then sterilize by high pressure steam at 121℃ for 20 min.

[0045] Preparation of the moisturizing culture dish: Pour 15 mL of sterilized water agar medium into a 2 cm high culture dish. After the medium cools and solidifies, invert the culture dish for later use.

[0046] Preparation of sweet potato slices: Wash the sweet potato tubers thoroughly with tap water and peel off the skin with a peeler. Disinfect the surface of the sweet potatoes by spraying with 75% ethanol. Place the sweet potatoes in a large beaker in a biosafety cabinet and immerse them in 1% (m / v) sodium hypochlorite solution for 10 minutes to sterilize. Remove the sweet potatoes and allow them to air dry with sterile air for 5 minutes. Wipe away any remaining sodium hypochlorite from the surface of the sweet potatoes with sterile, dry gauze. Use a sterile knife to cut slices approximately 1 cm thick from the equatorial region.

[0047] Washing sweet potato slices: Place sweet potato slices in a beaker, add sterile water, shake the slices, wash for 1 minute, discard the sterile water, then add sterile water again, washing the sweet potato slices 3 times. Use sterile tweezers to remove the sweet potato slices, and use sterilized dry filter paper to absorb the moisture on the surface of the sweet potato slices. Place the prepared sweet potato slices into a humidified petri dish using sterile tweezers, for later use.

[0048] Preparation of spore suspension: Pour 10 mL of 0.85% (m / v) physiological saline into a 7-day-old Fusarium solani culture plate, and scrape spores from the colony surface using a spreader. Filter the spores through sterile triple-layer lens paper into a 50 mL sterile centrifuge tube. Transfer 1 mL of the spore suspension to a 2 mL sterile centrifuge tube and centrifuge at 5000×g for 3 min to collect the spores. Resuspend the spores in 0.85% (m / v) physiological saline, and repeat this process three times to wash the spores. Calculate the concentration of the washed spore suspension using a hemocytometer. Dilute the spore concentration to 1×10⁻⁶ using 0.85% (m / v) physiological saline. 6 spores / mL, for later use.

[0049] Preparation of cinnamaldehyde solution: 0–128 μL of cinnamaldehyde was mixed with 0.1% (v / v) Tween 80 to obtain 1 mL of essential oil suspension. After vortexing and ultrasonic-assisted dissolution for 30 min, it was ready for use.

[0050] Pathogenicity control experiment procedure: 1 μL of spore suspension was dropped into the center of a sweet potato slice. 100 μL of cinnamaldehyde stock solution was spread onto the surface of water agar medium in a moistened culture dish, allowing cinnamaldehyde to evaporate into the air at final concentrations of 0.04 g / L, 0.08 g / L, and 0.12 g / L. The culture dishes were sealed with plastic wrap and incubated at 28℃ for 10 days. The effect of cinnamaldehyde on controlling sweet potato spoilage was then observed.

[0051] Experimental results showed that 0.12 g / L cinnamaldehyde could effectively inhibit sweet potato rot caused by Fusarium solani. Figure 5 In Example 1, 0.3 g / L cinnamaldehyde was needed to inhibit sweet potato rotting without washing the slices. Figure 3 This indicates that washing the cut surface of sweet potatoes with water to remove nutrients can enhance the preservative activity of cinnamaldehyde and accurately evaluate the effect of cinnamaldehyde in preventing sweet potato rot.

[0052] Example 3

[0053] The steps are basically the same as in Example 2, except that:

[0054] Preparation of 20% sterile glucose solution: Add 2g of glucose to an appropriate amount of deionized water, dissolve, and then dilute to 10mL with water. Filter the glucose through a 0.22μm filter membrane for sterilization, and set aside for later use.

[0055] Preparation of spore suspension: The spore solution, after surface nutrient washing, was mixed with 100 μL of 20% sterile glucose solution and physiological saline to a final volume of 1 mL, yielding a 1×10⁻⁶ spore suspension containing 2% glucose. 6 Prepare a spore suspension at a concentration of spores / mL for later use.

[0056] To verify the antibacterial and preservative activity of sweet potato components against cinnamaldehyde, the rot of sweet potatoes was analyzed after adding 2% glucose. The results showed that, under the condition of added glucose, cinnamaldehyde at concentrations of 0–0.12 g / L still exhibited strong antibacterial activity against *Fusarium solani*, and 0.12 g / L cinnamaldehyde effectively inhibited sweet potato rot caused by *Fusarium solani*. Figure 6 ). Figure 6 The # symbol in (b) indicates that, under conditions of 0.04 g / L and 0.08 g / L cinnamaldehyde, the spore group with added glucose resulted in a larger diameter of rotten spots on sweet potatoes compared to the group without added glucose. Figure 5 The diameter of the rotten spots on sweet potatoes caused by mechanical damage was larger (p<0.05). A significant factor in postharvest disease in sweet potatoes and other fruits and vegetables is the wounds created by mechanical injury. Pathogens infect through these wounds, leading to spoilage. However, the composition of fruit and vegetable juices is complex, including nutrients and immune components. Juice accumulated on plant wounds can interfere with the pathogenicity of pathogens and the evaluation of the preservative effect of preservatives. Therefore, by cleaning the cut surfaces of sweet potatoes to detect the nutrients, the control effect of preservatives on sweet potatoes and other fruits and vegetables can be accurately assessed. This method is particularly suitable for analyzing the effect of external nutrients on the preservative effect and mechanism of volatile antibacterial agents such as cinnamaldehyde in controlling the spoilage of sweet potatoes and other fruits and vegetables.

[0057] Comparison of the experimental results of Examples 1, 2, and 3 shows that a series of technical operations, such as using water agar as a humectant, using the plate coating method to assist the volatilization of cinnamon essential oil, using sweet potato slices instead of sweet potatoes for pathogenicity analysis, and washing the nutrients on the spore surface and the cut surface of sweet potatoes with sterile water, are beneficial for accurately evaluating the pathogenicity of Fusarium rot to sweet potatoes and the control effect of cinnamaldehyde on sweet potato rot.

[0058] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for evaluating the control effect of plant essential oils on sweet potato root rot, characterized in that, Includes the following steps: Step 1: Preparation of the moisturizing culture dish: Pour sterilized water agar medium into the culture dish in the biosafety cabinet. After the medium cools and solidifies, invert the culture dish for later use. Step 2: Preparation of sweet potato slices: Wash the sweet potato tubers with water, peel them, and disinfect the surface of the peeled sweet potatoes. Then, in a biosafety cabinet, immerse the sweet potatoes in sodium hypochlorite for sterilization. After removing them, remove the sodium hypochlorite from the surface of the sweet potatoes, dry them with sterile air, and use a sterile knife to cut sweet potato slices with a thickness of 0.8-1.2 cm from the equatorial region. Then transfer them to the inside of the lid of a humidified culture dish for later use. Step 3: Preparation of spore suspension: Pour physiological saline into a 7-day-old Fusarium solani culture plate on potato dextrose agar medium, scrape the spores from the surface of the colonies with a spreader; filter the spores into sterile centrifuge tubes using sterile lens paper. Step 4: Preparation of spore suspension for inoculation: Calculate the spore concentration of the filtered suspension using a hemocytometer, and dilute the spore concentration to a suitable level with physiological saline for later use; Step 5: Preparation of cinnamaldehyde solution: Cinnamaldehyde is mixed with a co-solvent to obtain an essential oil suspension, which is then vortexed and ultrasonically emulsified for 30 minutes for later use. Step 6: Pathogenicity control experiment: A small amount of spore liquid was added to the center of a sweet potato slice. 100 μL of cinnamaldehyde solution was spread onto the surface of the water agar medium in a moist culture dish, allowing cinnamaldehyde to evaporate into the air inside the dish to reach a certain concentration range. The culture dish was sealed and placed in an incubator at 28℃ for 10 days. The control effect of cinnamaldehyde on sweet potato spoilage was then observed.

2. The method for evaluating the effect of plant essential oil on the control of sweet potato root rot according to claim 1, characterized in that, It also includes the cleaning of sweet potato slices: wash the sweet potato slices three times with sterile water, remove them and pat dry the surface of the sweet potato slices, then transfer them to a humidified petri dish for later use.

3. The method for evaluating the effect of plant essential oil on the control of sweet potato root rot according to claim 2, characterized in that, This also includes spore washing: aspirate the spore suspension into a sterile centrifuge tube, centrifuge to collect the spores, resuspend the spores in physiological saline, and repeat this operation to wash the spores 3 times.

4. The method for evaluating the effect of plant essential oil on the control of sweet potato root rot according to claim 1, characterized in that, In step one, the water agar medium is prepared by the following steps: 15 g / L of agar powder is heated and dissolved in reverse osmosis water, and then sterilized by high-pressure steam at 121°C for 20 min.

5. The method for evaluating the effect of plant essential oil on the control of sweet potato root rot according to claim 1, characterized in that, In step two, the sweet potatoes are peeled to the extent that the outer skin and periderm are removed, while the flesh is retained; the sodium hypochlorite concentration is 1%, and the soaking time is 10 minutes.

6. The method for evaluating the effect of plant essential oil on the control of sweet potato root rot according to claim 3, characterized in that, The centrifugation conditions for spore collection were 5000×g centrifugation force for 3 min.

7. The method for evaluating the effect of plant essential oil on the control of sweet potato root rot according to claim 1, characterized in that, In step four, the spores are diluted with a 0.85% (m / v) physiological saline solution, and the concentration of the spore solution used for inoculation is 1×10⁻⁶. 6 spores / mL.

8. The method for evaluating the effect of plant essential oil on the control of sweet potato root rot according to claim 1, characterized in that, In step five, the cosolvent used is 0.1% (v / v) Tween 80, and the proportion of cinnamaldehyde added to 0.1% Tween 80 is 0% to 12.8%.

9. The method for evaluating the effect of plant essential oil on the control of sweet potato root rot according to claim 1, characterized in that, In step six, the inoculation volume of the spore solution is 1 μL, and the final concentration of cinnamaldehyde volatilized into the air inside the petri dish is 0.075 g / L to 0.3 g / L.

10. The application of the evaluation method according to any one of claims 1 to 9 in the evaluation of the pathogenicity of sweet potato and the effect of plant essential oil fumigation.

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