Application of thyme essential oil in prevention and treatment of wheat snow rot

Abstract

The invention belongs to the technical field of plant essential oil, and relates to application of thyme essential oil in prevention and treatment of wheat snow rot. Through a single factor test and a response surface optimization test, the optimal extraction process conditions of the thyme essential oil are as follows: an ultrasonic-assisted steam distillation method is adopted for extraction, the liquid-to-material ratio is 30: 1, the distillation time is 2.0 h, the ultrasonic power is 300W, and the ultrasonic time is 45min. A further test proves that the thyme essential oil prepared by the method has a remarkable inhibition effect on mycelial growth, spore germination and mycelial biomass accumulation of the microasperella nivalis. On the basis, the thyme essential oil is used as an active ingredient and is used for treating the wheat snow rot caused by the snow rot microseat spore, when the concentration is 0.5 microL / mL, the disease index is 26.2, the disease prevention effect is 60.42%, and the effect is remarkable. The invention provides a theoretical basis for resource development and utilization of thyme, and also provides a safe and efficient method for prevention and treatment of wheat snow rot.

Description

Technical Field

[0001] The invention belongs to the technical field of plant essential oils and relates to application of thyme essential oil in preventing and treating wheat snow rot. Background Art

[0002] Thyme, also known as ground pepper, ground ginger, thyme, ground corner flower, and mountain pepper, is a perennial herb or low subshrub. Thyme is widely distributed, typically growing in mountainous areas at altitudes of 1,000 to 2,500 meters, along riverbanks, and in grassy areas or sandy banks. Thyme essential oil, extracted from the thyme plant, is a volatile oil containing multiple active ingredients. It possesses natural antibacterial, antioxidant, insect repellent, and anti-cancer properties. It has a wide range of applications in food, medicine, cosmetics, and feed production, and holds promising prospects for development and utilization.

[0003] In addition, there are relevant reports and literature on the use of thyme essential oil in the field of plant disease control technology. Studies have shown that thyme essential oil has good in vitro antibacterial activity against certain postharvest pathogenic fungi of fruits and vegetables. For example, in the prevention and control of postharvest diseases of cherry tomatoes, thyme essential oil has a good inhibitory effect on diseases caused by Alternaria alternata. Furthermore, thyme essential oil also has a certain inhibitory effect on postharvest fungal diseases of other fruits and vegetables, such as dates, cherries, and grapes. Other studies have shown that thyme essential oil can effectively inhibit the growth of Rhizoctonia solani, a fungal fungus, using a mycelial growth rate inhibition test, and exhibits a synergistic antibacterial effect with polyoxin.

[0004] As a vital grain crop, wheat's economic value and social significance are self-evident. Without timely and effective prevention and control, wheat diseases such as stripe rust, head blight, and snow rot can spread rapidly and severely impact wheat growth and development, leading to significant yield declines. Snow rot primarily damages key wheat seedling parts, including the roots, stems, leaves, and sheaths, causing widespread seedling death and resulting in missing seedlings and broken ridges, directly impacting wheat yield and quality.

[0005] While chemical pesticides are an effective means of controlling wheat diseases, they pose challenges such as high residue levels, high toxicity, and the development of resistance in pathogens. Long-term pesticide use can lead to environmental pollution and ecosystem disruption. Therefore, finding safe, effective, and environmentally friendly alternatives to chemical pesticides is crucial. Thyme essential oil, as a natural fungicide, offers low toxicity and environmental friendliness, making it a promising alternative to chemical fungicides. However, the use of thyme essential oil for wheat disease control is rarely reported, and there are few proven case studies. Further development of Thyme resources for wheat disease control is needed. Summary of the Invention

[0006] To safely prevent and control wheat snow rot, reduce pesticide usage, and ensure healthy wheat growth and high yield and quality, the present invention replaces traditional chemical pesticides with thyme essential oil, achieving significant results in preventing and controlling wheat snow rot. Specifically, the present invention provides the following detailed technical solutions.

[0007] Firstly, the present invention provides application of thyme essential oil in preventing and treating wheat snow rot, wherein the pathogen of the wheat snow rot is Microdochium nivale.

[0008] Further, in the above application, the thyme essential oil is extracted by the following method:

[0009] Weigh 20 g of dry thyme powder and place it in a round-bottom flask. Add distilled water containing 1% NaCl at a liquid-to-solid ratio of 30:1, mix well, and sonicate at 300 W for 45 min.

[0010] Connect the distillation apparatus and start timing from the time the first drop of distillate is collected. End the distillation after 2.0 h, maintaining a slight boiling temperature of 100°C, and collect the distillate.

[0011] Take 10-30 mL of n-hexane to extract the essential oil from the distillate, repeat the extraction three times, and collect the extracts three times;

[0012] Add 3-4.5 g of anhydrous sodium sulfate to the extract to remove excess water, then filter to remove solids;

[0013] The n-hexane is removed by rotary evaporation to obtain the thyme essential oil.

[0014] Furthermore, in the above application, the collected thyme sample is dried in the shade, ultrafinely ground and then passed through a 60-mesh sieve to obtain the thyme dry powder.

[0015] On the other hand, the present invention provides a method for preventing and controlling wheat snow rot, which comprises: applying thyme essential oil as an active ingredient to wheat.

[0016] Furthermore, in the above-mentioned prevention and control method, the pathogen of wheat snow rot is Microdochium nivale.

[0017] Furthermore, in the above-mentioned prevention and control method, the thyme essential oil is extracted by the following method:

[0018] Weigh 20 g of dry thyme powder and place it in a round-bottom flask. Add distilled water containing 1% NaCl at a liquid-to-solid ratio of 30:1, mix well, and sonicate at 300 W for 45 min.

[0019] Connect the distillation apparatus and start timing from the time the first drop of distillate is collected. End the distillation after 2.0 h, maintaining a slight boiling temperature of 100°C, and collect the distillate.

[0020] Take 10-30 mL of n-hexane to extract the essential oil from the distillate, repeat the extraction three times, and collect the extracts three times;

[0021] Add 3-4.5 g of anhydrous sodium sulfate to the extract to remove excess water, then filter to remove solids;

[0022] The n-hexane is removed by rotary evaporation to obtain the thyme essential oil.

[0023] Furthermore, in the above prevention and control method, the collected thyme sample is dried in the shade, ultrafinely ground and then passed through a 60-mesh sieve to obtain the thyme dry powder.

[0024] Compared with the prior art, the invention "application of thyme essential oil in preventing and treating wheat snow rot" has at least the following beneficial effects:

[0025] Through single factor experiments and response surface optimization experiments, the present invention obtains the following optimal extraction process conditions for thyme essential oil: extraction by ultrasonic-assisted steam distillation, a liquid-to-solid ratio of 30:1, a distillation time of 2.0 h (timed from the collection of the first drop of distillate), an ultrasonic power of 300 W, and an ultrasonic treatment time of 45 min.

[0026] It has been verified that the thyme essential oil prepared by the above method has a significant inhibitory effect on the mycelial growth, spore germination and mycelial biomass accumulation of Microdochium nivale. Specifically, the minimum inhibitory concentration (MIC) for the mycelial growth of Microdochium nivale is 0.25 μL / mL, and the half effective concentration (EC 50 ) was 0.1545 μL / mL. The EC for inhibition of spore germination of Microdochium nivalis 50 The value was 0.1378 μL / mL. When the concentration of thyme essential oil reached 0.375 μL / mL, spore germination was completely inhibited. In terms of the inhibition of mycelial biomass accumulation of Microdochium nivale at a concentration of 0.3125 μL / mL, the fresh weight and dry weight of mycelium of Microdochium nivale were 0.08g and 0.01g, respectively, which were only 2.29% and 2.78% of the control group, showing a significant inhibitory effect.

[0027] Based on the above inhibitory effect, thyme essential oil was used as the active ingredient and used to treat wheat snow rot caused by Microdochium nivale. When the concentration of thyme essential oil was 0.5 μL / mL, the disease index of wheat snow rot was reduced to 26.2, and the disease prevention effect reached 60.42%, with significant results.

[0028] The present invention provides an optimized thyme essential oil extraction process, further improving the yield of thyme essential oil and providing a theoretical basis for fully utilizing thyme's active ingredients. Furthermore, the present invention provides a safe and efficient method for preventing and treating wheat snow rot, helping to reduce the use of chemical pesticides and ensuring wheat yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the yield of thyme essential oil under different liquid-to-solid ratios.

[0030] Figure 2 The yield of thyme essential oil under different distillation time conditions.

[0031] Figure 3 is the yield of thyme essential oil under different ultrasonic power conditions.

[0032] Figure 4 The yield of thyme essential oil under different ultrasonic time conditions.

[0033] Figure 5 Response surface diagram (left) and contour diagram (right) of the effects of liquid-to-solid ratio and ultrasonic power on the yield of thyme essential oil.

[0034] Figure 6 Response surface diagram (left) and contour diagram (right) showing the effects of liquid-to-solid ratio and distillation time on the yield of thyme essential oil.

[0035] Figure 7 Response surface diagram (left) and contour diagram (right) of the effects of ultrasonic power and ultrasonic time on the yield of thyme essential oil.

[0036] Figure 8 Response surface diagram (left) and contour diagram (right) showing the effects of ultrasonic power and distillation time on the yield of thyme essential oil.

[0037] Figure 9 The effect of different concentrations of thyme essential oil on the growth of Microdochium nivale colonies.

[0038] Figure 10 The inhibition rate of mycelial growth of Microdochium nivale (a) and the toxicity regression curve (b) under treatment with different concentrations of thyme essential oil.

[0039] Figure 11 The effect of different concentrations of thyme essential oil on the germination of Microdochium nivale spores.

[0040] Figure 12 The spore germination rate, germination inhibition rate (a) and toxicity regression curve (b) of Microdochium nivale under treatment with different concentrations of thyme essential oil.

[0041] Figure 13The effects of different concentrations of thyme essential oil on the mycelial biomass accumulation of Microdochium nivale.

[0042] Figure 14 Fresh weight, fresh weight inhibition rate (a), dry weight, dry weight inhibition rate (b) of mycelium of Microdochium nivale treated with different concentrations of thyme essential oil. DETAILED DESCRIPTION

[0043] The present invention is explained below with reference to the embodiments, and the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0044] Example 1

[0045] This example describes the extraction of thyme essential oil and optimization of the extraction process.

[0046] 1. Extraction of thyme essential oil

[0047] The collected thyme samples were dried in the shade, ultrafinely ground, and then passed through a 60-mesh sieve to obtain thyme dry powder; 20 g of thyme dry powder was weighed and placed in a 1000 mL round-bottom flask, and distilled water containing 1% NaCl was added according to different liquid-to-solid ratios (mL / g), mixed, and treated with a certain ultrasonic power and time;

[0048] Connect the distillation apparatus, start timing from the time the first drop of distillate is collected, and end the distillation after a certain period of time, during which the temperature is kept at a slight boiling point of 100°C, and collect the distillate mixed with oil and water;

[0049] Take 10-30 mL of n-hexane to extract the essential oil from the distillate, repeat the extraction three times, and collect the extracts three times;

[0050] Add 3-4.5 g of anhydrous sodium sulfate to the extract to remove excess water, then filter to remove solids;

[0051] The n-hexane is removed by rotary evaporation to obtain the thyme essential oil.

[0052] 2. Optimization of Thyme Essential Oil Extraction Process

[0053] 1. Single-factor experiment

[0054] General extraction conditions: liquid-to-solid ratio of 30:1, ultrasonic power of 300W, ultrasonic time of 45min, and distillation time of 2.0h.

[0055] On this basis, the liquid-to-solid ratio (10:1, 20:1, 30:1, 40:1, 50:1), ultrasonic power (250W, 300W, 350W, 400W, 450W), ultrasonic time (15min, 30min, 45min, 60min, 75min), and distillation time (0.5h, 1.0h, 1.5h, 2.0h, 2.5h) were selected for single factor experiments, and the thyme essential oil yield was used as the measurement index to determine the optimal value of each single factor condition.

[0056] 1) Effect of liquid-to-solid ratio on thyme essential oil yield

[0057] Figure 1 is the yield of thyme essential oil under different liquid-to-solid ratio conditions. Figure 1 As can be seen, as the liquid-to-solid ratio (mL / g) increases (10:1 to 30:1), the thyme essential oil yield shows a gradually increasing trend. When the liquid-to-solid ratio is 30:1, the essential oil yield reaches a maximum of 0.3164%. When the liquid-to-solid ratio increases from 30:1 to 50:1, the thyme essential oil yield gradually decreases. This may be because when the liquid-to-solid ratio is low, the solvent cannot fully penetrate the thyme powder, affecting the diffusion of essential oil molecules and preventing the essential oil components from being completely released from the cells. When the liquid-to-solid ratio is too high, the excessive solvent may dilute the essential oil, resulting in a decrease in the amount of essential oil distilled per unit time and a decrease in the essential oil yield. Therefore, a liquid-to-solid ratio of 30:1 is the best.

[0058] 2) Effect of distillation time on thyme essential oil yield

[0059] Figure 2 is the yield of thyme essential oil under different distillation time conditions. Figure 2 As can be seen, the yield of thyme essential oil increases rapidly with increasing distillation time (0.5-2.0 hours), reaching a maximum of 0.3111% at 2.0 hours. Beyond 2.0 hours, the yield decreases. This may be due to the dual effects of heat and steam, which carry the essential oil from its liquid or solid state out of the intercellular spaces and cells with the steam. Within a short period, the yield is proportional to the distillation time. As the distillation time increases, the majority of the essential oil components in the plant material are fully extracted, and the yield reaches its peak and stops increasing. However, due to the volatility and heat sensitivity of essential oils, the yield gradually decreases with increasing distillation time. Furthermore, experiments have shown that after 2.0 hours, non-essential components such as waxes, pigments, and resins are also largely distilled out, leading to an increase in impurities and a decrease in quality. Therefore, a distillation time of 2.0 hours is considered appropriate.

[0060] 3) Effect of ultrasonic power on the yield of thyme essential oil

[0061] Ultrasonic power can produce a strong cavitation effect, causing tiny bubbles in the liquid to be generated, expanded, and collapsed under the action of the ultrasonic field, generating high temperature, high pressure, and strong shock waves locally, thereby destroying the cell walls of the plants and making it easier for essential oils to be released from the cells.

[0062] Figure 3 is the yield of thyme essential oil under different ultrasonic power conditions. Figure 3 As can be seen, with increasing ultrasonic power (250-450W), the yield of thyme essential oil shows a trend of first increasing and then decreasing. When the ultrasonic power reaches 300W, the yield of thyme essential oil reaches a maximum of 0.3151%. When the ultrasonic power increases from 300W to 450W, the yield of thyme essential oil gradually decreases. This may be because lower ultrasonic power cannot fully destroy the plant cell walls, resulting in some components not being fully extracted. When the ultrasonic power is too high, it may cause localized excessive temperatures, resulting in the loss of volatile substances in the essential oil, thereby reducing the essential oil yield. Therefore, choosing an ultrasonic power of 300W is appropriate.

[0063] 4) Effect of ultrasonic time on the yield of thyme essential oil

[0064] Figure 4 is the yield of thyme essential oil under different ultrasonic time conditions. Figure 4 As can be seen, as the ultrasonic time increases (1 to 45 minutes), the thyme essential oil yield shows an upward trend. When the ultrasonic time is 45 minutes, the essential oil yield reaches a maximum of 0.3165%. As the ultrasonic time continues to extend within the range of 45 to 75 minutes, the thyme essential oil yield gradually decreases. This may be because the shorter ultrasonic time cannot allow the ultrasound to fully penetrate the interior of the plant material, and the essential oil components wrapped in the cell wall and cell membrane cannot be fully dissolved, resulting in a decrease in the essential oil yield. When the ultrasonic time reaches a certain level, further increasing the ultrasonic time will cause the already dissolved essential oil to decompose or volatilize, thereby causing a decrease in the essential oil yield. Therefore, it is advisable to select an ultrasonic time of 45 minutes.

[0065] 2. Response Surface Optimization Experiment

[0066] According to the results of the single factor experiment, the thyme essential oil yield (Y) was taken as the response value, and the Box-Behnken experimental scheme was designed using the Design-Expert13 software. The liquid-to-solid ratio (A), ultrasonic power (B), ultrasonic time (C), and distillation time (D) were selected as independent variables. The four-factor three-level response surface analysis method was used for experimental analysis. The factor levels are shown in Table 1. In this way, the thyme essential oil extraction process was optimized.

[0067] Table 1. Factor levels in Box-Behnken response surface experiments

[0068]

[0069] 1) Response surface experimental design and results.

[0070] The response surface design and results are shown in Table 2. Multiple regression analysis was performed on the data in Table 2 using Design-Expert 13 software to obtain a quadratic regression equation. The final equation obtained based on the coding factors is:

[0071] Y=0.3213+0.0006A+0.0008B-0.0014C+0.0019D-0.0011AB+0.0010AC+0.0012AD-0.0014BC+0.0013BD+0.0008CD-0.0145A 2 -0.0129B 2 -0.0152C 2 -0.0162D 2 .

[0072] The final equation based on practical factors is:

[0073] Y=-0.662377+0.008658A+0.003170B+0.006134C+0.235237D-2.25000E-06AB+6. 83333E-06AC+0.000235AD-1.90000E-06BC+0.000051BD+0.000110CD-0.000145A 2 -5.16967E-06B 2 -0.000068C 2 -0.064697D 2 .

[0074] Table 2. Response surface experimental design and results

[0075] Serial number A liquid-to-solid ratio (mL / g) B Ultrasonic power / W C Ultrasonic time / min D Distillation time / h Essential oil yield / % 1 -1 -1 0 0 0.2911 2 1 -1 0 0 0.2958 3 -1 1 0 0 0.2939 4 1 1 0 0 0.2941 5 0 0 -1 -1 0.2896 6 0 0 1 -1 0.2854 7 0 0 -1 1 0.2925 8 0 0 1 1 0.2916 9 -1 0 0 -1 0.2895 10 1 0 0 -1 0.2877 11 -1 0 0 1 0.2915 12 1 0 0 1 0.2944 13 0 -1 -1 0 0.2913 14 0 1 -1 0 0.2974 15 0 -1 1 0 0.2921 16 0 1 1 0 0.2925 17 -1 0 -1 0 0.2942 18 1 0 -1 0 0.2928 19 -1 0 1 0 0.2886 20 1 0 1 0 0.2913 21 0 -1 0 -1 0.2918 22 0 1 0 -1 0.2905 23 0 -1 0 1 0.2916 24 0 1 0 1 0.2954 25 0 0 0 0 0.3221 26 0 0 0 0 0.3203 27 0 0 0 0 0.3220 28 0 0 0 0 0.3208 29 0 0 0 0 0.3215

[0076] 2) Variance analysis and credibility analysis of regression models

[0077] Variance analysis and credibility analysis were performed on the regression model of thyme essential oil yield, and the results are shown in Tables 3 and 4.

[0078] Table 3. Results of variance analysis of regression model

[0079]

[0080] Note: “*” indicates significant difference (P<0.05); “**” indicates extremely significant difference (P<0.01); “#” indicates no significant difference (P>0.05).

[0081] Table 4. Results of the regression model credibility analysis

[0082]

[0083] From the results of variance analysis in Table 3, we can see that the P value of the regression model is <0.0001, which is extremely significant, while the lack of fit term P=0.3024>0.05 is not significant. The F value of the model is 282.72, which shows that the model is reliable, the regression equation has a high degree of fit with the actual situation, and the error is small. It can well analyze the relationship and change between the yield of thyme essential oil and the factor value. The P value <0.05 indicates that the simulation term is significant. The results of variance analysis of the regression model show that B, C, D, A 2 、B 2 、C 2 、D 2 The model showed that the influence of the four single factors on thyme oil yield was ranked as follows: D (distillation time) > C (ultrasound time) > B (ultrasound power) > A (liquid-to-solid ratio), which is consistent with the results of the variance analysis.

[0084] In Table 4, the coefficient of variation is 0.3269%, which is less than 10%, indicating that the accuracy of the regression model and the reliability of the test data are high; the coefficient of determination R 2 =0.9965, indicating that the model fits well and can explain 99.65% of the variability in the response surface; the adjusted R 2 =0.9930, and the difference between the two is less than 0.2, indicating that the measured value and the predicted value have a good correlation and the model is highly credible; the signal-to-noise ratio is 50.746>4, indicating that the model has a strong enough signal and can be used to determine the optimal extraction process conditions for thyme essential oil.

[0085] 3) Contour and response surface analysis

[0086] The response surface diagram can show the interaction patterns between the four single factors A, B, C, and D and their impact on the response value essential oil yield. Figures 5 to 8 They are the response surface curves and contour maps between liquid-to-material ratio and ultrasonic power (AB), liquid-to-material ratio and distillation time (AD), ultrasonic power and ultrasonic time (BC), and ultrasonic power and distillation time (BD). Figures 5 to 8It can be seen that the slopes of the response surface curves between liquid-to-material ratio and ultrasonic power (AB), liquid-to-material ratio and distillation time (AD), ultrasonic power and ultrasonic time (BC), and ultrasonic power and distillation time (BD) are relatively large, and the contour lines in the figure are all ellipses, indicating that the interaction between the two factors is significant and has a significant impact on the essential oil yield; at the same time, from the steepness of the response surface curve, it can be seen that BC>BD>AD>AB, which is consistent with the results of variance analysis.

[0087] 4) Experimental verification of optimal extraction process conditions

[0088] Through the above response surface test analysis, the optimal process conditions for extracting thyme essential oil are: liquid-to-material ratio of 30.2040:1, ultrasonic power of 301.8640W, ultrasonic time of 44.3370min, and distillation time of 2.0290h. Under these conditions, the maximum theoretical predicted value of thyme essential oil yield is 0.3210%. Taking into account the actual operability, according to the liquid-to-material ratio of 30:1, distillation time of 2.0h, ultrasonic power of 300W, and ultrasonic time of 45min, three verifications were carried out under these conditions, and the average value of thyme essential oil yield was 0.3200±0.0010%, which is close to the model prediction value, indicating that the model is reliable in simulating the process conditions of extracting thyme essential oil by ultrasound-assisted steam distillation.

[0089] 3. Comparative test

[0090] To verify whether ultrasound-assisted steam distillation significantly improves thyme essential oil yield compared to steam distillation alone, 20 g of dry thyme powder was weighed and extracted three times at a liquid-to-solid ratio of 30:1 and a distillation time of 2.0 h. The essential oil yield was calculated and the average results were taken (Table 5).

[0091] Table 5. Comparative test results

[0092]

[0093] As shown in Table 5, the yield of thyme essential oil without ultrasonic treatment was 0.2614%, while the yield with ultrasonic-assisted treatment was 0.3200%, an increase of 0.0586%, accounting for approximately 22.42% of the yield without ultrasonic treatment. This indicates that ultrasonic-assisted steam distillation can significantly increase the yield of thyme essential oil. The results in Table 5 further verify the feasibility of ultrasonic treatment in increasing the yield of thyme essential oil.

[0094] Example 2

[0095] This example describes the effect of thyme essential oil on the growth of Microdochium nivale.

[0096] 1. Determination of the inhibitory effect of thyme essential oil on the mycelial growth of Microdochium nivale

[0097] 1. Test methods

[0098] Thoroughly mix 120 μL of thyme essential oil and 48 μL of TW-80 solution, add 3832 μL of sterile water to prepare a 30 μL / mL thyme essential oil stock solution, and store it in a refrigerator at 4°C until use.

[0099] The mycelial growth rate method was used to determine the inhibitory effect of thyme essential oil on the mycelial growth of Microdochium nivale. The specific method was as follows: different volumes of thyme essential oil stock solution were added to 60 mL of sterilized potato dextrose agar (PDA) medium that had been naturally cooled to 50°C, and the mixture was thoroughly mixed to prepare thyme essential oil-containing culture media with final thyme essential oil concentrations of 0.0625, 0.125, 0.1875, 0.25, 0.3125, and 0.375 μL / mL. The culture media were then poured into plates and cooled for later use. PDA plates without any treatment were used as blank controls (CK); PDA plates added with 0.02% TW-80 solution were used as negative controls. Each treatment was repeated four times.

[0100] A 5mm diameter bacterial cake was obtained from the edge of a 5d activated Microdochium nivalis colony and placed in the center of a culture dish. The cake was cultured in a constant temperature incubator at 20°C. The colony diameter was measured by the cross method every 24h, and the mycelial growth inhibition rate was calculated. The essential oil concentration at which no mycelial growth was observed within 48h was taken as the minimum inhibitory concentration (MIC).

[0101]

[0102] The logarithm of thyme essential oil concentration was plotted on the x-axis and the probability of mycelial growth inhibition rate was plotted on the y-axis. Regression analysis was performed to determine the toxicity of thyme essential oil on mycelial growth of Microdochium nivale (y=ax+b) and the correlation coefficient R. 2 The effective concentration (EC) was calculated based on the toxicity regression equation. 50 .

[0103] 2. Test results

[0104] As shown in Table 6, no mycelial growth occurred within 48 h at thyme essential oil concentrations of 0.25 μL / mL or above. Therefore, the MIC of thyme essential oil against the mycelial growth of Microdochium nivale was 0.25 μL / mL.

[0105] Table 6. MIC values of thyme essential oil treatment against Microdochium nivale

[0106]

[0107]

[0108] Note: “+” indicates colony growth, “-” indicates colony no growth.

[0109] Figure 9 The following table shows the effects of different concentrations of thyme essential oil on the growth of Microdochium nivale colonies. Figure 9 As shown, mycelial growth in the CK and 0.02% TW-80 treatment groups continued throughout the culture period, while the addition of thyme essential oil to the culture medium effectively inhibited this growth, with this inhibitory effect becoming more pronounced with increasing concentration. Thyme essential oil at a concentration of 0.375 μL / mL completely inhibited the growth of M. nivalis mycelium. The colony size of the 0.02% TW-80 treatment group did not differ significantly from that of the CK control from day 1 to 5, indicating that 0.02% TW-80 as a solvent had no inhibitory effect on colony growth, and that the inhibitory effect on M. nivalis mycelial growth was contributed by thyme essential oil.

[0110] Figure 10 The following are the inhibition rates (a) and toxicity regression curves (b) of Microdochium nivale hyphae growth under different concentrations of thyme essential oil. Figure 10 As shown in (a), the mycelial growth inhibition rate showed a concentration-effect relationship with the concentration of thyme essential oil. After 5 days of PDA culture, the inhibition rate of 0.0625-0.3125 μL / mL thyme essential oil treatment was 16.13-84.95%; 0.375 μL / mL thyme essential oil completely inhibited mycelial growth. Figure 10 As shown in (b), a linear regression relationship was established between the thyme essential oil concentration and the mycelial growth inhibition rate to obtain the EC 50 The toxicity regression equation is: y = 2.7691x + 7.2456, R 2 =0.9702,EC 50 The value is 0.1545μL / mL.

[0111] 2. Determination of the inhibitory effect of li xiang essential oil on the germination of Microdochium nivale spores

[0112] 1. Solution Preparation

[0113] 1) Preparation of spore stock solution

[0114] Inject 3 mL of a 0.02% TW-80 solution into a PDA culture plate containing spores of Microdochium nivale. Elute the spores by repeatedly pipetting. Place the eluate into a 5 mL sterile syringe with a sterile cotton filter, filter out the culture medium and mycelium, and aliquot into 5 mL sterile centrifuge tubes. Draw out 100 μL of the solution, dilute it by the desired multiple, and count the spores under an optical microscope using a hemocytometer to determine the final concentration. This is the spore stock solution, which should be stored at 4°C until needed.

[0115] 2) Preparation of thyme essential oil mother liquor

[0116] Thoroughly mix 50 μL of thyme essential oil and 20 μL of TW-80 solution, add 930 μL of sterile water to prepare a 50 μL / mL thyme essential oil stock solution, and store at 4°C until use.

[0117] 3) Preparation of spore suspension containing thyme essential oil

[0118] 1.25, 2.5, 3.75, 5, 6.25, and 7.5 μL of thyme essential oil stock solution were pipetted into 1.5 mL sterile centrifuge tubes. 100 μL of spore stock solution was added, and the volume was made up to 1 mL with 0.02% TW-80 solution. The suspension was thoroughly mixed to prepare thyme essential oil-containing spore suspensions. The thyme essential oil concentrations were 0, 0.625, 0.125, 0.1875, 0.25, 0.3125, and 0.375 μL / mL, respectively. A control group (CK) was used without the addition of thyme essential oil stock solution, and all other treatments were the same.

[0119] 2. Test methods

[0120] Add 2-3 drops of thyme essential oil-containing spore suspension to the center of a concave glass slide. Place the concave glass slide in a sterile Petri dish containing moist filter paper and incubate in a 25°C incubator to maintain moisture. Microscopic observation is performed after 4 hours and every 2 hours thereafter. Spore germination is considered complete when the length of the germ tube exceeds half the spore diameter. Spore germination is counted. Spore germination statistics are collected when the spore germination rate in the control group (CK) exceeds 80%. Each treatment is replicated three times, with three fields of view examined microscopically in each replicate. Germination of at least 100 spores should be counted in each microscopic examination to calculate the spore germination rate and germination inhibition rate.

[0121] Spore germination rate (%) = number of germinated spores / total number of spores examined under the microscope × 100;

[0122] Spore germination inhibition rate (%) = [(spore germination rate of the control group - spore germination rate of the treatment group) / spore germination rate of the control group] × 100.

[0123] The logarithm of thyme essential oil concentration was plotted on the x-axis and the probability of spore germination inhibition rate on the y-axis. Regression analysis was performed to determine the toxicity of thyme essential oil on the spore germination of Microspore nivalis, and the regression equation y = ax + b and the correlation coefficient R were obtained. 2 , the half effective concentration EC was obtained according to the regression equation 50 .

[0124] 3. Test results

[0125] At 6 h, the spore germination rate of the control group (CK) exceeded 80%, so this time point was selected for the calculation of the spore germination rate and germination inhibition rate. Figure 11The following table shows the effects of different concentrations of thyme essential oil on the germination of Microdochium nivale spores. Figure 11 As shown in the figure, at 6 h, more spores in the CK group had fully germinated and the germ tubes were longer. As the concentration of thyme essential oil increased, the germination rate of Microdochium nivalis spores gradually decreased and the germ tube length shortened. When the essential oil concentration reached 0.375 μL / mL, most spores lost their germination ability, became sickle-shaped, and no germ tubes were observed.

[0126] Figure 12 The spore germination rate, germination inhibition rate (a) and toxicity regression curve (b) of Microdochium nivale under different concentrations of thyme essential oil treatment. Figure 12 As shown in (a), thyme essential oil has an inhibitory effect on the spore germination of Microdochium nivale, and the effect is concentration-dependent. After 6 hours of culture, the spore germination rate of the CK group was 93%. As the concentration of thyme essential oil increased, the spore germination rate gradually decreased. The spore germination rate of the 0.0625-0.3125 μL / mL thyme essential oil treatment was 3.67-84%, and the spore germination inhibition rate was 9.68-96.06%. When the concentration of thyme essential oil reached 0.375 μL / mL, spore germination was completely inhibited. Figure 12 As shown in (b), the toxicity regression equation of thyme essential oil in inhibiting the germination of Microdochium nivale spores is y=4.1729x+8.5914, and the correlation coefficient R 2 =0.9717,EC 50 The value is 0.1378μL / mL.

[0127] 3. Determination of the inhibitory effect of thyme essential oil on the accumulation of mycelial biomass of Microdochium nivale

[0128] 1. Test methods

[0129] Preparation of PDB culture medium (1000 mL): 200 g potato, 20 g glucose, 1000 mL deionized water, sterilize at 121°C for 20 min.

[0130] Different volumes of thyme essential oil were added to 150 mL of PDB medium so that the final concentrations of thyme essential oil in PDB medium were 0.0625, 0.125, 0.1875, 0.25, and 0.3125 μL / mL. Ten cakes of Microsporum nivale (6 mm in diameter) were used to inoculate the PDB medium, and each concentration was repeated 3 times. TW-80 containing 0.02% was used instead of thyme essential oil as a control (CK), and the rest of the treatments were the same. Each group was placed in a constant temperature shaking incubator and cultured at 20°C, 12h light / 12h dark for 15 days. After that, the mycelia were collected, the fresh weight and dry weight of the mycelia were weighed, and the fresh weight and dry weight inhibition rates of the mycelia were calculated respectively. The calculation formula is as follows:

[0131] Fresh weight inhibition rate (%) = [(fresh weight of mycelia in the control group - fresh weight of mycelia in the experimental group) / fresh weight of mycelia in the control group] × 100;

[0132] Dry weight inhibition rate (%) = [(dry weight of mycelia in the control group - dry weight of mycelia in the experimental group) / dry weight of mycelia in the control group] × 100.

[0133] 2. Test results

[0134] The inhibitory effect of thyme essential oil on the biomass accumulation of Microdochium nivale mycelium can be reflected by measuring the dry weight of mycelium. Figure 13 The following table shows the effects of different concentrations of thyme essential oil on the accumulation of mycelial biomass of Microdochium nivale. Figure 13 As shown in the figure, with the increase of thyme essential oil concentration, the mycelial mass gradually became sparse. When the essential oil concentration was 0.3125 μL / mL, only scattered mycelia were visible. This shows that thyme essential oil treatment can significantly inhibit the growth of Microdochium nivalis and reduce mycelial biomass.

[0135] Figure 14 The fresh weight, fresh weight inhibition rate (a), dry weight, and dry weight inhibition rate (b) of the mycelium of Microdochium nivale under different concentrations of thyme essential oil treatment. Figure 14 As shown in Figures (a) and (b), with increasing thyme essential oil concentration, both the fresh and dry weights of mycelium decreased significantly in a concentration-dependent manner. While the fresh and dry weights of mycelium in the control group were 3.49 g and 0.36 g, respectively, at a concentration of 0.3125 μL / mL, the fresh and dry weights of mycelium in Microdochium nivale were 0.08 g and 0.01 g, respectively, representing only 2.29% and 2.78% of the control group. These results demonstrate that thyme essential oil significantly inhibits mycelial biomass accumulation in Microdochium nivale, with the inhibitory effect increasing with increasing concentration.

[0136] Example 3

[0137] This example provides a potted plant experiment on the control effect of thyme essential oil on snow rot of wheat seedlings.

[0138] In this example, the wheat variety used in the experiment is Xindong 20; the pathogen of wheat snow rot used in the experiment is Microdochium nivale, which was isolated and identified by this laboratory.

[0139] 1. Experimental Preparation

[0140] Preparation of wheat grain culture medium: Wash the wheat grains and soak them overnight, boil them in boiling water for 30 minutes, remove them and rinse them with cold water for 3 to 4 times, dry them and divide them into conical flasks, place the conical flasks filled with wheat grains in a wet heat sterilizer, and sterilize them at 0.1 MPa and 121°C for 20 minutes.

[0141] Propagation of pathogens using wheat grain culture medium: First, inoculate PDA medium with Microdochium nivale. After 3 days of culture, remove a 5mm diameter cake from the edge of the growing mycelium and inoculate it into cooled sterilized wheat grain culture medium. Inoculate 10 cakes into each conical flask with the same inoculum volume. Incubate in a 20°C incubator for 10 days, shaking the conical flask regularly to ensure that the mycelium is evenly distributed on the wheat grain culture medium. Once the mycelium evenly covers the surface of the wheat grain culture medium, pour it out, spread it flat on a clean paper, and place it in a cool, dry, well-ventilated place to dry until ready for use.

[0142] The drug solution settings for the experimental group and the control group were: the concentrations of thyme essential oil in the experimental group were 0.0625, 0.125, 0.25, and 0.5 μL / mL, respectively, and the drug solutions used in the experimental groups all contained 0.02% TW-80; the negative control group: 0.02% TW-80 solution; the positive control group: 50% carbendazim (1000 times solution).

[0143] 2. Experimental steps

[0144] Select healthy, plump, and uniform-sized wheat seeds, disinfect them with 1% sodium hypochlorite for 15 minutes, rinse them three times with sterile water, place them in a germination box covered with two layers of filter paper, and germinate them at 25°C for 24 hours.

[0145] Fill the bottom of a 15 cm diameter pot halfway with sterile nutrient soil (peat soil: vermiculite = 3:1), flatten it, and sow nine wheat seeds of uniform germination status per pot. Separately, mix wheat grain culture medium containing Microdochium nivalis and sterile nutrient soil at a mass ratio of 0.75:100 and cover the wheat seeds to a thickness of 3 cm. Irrigate each pot with 30 mL of the solution, followed by a second irrigation at the same volume 24 hours later. Cultivate for 30 days at 25°C / 20°C, 14 hours light / 10 hours dark, and 60-70% humidity. A 0.02% TW-80 solution was substituted for the thyme essential oil solution, with all other treatments remaining the same, as a negative control. A 1000x dilution of 50% carbendazim was substituted for the thyme essential oil solution, with all other treatments remaining the same, as a positive control. Wheat seeds were sown and covered with sterile nutrient soil and irrigated with an equal volume of water as a blank control. Six replicates were used for each treatment.

[0146] After the incubation period, the wheat seedlings were removed from the pots and carefully rinsed with clean water. The incidence of wheat stem rot was recorded according to the grading standards of Poole et al. and He Xiaolun, and the disease index and control effect (%) were calculated. The disease grading standards are as follows:

[0147] Level 0: Plants are not diseased;

[0148] Level 1: The stem in the ground turns obviously brown or the first leaf sheath shows mild symptoms;

[0149] Level 3: The first leaf sheath turns obviously brown, but the leaf sheath does not turn black;

[0150] Level 5: The first leaf sheath turns black or the second leaf sheath turns brown;

[0151] Level 7: Browning symptoms appear on the third leaf sheath, or the plant is stunted or close to death due to the disease;

[0152] Level 9: The plant dies due to the disease.

[0153] Disease index and prevention and treatment effect calculation formula:

[0154]

[0155] 3. Test results

[0156] The disease index and prevention and treatment effects of the experimental group and the control group are shown in Table 7.

[0157] Table 7. Thyme essential oil treatment on wheat snow rot pot control effect

[0158]

[0159] As shown in Table 7, the negative control group, irrigated with only 0.02% TW-80, had a more severe disease. In the experimental group, the disease index of wheat snow rot gradually decreased with increasing thyme essential oil concentration. After applying 0.0625, 0.125, 0.25, and 0.5 μL / mL of thyme essential oil, the disease index of wheat snow rot was 60.6, 55.2, 41.8, and 26.2, respectively. The disease prevention efficacy was 8.46%, 16.62%, 36.86%, and 60.42%, respectively, which were significantly different from the negative control group (P < 0.05). This indicates that thyme essential oil can effectively inhibit the occurrence and development of Microdochium nivalis and is significantly effective in preventing and controlling wheat snow rot caused by Microdochium nivalis.

[0160] To sum up, the present invention, by single factor experiment and response surface optimization test, draws that thyme essential oil optimal extraction process condition is: adopt ultrasound-assisted steam distillation method to extract, liquid-to-material ratio 30:1, distillation time 2.0h (starting timing from collecting the first distillate), ultrasonic power 300W, ultrasonic treatment time 45min.Further test is learned, the thyme essential oil prepared by the above method has a significant inhibitory effect on mycelial growth, spore germination, mycelial biomass accumulation of Microdochium nivale.Based on this, using thyme essential oil as active ingredient, it is used for the wheat snow rot caused by Microdochium nivale, when thyme essential oil concentration 0.5 μ L / mL, the disease index of wheat snow rot is reduced to 26.2, and disease prevention effect is 60.42%, and is effective.

[0161] The embodiments described above are only some of the embodiments of the present invention, not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but merely represents selected embodiments of the present invention. All other embodiments obtained without creative effort and through deduction and substitution by a person of ordinary skill in the art based on the concept of the present invention are within the scope of protection of the present invention.

Claims

1. The application of thyme essential oil in preventing and treating wheat snow rot is characterized in that, The pathogen of wheat snow rot is Microdochium nivale.

2. The use according to claim 1, characterized in that The thyme essential oil is extracted by the following method: 20 g of dry thyme powder is weighed and placed in a round-bottom flask, distilled water containing 1% NaCl is added at a liquid-to-solid ratio of 30:1, mixed, and ultrasonically treated at an ultrasonic power of 300 W for 45 minutes; Connect the distillation apparatus and start timing from the time the first drop of distillate is collected. End the distillation after 2.0 h, maintaining a slight boiling temperature of 100°C, and collect the distillate. Extract the essential oil from the distillate with 10-30 mL of n-hexane, repeat the extraction three times, and collect the extracts three times; add 3-4.5 g of anhydrous sodium sulfate to the extract to remove excess water, and then filter to remove solids; The n-hexane is removed by rotary evaporation to obtain the thyme essential oil.

3. The use according to claim 2, characterized in that The collected thyme sample is dried in the shade, ultrafinely ground, and then passed through a 60-mesh sieve to obtain the thyme dry powder.

4. A method for preventing and treating wheat snow rot, characterized in that: include: Thyme essential oil was used as the active ingredient in wheat.

5. The control method according to claim 4, characterized in that: The pathogen of wheat snow rot is Microdochium nivale.

6. The control method according to claim 4, characterized in that: Described thyme essential oil is extracted by the following method: Weigh 20 g of dry thyme powder and place it in a round-bottom flask. Add distilled water containing 1% NaCl at a liquid-to-solid ratio of 30:1, mix well, and sonicate at 300 W for 45 min. Connect the distillation apparatus and start timing from the time the first drop of distillate is collected. End the distillation after 2.0 h, maintaining a slight boiling temperature of 100°C, and collect the distillate. Take 10-30 mL of n-hexane to extract the essential oil from the distillate, repeat the extraction three times, and collect the extracts three times; Add 3-4.5 g of anhydrous sodium sulfate to the extract to remove excess water, then filter to remove solids; The n-hexane is removed by rotary evaporation to obtain the thyme essential oil.

7. The control method according to claim 6, characterized in that: The collected thyme sample is dried in the shade, ultrafinely ground, and then passed through a 60-mesh sieve to obtain the thyme dry powder.

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