Method for preventing and controlling alternaria alternata by using tyrosol

Abstract

The application provides a method for preventing and controlling pear ring rot by using tyrosol, and the pear ring rot is prevented and controlled by spraying tyrosol aqueous solution externally; the concentration of tyrosol in the tyrosol aqueous solution is 1-5 mg / ml. The application takes 'Dangshan' pear as experimental material, and proves that tyrosol has a significant inhibitory effect on pear ring rot fungus through in-vitro antibacterial experiment and ring rot fungus inoculation experiment, can effectively inhibit the development speed of ring rot fungus spots, reduces the occurrence and spread of diseases, and thus reduces the invasion of ring rot fungus on plants. Tyrosol is a natural compound in plants, has low toxicity, and when applied to the prevention and control of pear ring rot, can enhance the defense mechanism of the plants, improve the natural resistance of the plants to ring rot, simultaneously reduces or replaces the use of chemical pesticides, and reduces the potential risks to the environment and human health.

Description

Technical Field

[0001] This invention belongs to the field of fruit tree disease control technology, and relates to natural fungicidal active compounds in plants, specifically a method for controlling pear ring rot using tyrosol. Background Technology

[0002] Pears are deciduous trees belonging to the Rosaceae family and the Pyrus genus. They are the third most popular fruit in my country, after apples and citrus fruits. Pears contain metabolites such as flavanols, phenolic acids, and arbutin, which play a crucial role in the fruit's adaptation to its environment and provide essential resources for human health. Pears are fragrant, crisp, juicy, and nutritious. Their main active chemical components are phenolic substances, primarily phenolic acids and flavonoids, such as arbutin, chlorogenic acid, and caffeic acid, which possess medicinal and edible properties, including moisturizing the lungs and relieving coughs, scavenging free radicals, and inhibiting bacteria. Pears are widely cultivated in my country, mainly in Hebei, Shandong, Shaanxi, and Gansu provinces. However, with the increase in cultivation area, various pests and diseases have also increased. Pear ring rot is the most common disease in my country, spreading throughout the country and seriously hindering the development of the pear industry.

[0003] Pear ring rot is a fungal disease caused by *Botryosphaeria dothidea* (B. dothidea), the main pathogen causing the disease. It typically enters the plant through wounds or natural openings, leading to infection. This disease not only weakens pear trees but can also cause fruit rot in severe cases, even destroying orchards and causing significant economic losses for pear growers, hindering the sustainable development of the pear industry. The occurrence of pear ring rot is closely related to environmental factors such as temperature, humidity, and climatic conditions, as well as the physiological condition of the pear tree itself. The pathogen mainly enters through wounds or pruning cuts on the pear tree and grows rapidly in humid environments, forming irregular brown spots centered on lenticels, ultimately leading to fruit rot. *Botryosphaeria dothidea* can infect 312 species of plants in 24 genera, including pome fruits (such as apples, pears, and hawthorns), stone fruits (such as peaches, plums, and apricots), berries (such as kiwifruit, pomegranates, and grapes), and trees (such as eucalyptus and locust trees). Studies show that the pathogens causing pear ring rot are very similar to those causing apple ring rot. During infection, they release a large number of effectors, which can weaken the plant's defense mechanisms and enhance the infectivity of the pathogens.

[0004] Currently, the main control measures for pear ring rot are chemical control. This is combined with exogenous spraying of fungicides to control other diseases, such as alternating applications of 50% carbendazim, 50% thiophanate-methyl, and 70% mancozeb. However, the use of chemical agents not only causes irreversible damage to the environment but also has a direct impact on human health. Long-term exposure can easily lead to chronic or acute poisoning and increase the risk of cancer. Furthermore, prolonged overuse or continuous use of a single pesticide can easily lead to resistance. Therefore, a natural, non-toxic, and highly effective fungicide is of great significance for resisting pear ring rot and improving pear yield and quality.

[0005] Tyrosol, a naturally occurring compound, is an environmentally friendly, natural antifungal agent that helps reduce the use of chemical pesticides. Compared to pesticides, tyrosol, as a plant-derived fungicide, is less likely to induce resistance in pathogens, which is crucial for long-term disease management. Tyrosol can be formulated into various forms, such as aqueous solutions, powders, and granules, facilitating its application in diverse agricultural production scenarios.

[0006] Tyrosol belongs to the phenol family and was originally derived from olive oil. Its molecular formula is C8H10O2, and its molecular weight is 138.164. CAS number: 501-94-0. Other names: p-hydroxyphenylethanol; English name: Tyrosol. Molecular structure:

[0007]

[0008] Tyrosol, a natural polyphenol compound, possesses a variety of biological and pharmacological activities. Currently, tyrosol has received widespread attention in the medical field, exhibiting multiple physiological activities such as antioxidant, anti-inflammatory, anti-cancer, cardiovascular disease prevention, and neuroprotective effects. In the food industry, it can be used as a food additive to improve flavor.

[0009] There are currently no studies or reports on the use of tyrosol in treating pear ring rot, either domestically or internationally. Summary of the Invention

[0010] To address the above problems, this invention provides a method for controlling pear ring rot using tyrosol.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] In a first aspect, the present invention claims protection for the use of tyrosol in the control of pear ring rot or in the preparation of fungicides for the control of pear ring rot. Further, the aforementioned use refers to the control of pear ring rot by exogenously spraying an aqueous solution of tyrosol.

[0013] Secondly, this invention claims protection for a method for controlling pear ring rot, which uses tyrosol to control pear ring rot. Further, the above method involves controlling pear ring rot by exogenously spraying an aqueous tyrosol solution.

[0014] In the technical solution of this invention, the concentration of tyrosol in the tyrosol aqueous solution is 1-5 mg / ml; preferably, 3-5 mg / ml; most preferably 5 mg / ml. Further, the tyrosol aqueous solution also contains 0.5%-5% (v / v) of Tween 80; preferably 0.5%-2% of Tween 80.

[0015] Furthermore, the pear ring rot disease mentioned above is a pear disease caused by *Botrytis cinerea*.

[0016] This invention demonstrates, through in vitro antibacterial experiments and exogenous spraying of tyrosol solution, that tyrosol can inhibit the infection of *Cladosporium clavatum*.

[0017] Furthermore: the tyrosol required for the experiment was purchased from a biotechnology company, and the B. dothidea strain required for the experiment was a conventional strain, model FJ-54, provided by the Pear Engineering Technology Research Center of Nanjing Agricultural University.

[0018] During the study, the effective concentration range of the tyrosol aqueous solution was 0-5 mg / ml. The results showed that the inhibition of mycelial growth of *Pyrus pyrifolia* by tyrosol was dose-dependent. When the concentration of the tyrosol solution was 5 mg / ml, the inhibition rate of tyrosol on the mycelial growth of *Pyrus pyrifolia* reached 70.73%.

[0019] The detection method for controlling pear ring rot using tyrosol involved spraying 5 mg / ml tyrosol aqueous solution onto detached 'Dangshan' pear leaves and inoculating them with ring rot pathogens, and then regularly observing the diameter of the lesions.

[0020] In a specific embodiment of this invention, the leaves of 'Dangshan' pear are in vitro inoculated with mycelium using a puncture method, with two mycelial cakes attached to each leaf. Finally, the treated leaves are wrapped with cotton around the petioles, placed back on a tray, covered with a black plastic bag, and incubated in a dark incubator at 25°C and 80% humidity. Inoculated mycelial cakes are obtained by punching holes in the culture medium containing *B. dothidea* mycelium using a 5mm punch. 25°C is the optimal temperature for the activity of the ring rot pathogen; at this temperature, it is easy to observe the diameter of lesions.

[0021] This invention uses 'Dangshan' pear as the experimental material. Through in vitro antibacterial experiments and inoculation experiments with *Pyrus pyrifolia*, it was demonstrated that tyrosol has a significant inhibitory effect on *Pyrus pyrifolia*, effectively inhibiting the development rate of *Pyrus pyrifolia* lesions, reducing the occurrence and spread of the disease, and thus reducing the damage caused by *Pyrus pyrifolia* to the plant. Tyrosol is a natural compound in plants with low toxicity. Its application in the control of pear *Pyrus pyrifolia* can enhance the plant's own defense mechanisms, improve its natural resistance to the disease, and reduce or replace the use of chemical pesticides, thereby reducing potential risks to the environment and human health. As a naturally derived plant fungicide, tyrosol emphasizes green environmental protection and conforms to the principles of organic agriculture; therefore, it is expected to be more widely used in this field.

[0022] The beneficial effects of this invention are:

[0023] This invention provides the application of tyrosol in the preparation of a fungicide for controlling pear ring rot. In vitro antibacterial experiments and exogenous tyrosol spraying experiments demonstrated that tyrosol exhibits significant resistance to *Rhizoctonia solani*, and remains effective over a wide concentration range, making it suitable for widespread application. Attached Figure Description

[0024] Figure 1 The results of the antibacterial experiment on the hyphal growth of *Rhizoctonia solani* by different concentrations of tyrosol on PDA medium.

[0025] Figure 2 The results of diameter measurement in the antibacterial experiment of different concentrations of tyrosol on the mycelial growth of *Rhizoctonia solani* on PDA medium.

[0026] Figure 3 The study aimed to investigate the inhibitory effect of exogenous spraying of tyrosol at a concentration of 5 mg / ml on ring spot lesions on the leaves of 'Dangshan' pear.

[0027] Figure 4 The results are the diameter measurements of ring rot lesions on the leaves of 'Dangshan' pear after exogenous spraying of tyrosol at a concentration of 5 mg / ml.

[0028] Figure 5 The SOD enzyme activity of tyrosol-treated leaves of 'Dangshan' pear inoculated with ring rot disease was measured 6 days after inoculation.

[0029] Figure 6 The activity of POD enzyme in tyrosol-treated leaves of 'Dangshan' pear inoculated with ring rot for 6 days was measured.

[0030] Figure 7 The activity of CAT enzyme in tyrosol-treated leaves of 'Dangshan' pear inoculated with ring rot for 6 days was measured.

[0031] Figure 8 OFR content of tyrosol-treated leaves of 'Dangshan' pear inoculated with ring rot disease 6 days after treatment.

[0032] Figure 9 The effects of tyrosol treatment on H2O2 content in 'Dangshan' pear leaves 6 days after inoculation with ring rot were investigated. Detailed Implementation

[0033] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] The tyrosol required for the experiment was purchased from a biotechnology company, and the B. dothidea strain required for the experiment was a conventional strain, model FJ-54, provided by the Pear Engineering Technology Research Center of Nanjing Agricultural University.

[0036] Eight concentrations of tyrosol aqueous solutions (0, 0.1, 0.5, 1, 2, 3, 4, and 5 mg / ml) were prepared. The tyrosol was dissolved in water and sterilized by passing it through a 0.22 μm aqueous filter in a clean bench. These solutions were then added to PDA medium to prepare PDA media of different concentrations. Inoculated mycelial discs were obtained by punching holes in the medium containing *B. dothidea* hyphae using a 5 mm punch. The mycelial discs were incubated in a dark incubator at 25°C and 80% humidity, and colony growth was subsequently observed.

[0037] Example 1: In vitro antibacterial experiment of different concentrations of tyrosol against *Rhizoctonia solani*.

[0038] The method for preparing potato dextrose agar (PDA) medium for the culture and propagation of Aureobasidium is as follows: 200g peeled potato, 20g glucose, 15g agar, pH 5.8, distilled water to a final volume of 1000ml, sterilize at 121℃ for 20min, and cool before use.

[0039] Once the plate without tyrosol has been almost fully covered, take a picture and measure the growth diameter of the hyphae to calculate the inhibition rate.

[0040] Inhibition rate (%) = (diameter of newly grown hyphae in control - diameter of newly grown hyphae in treatment) / diameter of newly grown hyphae in control × 100. Each treatment was repeated five times.

[0041] Tyrosol's inhibitory effect on the mycelial growth of *Pyrus pyrifolia* is as follows: Figure 1 and Figure 2 As shown, tyrosol at 5 mg / ml significantly inhibited the mycelial growth of pathogenic fungi. Table 1 summarizes the inhibition rate of tyrosol on the mycelial growth of plant pathogenic fungi. Table 1 shows that when the tyrosol concentration was 5 mg / ml, the inhibition rate on the mycelial growth of *Botrytis cinerea* reached 70.73%, indicating that tyrosol has a significant inhibitory effect on *Botrytis cinerea* growing on PDA total nutrient medium.

[0042] Table 1. Inhibition rate of tyrosol on the growth of mycelia of the ring spot pathogen on solid culture medium.

[0043]

[0044] Example 2: Exogenous application of tyrosol to detect the inhibitory effect of tyrosol on pear ring rot pathogen.

[0045] Leaves of Dangshan pear (Dangshan su li) were used as the research material. Leaves that were uniform in size, smooth in surface, and undamaged were selected. First, the leaves were washed with distilled water, and excessively long petioles were trimmed to prevent air plugging, which could affect the normal function of the xylem vessels. Next, the leaves were soaked in a 0.1% sodium hypochlorite solution for 3 to 5 minutes to disinfect and remove microorganisms from the leaf surface. After disinfection, the leaves were rinsed repeatedly with distilled water 3 to 5 times to ensure that any residual disinfectant was completely removed. After treatment, the base of the leaves was covered with damp cotton and placed on a tray lined with damp gauze to keep the leaves moist.

[0046] Tyrosol was prepared as a 5 mg / ml aqueous solution, and 1% (v / v) of Tween 80 was added. This solution was sprayed onto the leaf surface, with water spraying serving as a control group. The leaves were then inoculated in vitro using a puncture method, with two mycelial cakes attached to each leaf. Finally, the treated leaves were placed back in trays, covered with black plastic bags, and incubated in a dark incubator at 25°C and 80% humidity. The diameter of the lesions was measured at 0, 2, 4, 6, and 8 days.

[0047] After treating Dangshan pear leaves with a 5 mg / ml tyrosol aqueous solution, and then inoculating them with the pathogen of *Rhizoctonia solani*, the following results were observed: Figure 3 and Figure 4 As shown in the lesion diameter, the lesion diameter was significantly smaller than that of the CK treatment.

[0048] Exogenous application of 5 mg / ml tyrosol resulted in a 23.35% inhibition rate against pear ring rot pathogen on day 8.

[0049] A series of biochemical parameters were measured on pear leaves inoculated with the pathogen of *Rhizoctonia solani*, including the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as the levels of hydrogen peroxide (H2O2) and superoxide anion (OFR). Figure 5-9 The data showed that, 6 days after inoculating 'Dangshan' pear leaves with the pathogen of ring rot, the activities of SOD, POD, and CAT significantly increased compared with the control group (CK), with increases of 31.98%, 59.79%, and 38.49%, respectively. Meanwhile, the levels of H2O2 and OFR significantly decreased compared with the control group, with decreases of 22.27% and 32.86%, respectively.

[0050] In summary, tyrosol enhances the activity of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), thereby increasing the scavenging efficiency of hydrogen peroxide (H2O2) and superoxide anion (OFR) in pear trees when facing pear ring rot pathogens. This effect strengthens the pear tree's resistance to ring rot pathogens and slows down the infection process. Therefore, tyrosol has a significant effect on inhibiting the development of pear ring rot and can be used as an effective means of controlling the disease.

Claims

1. The application of tyrosol in the control of pear ring rot or in the preparation of fungicides for the control of pear ring rot, characterized in that, Pear ring rot is controlled by spraying an exogenous tyrosol aqueous solution; the concentration of tyrosol in the tyrosol aqueous solution is 1-5 mg / ml; pear ring rot is a pear disease caused by *Botrytis cinerea*.

2. The application according to claim 1, characterized in that, The concentration of tyrosol in the tyrosol aqueous solution is 3-5 mg / ml.

3. The application according to claim 2, characterized in that, The concentration of tyrosol in the tyrosol aqueous solution is 5 mg / ml.

4. The application according to claim 3, characterized in that, The tyrosol aqueous solution also contains 0.5%-5% Tween 80.

5. A method for controlling pear ring rot, characterized in that, Tyrosol is used to control pear ring rot; pear ring rot is controlled by spraying an aqueous solution of tyrosol; the concentration of tyrosol in the aqueous solution is 1-5 mg / ml; pear ring rot is a pear disease caused by *Botrytis cinerea*.

6. The method according to claim 5, characterized in that, The concentration of tyrosol in the tyrosol aqueous solution is 3-5 mg / ml.

7. The method according to claim 6, characterized in that, The concentration of tyrosol in the tyrosol aqueous solution is 5 mg / ml.

8. The method according to claim 7, characterized in that, The tyrosol aqueous solution also contains 0.5%-5% Tween 80.

Images