Application of nerol in prevention and treatment of rubber anthracnose

Abstract

The invention belongs to the technical field of plant disease and insect pest prevention and control, and particularly relates to application of nerol in prevention and control of rubber anthracnose. It is found for the first time that nerol with low concentration (0.5 [mu] L / mL) can effectively inhibit colletotrichum gloeosporioides, phytophthora capsici and colletotrichum gloeosporioides, and especially the inhibition rate of nerol to colletotrichum gloeosporioides and colletotrichum gloeosporioides can reach 100%. Besides, nerol also has obvious inhibitory activity on rubber anthrax bacteria inoculated on in-vitro leaves, and can obviously reduce the area of scabs, and the inhibition rate reaches 98.4%. The results show that the nerol has a good prevention and treatment effect on the rubber anthracnose, the nerol belongs to a plant-sourced volatile compound, and few residues are left when the nerol is used for preventing and treating the rubber tree anthracnose, so that the nerol can be used as a potential green pesticide for preventing and treating the rubber anthracnose, and a new way is provided for preventing and treating the rubber anthracnose.

Description

Technical Field

[0001] This invention belongs to the field of plant disease and pest control technology, specifically involving the application of nerol in the control of rubber anthracnose. Background Technology

[0002] Anthracnose can infect a variety of plants, causing anthracnose disease. This type of disease is widespread and causes serious damage, making it a key research focus for plant pathologists. Rubber tree anthracnose is one of the most severe types. Caused by *Colletotrichum gloeosporioides*, rubber tree anthracnose can lead to leaf drop and even the death of the entire tree, causing significant losses to the rubber industry.

[0003] Anthracnose primarily overwinters as mycelium in diseased branches, producing conidia under suitable conditions as the primary source of infection. Rubber tree seedlings, young trees, and even mature tapping trees can be infected by anthracnose. The pathogen can infect young leaves, petioles, shoots, and latex fruits, causing leaf drop, shoot dieback, and fruit rot, even resulting in mummified fruits hanging on the tree. In severe cases, it can cause repeated leaf drop and shoot dieback, delay tapping time, and even reduce the efficiency of latex synthesis and accumulation. This disease is considered one of the main causes of reduced yields in natural rubber trees.

[0004] Currently, the control of rubber anthracnose still relies mainly on chemical fungicides. However, long-term and excessive application of chemical agents can lead to drug resistance and pesticide residues, causing environmental pollution. These problems have become major obstacles to the sustainable development of agriculture. Therefore, there is an urgent need to develop a safe and green antifungal agent for the control of rubber anthracnose. Furthermore, using a green antifungal agent to control rubber tree anthracnose is of great significance for achieving sustainable agricultural development.

[0005] Nerol is a noncyclic monoterpene alcohol found naturally in plants such as orange leaves, lemons, roses, and grapefruits. As a safe and edible flavoring agent, nerol is widely used in medicine, cosmetics, and food due to its unique rose and orange blossom fragrance. Studies have found that natural rubber latex contains abundant nerol; however, no research has yet been reported on the use of nerol in the prevention and treatment of rubber anthrax. Summary of the Invention

[0006] The purpose of this invention is to provide new applications of nerol in response to practical problems and needs in production practice, specifically in the prevention and control of rubber anthrax or the inhibition of rubber anthrax bacteria.

[0007] The technical solution of this invention is implemented as follows: Application of nerol in the preparation of fungi that inhibit anthracnose of rubber trees, Phytophthora capsici and / or anthracnose of bananas.

[0008] Application of nerol in the prevention and control of rubber anthrax.

[0009] Furthermore, in the aforementioned application, the volume concentration of nerol is 10 μL / mL.

[0010] Application of nerol in the preparation of formulations that inhibit anthracnose of rubber trees, Phytophthora capsici and / or anthracnose of bananas.

[0011] Application of nerol in the preparation of formulations for the prevention and control of diseases caused by Phytophthora capsici and / or Anthracnose moniliforme.

[0012] Furthermore, in the aforementioned application, the volume concentration of nerol is 0.5 μL / mL.

[0013] Application of nerol in the preparation of formulations that inhibit the mycelial growth of rubber anthrax fungus.

[0014] Furthermore, in the aforementioned application, the volume concentration of nerol is 0.25 μL / mL.

[0015] Furthermore, in the aforementioned application, the nerol causes the hyphae of *Anthrax rubrum* to shrink and / or become thinner.

[0016] A method for preventing and controlling anthracnose in rubber trees includes the following steps: Dissolve nerol in a co-solvent to prepare a solution, and spray the solution onto the leaves of the rubber tree after sterilization.

[0017] Furthermore, the volume concentration of nerol in the solution is 10 μL / mL, and the co-solvent is a 1% Tween-80 aqueous solution. Beneficial effects

[0018] This invention marks the first discovery that low concentrations (0.5 μL / mL) of nerol can effectively inhibit *Anthracnose rubrum*, *Phytophthora capsici*, and *Anthracnose citrinum*, particularly achieving a 100% inhibition rate against *Anthracnose rubrum* and *Anthracnose citrinum*. The mycelia treated with nerol showed wrinkling and thinning, indicating that nerol affected the mycelial growth of *Anthracnose rubrum*. Furthermore, nerol also exhibited significant inhibitory activity against *Anthracnose rubrum* inoculated on detached leaves, significantly reducing lesion area with an inhibition rate of 98.4%. These experimental results demonstrate that nerol has significant inhibitory activity against *Anthracnose citrinum*, making it an effective method for controlling rubber anthracnose. These results also indicate that nerol has a good control effect on rubber anthracnose. As nerol is a plant-derived volatile compound, it leaves minimal residue when used for the control of rubber anthracnose. Therefore, nerol can serve as a potential green pesticide for controlling *Anthracnose rubrum*, providing a new approach to the prevention and control of this disease. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 It is the structural formula of nerol.

[0021] Figure 2 The results show the in vitro antibacterial activity of nerol.

[0022] Figure 3 This study aims to determine the indoor toxicity of nerol against *Bacillus anthracis*.

[0023] Figure 4 The effect of nerol on the mycelial growth of *Amanita muscaria*, the pathogen of rubber anthracnose.

[0024] Figure 5 The results show the antibacterial activity of nerol against anthracnose in detached rubber leaves. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to better understand the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0026] Example 1 Determination of broad-spectrum antibacterial activity of nerol Neroli ol was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with a purity of 97%, and its chemical structure is as follows: Figure 1 .

[0027] Preparation of nerol solution: Dissolve nerol in a co-solvent (1 v / v% Tween-80 aqueous solution) to prepare a stock solution of 10 μL / mL, then dilute with sterile water to 0.5 μL / mL, and filter sterilize using a 0.22 μm microporous membrane.

[0028] Test strain: Rubber anthracnose fungus ( Colletotrichum gloeosporioides (Penz.) Sacc ), Phytophthora capsici ( Phytophthora capsici Longan rot pathogen ( Lasiodiplodia theobromae ), Rubber brown root rot fungus ( Phellinus noxius ) and banana anthrax bacteria ( Colletotrichum musae All of these were provided by the Rubber Tree Diseases and Control Team Laboratory of Hainan University.

[0029] (1) Plate activity detection The inhibitory effect of nerol on the growth of plant pathogens was determined using the mycelial growth rate method.

[0030] Potato dextrose agar (PDA) was poured into sterile Petri dishes (90 mm in diameter). Nerol was added to the PDA medium at concentrations of 0 and 0.5 μL / mL. A 5 mm mycelial disc was taken from the edge of the colony and placed in the center of the Petri dish. After incubation in the dark at 25–28 °C for 4 days, mycelial length was measured using the cross-cross method. Each treatment was repeated at least three times. The mycelial growth inhibition rate was calculated using the following formula: Mycelial growth inhibition rate (%) = (Control colony growth diameter - Treatment colony growth diameter) / Control colony growth diameter × 100%.

[0031] The inhibition rate of nerol against five tested pathogenic fungi was determined using the growth rate method. The results are shown in Table 1 and 2. Figure 2 .

[0032] Table 1 Results of the in vitro antibacterial activity of nerol Pathogens Inhibition rate (%) Pathogens Inhibition rate (%) Rubber anthrax bacteria 100±0 Phytophthora capsici 46.44±0.03 Banana anthrax bacteria 100±0.02 Longan rot fungus 0±0 Rubber brown root causal agent 0±0 Note: The test concentration of nerol was 0.5 μL / mL.

[0033] From Table 1 and Figure 2 It is evident that, under in vitro conditions, nerol exhibits certain inhibitory activity against *Anthracis rubrum*, *Anthracis banana*, and *Phytophthora capsici* at a dose of 0.5 μL / mL. In particular, the inhibition rate against *Anthracis rubrum* and *Anthracis banana* is 100%, and the inhibition against *Anthracis* is complete, demonstrating significant development potential.

[0034] (2) Indoor toxicity test of nerol against *Anthrax rubrum* To determine the antibacterial effect of nerol against *Anthracis rubberii*, six nerol concentrations (0 μL / mL, 0.1 μL / mL, 0.2 μL / mL, 0.3 μL / mL, 0.4 μL / mL, and 0.5 μL / mL) were prepared and incubated upside down at 28℃. The results showed that as the nerol concentration gradually increased, the colony diameter decreased significantly, indicating that nerol had a lower EC50 against *Anthracis rubberii*. 50 It is 0.218 μL / mL (see) Figure 3 (and Table 2).

[0035] Table 2. Indoor toxicity test of nerol against *Anthrax rubrum*. medicine virulence regression equation Correlation coefficient <![CDATA[EC 50 ]]> 95% confidence zone Neroli y = 1.89 + 2.85 * X 0.983 0.218 0.195-0.243 Note: X is the logarithm of concentration, and y is the inhibition rate.

[0036] (4) Observe the morphology and structure of mycelia treated with nerol (scanning electron microscope) Fresh *Anthracnose rubrum* mycelial cakes were placed in PDB medium containing 0.25 μL / mL nerol and cultured at 28°C with shaking at 120 rpm for 72 h, with a blank control. Mycelial samples were fixed overnight in 4% glutaraldehyde buffer at 4°C, washed for 15 min with PBS buffer (0.1 mol / L; pH 7.0), repeated three times. At room temperature, the samples were fixed in 1% osmium tetroxide for 2 h. The mycelia were washed again four times with PBS buffer (0.1 mol / L; pH 7.0), 10 min each time. A gradient elution was performed in fractionated ethanol (30%, 50%, 70%, 80%, 90%, 95%, 100%), 15 min each time, with 100% ethanol elution repeated twice to ensure complete dehydration. The samples were dried in a critical point desiccator. The samples were then placed tightly on conductive carbon film double-sided adhesive and sputtered with gold for approximately 30 s on an ion sputtering stage. Images were captured and observed using a scanning electron microscope.

[0037] like Figure 4 As shown, under normal cultivation conditions, the hyphae of *Anthracis rubberii* have smooth surfaces, normal morphology, and intact outer walls; under treatment with 0.25 μL / mL nerol, the hyphae become wrinkled and thinner, indicating that nerol affects the growth of *Anthracis rubberii* hyphae.

[0038] Example 2 Inhibitory effect of nerol on detached leaves of *Anthracis rubberii* Several leaves of similar size during the pale green stage were selected and their surfaces were disinfected with 75% ethanol. A 3 mm × 3 mm wound was made in each leaf using a sterile toothpick. A 5 mm piece of fresh mycelium was inoculated onto the wound. The leaves were placed in a plastic insulated box lined with moist gauze, and cotton soaked in sterile water was wrapped around the petiole. The boxes were then placed in an artificial climate chamber at 28℃ and 90±5% humidity. After 24 h, the control (CK) group was sprayed with a 1% Tween-80 solution, and the nerol (Ner-10) group was sprayed with 10 μL / mL nerol (dissolved in a 1% Tween-80 solution and sterilized before use). Incubation continued, and leaf disease was observed and photographed after 5-6 hours. Ten leaves were used for each treatment.

[0039] Ex vivo leaf analysis showed that, compared with the control, the application of 10 μL / mL nerol significantly reduced the lesion area of ​​anthracnose, with an inhibition rate of 98.4% against rubber anthracnose. Figure 5 This indicates that nerol has a good control effect on rubber anthracnose and can be used as a potential green pesticide for the control of rubber anthracnose.

[0040] Inhibition rate (%) = (Leg area in experimental group - lesion area in control group) / lesion area in control group × 100% As demonstrated by the above examples, nerol has a good control effect on rubber anthracnose. Compared with existing anthracnose inhibitors on the market, nerol is a plant-derived volatile compound, leaving less residue and exhibiting significant control effects when used to control rubber anthracnose.

[0041] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. Application of nerol in the prevention and control of rubber anthrax.

2. The application as described in claim 1, characterized in that, The volume concentration of nerol is 10 μL / mL.

3. Application of nerol in the preparation of formulations that inhibit anthracnose fungi of rubber trees, Phytophthora capsici and / or anthracnose fungi of bananas.

4. Application of nerol in the preparation of formulations for the prevention and control of diseases caused by Phytophthora capsici and / or Anthracnose moniliforme.

5. The application as described in claim 3 or 4, characterized in that, The volume concentration of nerol is 0.5 μL / mL.

6. Application of nerol in the preparation of formulations that inhibit the mycelial growth of rubber anthrax fungus.

7. The application as described in claim 6, characterized in that, The volume concentration of nerol is 0.25 μL / mL.

8. The application as described in claim 6, characterized in that, The nerol described above causes the hyphae of rubber anthrax fungus to shrink and / or become thinner.

9. A method for preventing and controlling rubber anthracnose, characterized in that, Includes the following steps: Dissolve nerol in a co-solvent to prepare a solution, and spray the solution onto the leaves of the rubber tree after sterilization.

10. The method as described in claim 9, characterized in that, The solution contains 10 μL / mL of nerol, and the co-solvent is a 1% Tween-80 aqueous solution.

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