A growth inhibitor of rice bacterial brown spot

Abstract

The present application provides a kind of growth inhibitor of rice green core bacteria, which contains one or more of eugenol, carvacrol or 2,4-di-tert-butyl phenol.The growth inhibitor of rice green core bacteria provided by the present application, wherein eugenol, carvacrol or 2,4-di-tert-butyl phenol are all natural products, low toxicity and pollution-free, and have a significant inhibitory effect on the mycelial growth and spore germination of the pathogen of rice false smut, rice green core bacteria, at low concentration, providing an efficient, low-toxicity and pollution-free bacteriostatic agent for the prevention and control of rice false smut.

Description

[0001] This application is a divisional application of application number 202211308328.0, filed on October 25, 2022, entitled "An Inhibitor for the Growth of Rhizoctonia solani". Technical Field

[0002] This invention relates to the field of agricultural product quality and safety, and more specifically to a growth inhibitor of Rhizoctonia solani. Background Technology

[0003] Rice false smut, caused by *Ustilaginoidea virens* (Cook) Tak, is a panicle disease of rice and one of the three major rice diseases worldwide. It is widely distributed in major rice-producing areas across Asia, Africa, South America, and Europe. Since the 1980s, with improved irrigation and fertilization levels, the widespread adoption of high-yield varieties, and changes in climate, the incidence and severity of rice false smut in my country have continuously expanded. *Ustilaginoidea virens* not only causes rice false smut, significantly increasing the rate of empty grains and reducing grain weight, resulting in a 20%–30% yield loss, but its chlamydospores also produce fungal toxins such as uttiloxin and uttilaginoidins, which are highly toxic to plants and animals, seriously threatening rice quality and safety.

[0004] To control rice false smut, the use of fungicides in the early booting stage of rice has been proven to be effective. However, the long-term application of commonly used chemical reagents such as benzoyl propiconazole, hexaconazole-pyraclostrobin, propiconazole-prochloraz, and carbendazim can lead to a series of problems, including environmental pollution, increased drug resistance of pathogens, and the introduction of new food safety risks. Therefore, it is urgent to develop a new type of green, environmentally friendly, and highly efficient fungicide to control rice false smut caused by Rhizoctonia solani. Summary of the Invention

[0005] This invention provides a growth inhibitor of *Rhizoctonia solani* containing one or more of eugenol, carvacrol, or 2,4-di-tert-butylphenol.

[0006] Specifically, in the present invention, a growth inhibitor of *Rhizoctonia solani* is prepared by dissolving eugenol, carvacrol, or 2,4-di-tert-butylphenol in a small amount of dimethyl sulfoxide before adding water to form an aqueous solution.

[0007] The concentrations of eugenol, carvacrol, or 2,4-di-tert-butylphenol are each at least 0.1 mmol / L.

[0008] More specifically, the concentrations of eugenol, carvacrol, or 2,4-di-tert-butylphenol are 0.1-5 mmol / L. When used to treat rice false smut in rice, the concentrations of eugenol, carvacrol, or 2,4-di-tert-butylphenol may be higher than those under laboratory conditions due to the complex rice growing environment.

[0009]

[0010] The structural formula of 2,4-di-tert-butylphenol

[0011] The *Rhizoctonia solani* growth inhibitor provided by this invention has the following advantages:

[0012] (1) This invention first discovered that eugenol, carvacrol and 2,4-di-tert-butylphenol can effectively inhibit the colony growth and spore germination of Rhizoctonia solani, and the inhibition rate can reach 100% at low concentrations;

[0013] (2) Eugenol, carvacrol, and 2,4-di-tert-butylphenol are all natural products, safe, easily degradable, pollution-free, and low in toxicity. Eugenol is also a plant-derived natural product, and it can be legally used as a flavoring and food additive in many countries and regions such as China, the United States, and the European Union, without any maximum residue limit or usage limit set (GB 1886.129-2022 National Food Safety Standard - Food Additives - Eugenol, GB 2760-2011 National Food Safety Standard - Standard for the Use of Food Additives);

[0014] Carvacrol is also a plant-derived natural product with a spicy, cool, and herbal aroma. It has low toxicity and has been included in the list of permitted natural flavorings for food use (GB 2760-2011 National Food Safety Standard - Standard for the Use of Food Additives).

[0015] (3) Wide range of sources. Eugenol can be isolated from eugenol-rich plant essential oils through alkali treatment, or it can be chemically synthesized from raw materials such as eugenol and allyl chloride, resulting in low production costs; carvacrol is naturally found in plants such as thyme, with wide sources and simple extraction, and can also be obtained by sulfonation of thymol followed by alkali treatment, resulting in low production costs; 2,4-di-tert-butylphenol is a component of volatile metabolites of many plants and microorganisms, with wide sources and simple extraction, and can also be synthesized on a large scale through esterification reactions, resulting in low costs.

[0016] (4) The antibacterial agent of the present invention is simple to use, requires no complicated operation, and is easy to apply on a large scale. Attached Figure Description

[0017] Figure 1 Eugenol's inhibitory effect on the mycelial growth of Rhizoctonia solani

[0018] Figure 2Eugenol's inhibitory effect on the germination of *Rhizoctonia solani* spores

[0019] Figure 3 Inhibitory effect of carvacrol on the mycelial growth of Rhizoctonia solani

[0020] Figure 4 Inhibitory effect of 2,4-di-tert-butylphenol on the mycelial growth of Rhizoctonia solani Detailed Implementation

[0021] The materials used in the following examples and their sources or preparation methods:

[0022] (1) Reagents and materials

[0023] Eugenol, carvacrol, 2,4-di-tert-butylphenol, dimethyl sulfoxide (Sinopharm Chemical Reagent Co., Ltd.)

[0024] Sucrose, agar, Tween 20 (Shanghai Yuanye Biotechnology Co., Ltd.);

[0025] Potatoes (common commercially available product);

[0026] Rhizoctonia solani strain HWD-2 (China Center for Type Culture Collection, NO: 2011023).

[0027] (2) Instruments

[0028] Milli-Q ultrapure water system (Millipore, USA);

[0029] AL104 analytical balance (Mettler-Toledo Instruments GmbH, Switzerland);

[0030] SX-500 Autoclave (TOMY Corporation, Japan);

[0031] RI 250 incubator (Thermo Fisher Scientific, USA);

[0032] Heraeus Multifuge X3 high-speed centrifuge (Thermo Fisher Scientific, USA);

[0033] BX-2 microscope (Olympus Corporation, Japan).

[0034] (3) Preparation of culture medium for strains

[0035] PSA medium: Cut 200g of peeled potatoes into small pieces, boil for 30 minutes, and then filter the liquid. Add 20g of sucrose and 16g of agar, and bring the volume to 1000mL with distilled water. Autoclave at 115℃ for 30 minutes, cool to about 55℃, and pour into plates, 20mL per plate.

[0036] PSB liquid culture medium: Cut 200g of peeled potatoes into small pieces, boil for 30 minutes, take the filtrate, add 20g of sucrose, and make up to 1000mL with distilled water. Autoclave at 115℃ for 30 minutes.

[0037] Example 1: Inhibitory effect of eugenol on the mycelial growth of Rhizoctonia solani

[0038] A suitable amount of eugenol was dissolved in 10 mL of sterile ultrapure water containing 0.5% Tween 20. After filtration and sterilization, 90 mL of sterilized PSA medium was added to achieve final concentrations of 0.1, 0.2, 0.5, 1, 2, and 5 mmol / L, respectively, to obtain eugenol-containing PSA medium. *Rhizoctonia solani* strain HWD-2 was inoculated into PSA medium and activated in the dark at 28°C for 14 days. The outer edge of the mycelial cake (approximately 5 mm in diameter) was cut and placed in the center of an eugenol-containing medium plate. After culturing in the dark at 28°C for 14 days, the colony diameter of *Rhizoctonia solani* was measured using the vertical cross method. With PSA medium without eugenol as the control group, the growth inhibition rate was calculated.

[0039] Growth inhibition rate / % = [(control colony diameter - treated colony diameter) / (control colony diameter)] × 100%

[0040] like Figure 1 As shown, when the concentration reached 0.5 mmol / L, eugenol significantly inhibited the growth of *Rhizoctonia solani*, with an inhibition rate of 30.38%. The inhibitory effect increased with increasing eugenol concentration, reaching 76.59% when the concentration was 1 mmol / L. When the concentration reached 2 mmol / L, the growth of *Rhizoctonia solani* was completely inhibited.

[0041] Example 2: Inhibitory effect of eugenol on spore germination of *Rhizoctonia solani*.

[0042] The activated *Rhizoctonia solani* mycelium was added to PSB medium and incubated in the dark at 28°C and 150 rpm for 7 days until the culture medium turned green. The mycelium was filtered, and the spore suspension was collected. The spore concentration was observed under a microscope, and the spores were counted using a hemocytometer. The concentration was adjusted to 10⁻¹⁰ with sterile water. 6 spores / mL. Take 30 μL of spore suspension and evenly spread it on the surface of PSA medium containing different concentrations of eugenol (0.1, 0.2, 0.5, 1, 2, and 5 mmol / L). Incubate in the dark at 28°C for 24 hours, and observe spore germination under a microscope. The spore germination criterion is that the germ tube length exceeds the short side diameter of the spore. Use PSA medium without eugenol as a control group and calculate the spore germination inhibition rate.

[0043] Spore germination inhibition rate / % = [(Control spore germination rate - Treated spore germination rate) / (Control spore germination rate)] × 100%

[0044] The results show ( Figure 2 When the concentration reaches 0.1 mmol / L, eugenol inhibits the spore germination of Rhizoctonia solani with an inhibition rate of 83.8%; at 0.5 mmol / L, the inhibition rate is 98.3%; and at concentrations above 1 mmol / L, the inhibition rate is 100%.

[0045] Example 3

[0046] (1) Inhibitory effect of carvacrol on the mycelial growth of Rhizoctonia solani

[0047] The experimental method is the same as in Example 1, and the experimental results are as follows:

[0048] The results show that: Figure 3 As shown, when the concentration reached 0.2 mmol / L, carvacrol significantly inhibited the growth of *Rhizoctonia solani* (P<0.01), with an inhibition rate of 44.32%. The inhibitory effect increased with the increase of carvacrol concentration, and the inhibition rate reached 62.31% when the carvacrol concentration was 0.5 mmol / L. When the carvacrol concentration reached 1 mmol / L, the growth of *Rhizoctonia solani* was completely inhibited.

[0049] (2) Inhibitory effect of carvacrol on the germination of Rhizoctonia solani spores

[0050] The experimental method is the same as in Example 2.

[0051] The results showed that when the concentration reached 0.1 mmol / L and 0.2 mmol / L, carvacrol almost completely inhibited the spore germination of Rhizoctonia solani, with inhibition rates of 99.0% and 99.5%, respectively. At concentrations above 0.2 mmol / L, spore germination was completely inhibited (inhibition rate of 100%).

[0052] Example 4

[0053] (1) Inhibitory effect of 2,4-di-tert-butylphenol on the mycelial growth of Rhizoctonia solani

[0054] The experimental method is the same as in Example 1.

[0055] The results are as follows Figure 4 As shown, when the concentration reached 0.1 mmol / L, 2,4-di-tert-butylphenol significantly inhibited the growth of *Rhizoctonia solani* (P<0.01), with an inhibition rate of 52.29%. The inhibitory effect increased with increasing concentration of 2,4-di-tert-butylphenol, reaching an inhibition rate of 86.87% at a concentration of 0.2 mmol / L. When the concentration of 2,4-di-tert-butylphenol reached 0.5 mmol / L, it completely inhibited the growth of *Rhizoctonia solani*.

[0056] (2) Inhibitory effect of 2,4-di-tert-butylphenol on the germination of *Rhizoctonia solani* spores

[0057] The experimental method is the same as in Example 2.

[0058] The results showed that when the concentration reached 0.1 mmol / L or above, 2,4-di-tert-butylphenol completely inhibited the spore germination of Rhizoctonia solani, with an inhibition rate of 100%.

Claims

1.2,4-Di-tert-butylphenol aqueous solution is used to prepare a growth inhibitor of Rhizoctonia solani, wherein the concentration of 2,4-di-tert-butylphenol in the aqueous solution is 0.1-5 mmol / L. 2.2,4-Di-tert-butylphenol aqueous solution is used to inhibit the germination of Rhizoctonia solani spores, wherein the concentration of 2,4-di-tert-butylphenol in the aqueous solution is 0.1-5 mmol / L.

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