阅读说明：本技术 盐酸苄达明在制备用于防治由植物病原菌引起的植物病害的杀菌剂中的应用 (Application of benzydamine hydrochloride in preparation of bactericide for preventing and treating plant diseases caused by plant pathogenic bacteria ) 是由 梁文星 朱明怡 王光远 刘炳辰 范慧珍 于 2021-01-26 设计创作，主要内容包括：本发明提供了盐酸苄达明在制备用于防治由植物病原菌引起的植物病害的杀菌剂中的应用。本发明通过测试盐酸苄达明对番茄灰霉病菌和尖孢镰刀菌番茄专化型的菌丝生长、孢子萌发和芽管伸长的抑制实验,证明盐酸苄达明对番茄灰霉病菌和尖孢镰刀菌番茄专化型具有较强的抑制作用。传统的杀菌剂对环境污染大、且残留高,直接威胁着食品安全。盐酸苄达明作为杀菌剂,具有高效和低毒的优点,适合于植物病害化学防治的要求。盐酸苄达明是一种无污染、对环境友好的小分子化合物,对非靶标生物及人畜安全,能够保证农产品及果蔬的高品质,符合可持续发展的要求,其研究和市场应用前景广阔。(The invention provides application of benzydamine hydrochloride in preparing a bactericide for preventing and treating plant diseases caused by plant pathogenic bacteria. According to the invention, the inhibition experiment of the benzydamine hydrochloride on the growth of hyphae, spore germination and germ tube elongation of the tomato specialization types of botrytis cinerea and fusarium oxysporum proves that the benzydamine hydrochloride has a strong inhibition effect on the tomato specialization types of botrytis cinerea and fusarium oxysporum. The traditional bactericide has large environmental pollution and high residue, and directly threatens the food safety. The benzydamine hydrochloride is used as a bactericide, has the advantages of high efficiency and low toxicity, and is suitable for the requirements of chemical prevention and control of plant diseases. The benzydamine hydrochloride is a small molecular compound which is pollution-free and environment-friendly, is safe to non-target organisms, people and livestock, can ensure the high quality of agricultural products, fruits and vegetables, meets the requirement of sustainable development, and has wide research and market application prospects.)

1. Application of benzydamine hydrochloride in preparing bactericide for preventing and treating plant diseases caused by plant pathogenic bacteria.

2. The use of benzydamine hydrochloride according to claim 1 for the preparation of a fungicide for the control of plant diseases caused by phytopathogens: the plant pathogenic bacteria are tomato botrytis cinerea and fusarium oxysporum tomato specialization types.

3. Use of benzydamine hydrochloride according to claim 1 or 2 for the preparation of a fungicide for the control of plant diseases caused by phytopathogens: the effective using concentration of the benzydamine hydrochloride in the bactericide is 0.1 mM-1.5 mM.

4. The use of benzydamine hydrochloride according to claim 3 for the preparation of a fungicide for the control of plant diseases caused by phytopathogens: the inhibition rate of the benzydamine hydrochloride on the growth of tomato special hyphae of botrytis cinerea and fusarium oxysporum is dose-dependent.

5. The use of benzydamine hydrochloride according to claim 4 in the preparation of a fungicide for the control of plant diseases caused by phytopathogens, characterized in that: when the concentration of the benzydamine hydrochloride in the bactericide is 1.5 mM, the inhibition rate of the benzydamine hydrochloride on the growth of the special tomato hyphae of botrytis cinerea and fusarium oxysporum exceeds 94.1%.

6. The use of benzydamine hydrochloride according to claim 3 for the preparation of a fungicide for the control of plant diseases caused by phytopathogens: the inhibition rate of the benzydamine hydrochloride on the tomato special spore germination of botrytis cinerea and fusarium oxysporum is dose-dependent.

7. The use of benzydamine hydrochloride according to claim 6 in the preparation of a fungicide for the control of plant diseases caused by phytopathogens, characterized in that: when the concentration of the benzydamine hydrochloride in the bactericide is 0.2 mM, the inhibition rate of the benzydamine hydrochloride on the spore germination of the tomato botrytis cinerea and fusarium oxysporum tomato specialization type exceeds 87.0%.

8. The use of benzydamine hydrochloride according to claim 3 for the preparation of a fungicide for the control of plant diseases caused by phytopathogens: the inhibition rate of the benzydamine hydrochloride on the elongation of tomato special germ tubes of botrytis cinerea and fusarium oxysporum is dose-dependent.

9. The use of benzydamine hydrochloride according to claim 8 in the preparation of a fungicide for the control of plant diseases caused by phytopathogens: when the concentration of the benzydamine hydrochloride in the bactericide is 0.2 mM, the inhibition rate of the benzydamine hydrochloride on the elongation of the special tomato tubes of botrytis cinerea and fusarium oxysporum is over 95.5%.

10. The use of benzydamine hydrochloride according to claim 1 for the preparation of a fungicide for the control of plant diseases caused by phytopathogens: the plant is selected from fructus Lycopersici Esculenti, fructus Solani Melongenae, Capsici fructus, fructus Cucumidis Sativi, fructus Vitis Viniferae, and strawberry.

Technical Field

The invention belongs to the technical field of agricultural biology, relates to a bactericidal active compound, and particularly relates to an application of benzydamine hydrochloride in preparation of a bactericide for preventing and treating plant diseases caused by plant pathogenic bacteria.

Background

The tomato gray mold is a common disease caused by botrytis cinerea infection, is one of the most harmful diseases, and is common. In addition to tomato, botrytis cinerea can also damage eggplant, pepper, cucumber, grape, strawberry and other important economic crops, and can cause fruit and vegetable botrytis. The disease may occur not only in the growing season of the host plant but also during the storage of the agricultural product, and thus is seriously harmful. Especially, due to the large-area popularization of the facility cultivation mode in recent years, the pathogen infection sources are wide, the propagation speed is high, the gray mold of fruits and vegetables is more and more serious, and the occurrence and the prevalence of the gray mold often cause serious economic loss. The occurrence of gray mold of tomatoes in northern facilities is generally reported to be serious, the economic loss caused by general years can reach 20% -30%, and more than 50% of serious years can even be no more than absolutely harvested.

The pathogen of the plant fusarium wilt is fusarium oxysporum, which is a soil-borne pathogenic fungus distributed worldwide, has a wide host range, and can cause the fusarium wilt of more than 100 fusarium wilt of melons, solanaceae, bananas, cotton, leguminous, flowers and the like. After the host is infected by fusarium, the symptoms are diversified, which generally causes the browning of vascular bundles, the wilting and withering of plants, the rotting of bulbs and roots, the weak growth of plants and the like. Especially, fusarium oxysporum tomato specialization causes tomato blight, and seriously threatens tomato production. Fusarium wilt is also an important fungal soil-borne disease causing harm to agricultural production in recent years, and has been generated in many areas, particularly in crop areas with higher serious influence on crop yield and economic benefit.

The botrytis cinerea and fusarium oxysporum have very high propagation speed, and produce a great number of conidia, so that the conidia are easy to be mutated to generate drug resistance, and various bactericides such as dicarboximides, carbamates and the like are commonly used for preventing and treating in the agricultural production at present. However, pathogenic fungi are susceptible to resistance, which not only results in reduced control efficacy, but also results in an increasing amount of the drug. Excessive use of chemical agents not only causes the pesticide residues of agricultural products to exceed the standard and harm the physical health of consumers, but also causes environmental pollution and damages ecological balance and influences the sustainable and healthy development of agriculture. Therefore, there is a need to develop a bactericide which is highly efficient, low toxic, low in residue and environmentally friendly, and to apply it to production practices. Agriculture in China is to be developed towards a healthy and sustainable direction, and the development of bactericide products is oriented to solving the problems of resistance, plant immunity enhancement, safety, high efficiency, good environmental compatibility and the like.

Benzydamine hydrochloride, also known as yantongjing; the Chinese synonyms are: 1-benzyl-3- (3- [ dimethylamino group)]Propoxy) -1H-indazole hydrochloride; english name: benzydamine hydrochloride; CAS number: 132-69-4; the molecular formula is as follows: c₁₉H₂₃N₃O.HCl; molecular weight: 345.87. the molecular structural formula is:

the benzydamine hydrochloride is an antipyretic analgesic, and has the effects of diminishing inflammation, relieving fever and easing pain. It is effective in relieving pain caused by inflammation.

At present, no report about the inhibition effect of benzydamine hydrochloride on agricultural plant pathogenic bacteria exists at home and abroad.

Disclosure of Invention

The invention aims to provide application of benzydamine hydrochloride in preparing a bactericide for preventing and treating plant diseases caused by phytopathogens. The toxicity test proves that the benzydamine hydrochloride has good inhibitory activity on tomato botrytis cinerea and fusarium oxysporum tomato specialization types. The benzydamine hydrochloride is used as a bactericide, has high efficiency and low toxicity, and is suitable for the requirements of chemical prevention and control of plant diseases.

In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:

the invention provides application of benzydamine hydrochloride in preparing a bactericide for preventing and treating plant diseases caused by plant pathogenic bacteria.

Further: the plant pathogenic bacteria are tomato botrytis cinerea and fusarium oxysporum tomato specialization types.

Further: the effective using concentration of the benzydamine hydrochloride in the bactericide is 0.1 mM-1.5 mM.

Further: the inhibition rate of the benzydamine hydrochloride on the growth of tomato special hyphae of botrytis cinerea and fusarium oxysporum is dose-dependent.

Further: when the concentration of the benzydamine hydrochloride in the bactericide is 1.5 mM, the inhibition rate of the benzydamine hydrochloride on the growth of the special tomato hyphae of botrytis cinerea and fusarium oxysporum exceeds 94.1%.

Further: the inhibition rate of the benzydamine hydrochloride on the tomato special spore germination of botrytis cinerea and fusarium oxysporum is dose-dependent.

Further: when the concentration of the benzydamine hydrochloride in the bactericide is 0.2 mM, the inhibition rate of the benzydamine hydrochloride on the spore germination of the tomato botrytis cinerea and fusarium oxysporum tomato specialization type exceeds 87.0%.

Further: the inhibition rate of the benzydamine hydrochloride on the elongation of tomato special germ tubes of botrytis cinerea and fusarium oxysporum is dose-dependent.

Further: when the concentration of the benzydamine hydrochloride in the bactericide is 0.2 mM, the inhibition rate of the benzydamine hydrochloride on the elongation of the special tomato tubes of botrytis cinerea and fusarium oxysporum is over 95.5%.

Further: the plant is selected from fructus Lycopersici Esculenti, fructus Solani Melongenae, Capsici fructus, fructus Cucumidis Sativi, fructus Vitis Viniferae, and strawberry.

Compared with the prior art, the invention has the advantages and the technical effects that: at present, the prevention and control of plant pathogenic bacteria mainly depend on pesticides. The variety of the pesticide bactericide has a single action mechanism, has the problem of pesticide residue, and directly threatens the environment and food safety of human beings. Therefore, the development of pollution-free and pollution-free green bactericides is an important prevention and control method in agricultural production. Many green bactericides have been developed and widely used in production, and have achieved remarkable effects. The invention proves that the benzydamine hydrochloride has good inhibitory activity to plant pathogenic fungi by indoor toxicity measurement. Moreover, the benzydamine hydrochloride is an environment-friendly small molecular compound, has poor drug resistance, is safe to non-target organisms, people and livestock, can ensure the high quality of agricultural products, fruits and vegetables, meets the requirement of sustainable development, and has wide research and market application prospects.

Drawings

FIG. 1 shows the results of experiments on the inhibition of mycelial growth of Botrytis cinerea by benzydamine hydrochloride in the present invention;

FIG. 2 is the result of bacteriostatic experiments on the growth of fusarium oxysporum tomato specialized hyphae with benzydamine hydrochloride in the present invention;

FIG. 3 is the results of experiments on the inhibition of germination of tomato botrytis cinerea and fusarium oxysporum tomato obligate conidia by benzydamine hydrochloride in the present invention;

FIG. 4 shows the results of experiments on inhibition of Botrytis cinerea germ extension by benzydamine hydrochloride according to the present invention;

FIG. 5 shows the results of experiments on inhibition of elongation of tomato specialized germ tube of Fusarium oxysporum by benzydamine hydrochloride of the present invention.

Detailed Description

The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

Example 1

First, experimental material

1. Benzydamine hydrochloride was purchased from Sigma and stored at 4 ℃ until use.

2. In the experiment, the plant pathogenic bacteria are stored in a refrigerator at 4 ℃, and the culture medium is a potato glucose culture medium (PDA for short). The PDA formula comprises: potato (potato) 200 g, glucose 20g, agar 20g, distilled water 1000 ml, natural pH. The preparation method comprises the following steps: cleaning and peeling potatoes, weighing 200 g of potatoes, cutting the potatoes into small pieces, adding 1000 g of distilled water, boiling for 20 min, filtering the small pieces of distilled water in a big beaker by using four layers of gauze, adding 20g of agar according to experimental requirements, adding 20g of glucose, uniformly stirring, supplementing water to a constant volume of 1000 ml, subpackaging, sterilizing at 121 ℃ for 20 min, and cooling for later use.

3. In this experiment, yeast extract peptone glucose medium (YPD for short) was used for determining spore germination and sprout tube elongation of plant pathogenic bacteria. YPD medium formula: 1% Yeast Extract (Yeast Extract), 2% Peptone (Peptone), 2% Dextrose (glucose). The preparation method comprises the following steps: 10 g of yeast extract, 20g of peptone, 20g of glucose and 1000 ml of distilled water were weighed out, respectively, at natural pH. Subpackaging, sterilizing at 121 ℃ for 20 minutes, and cooling for later use.

Biological control experiment of benzydamine hydrochloride on botrytis cinerea and fusarium oxysporum

1. Inhibition of growth of plant pathogenic bacteria hyphae by benzydamine hydrochloride

The experimental method comprises the following steps: respectively activating and culturing pathogenic fungi on a PDA (personal digital assistant) plate at 25 ℃ for about 5 days for later use; heating PDA culture medium to melt, cooling to 45-50 deg.C, adding different amounts of benzydamine hydrochloride to obtain culture medium containing 0, 0.5, 1.0 and 1.5 mM medicinal liquid, and cooling in culture dish; a round fungus cake (diameter 0.60 cm) is punched at the edge of activated plant pathogenic bacteria strain hypha (growth condition is consistent as much as possible), then an inoculating needle is used for picking to the center of a medicine-containing flat plate, and then the culture dish is placed in an incubator (25 ℃) for culture.

And observing and measuring the growth condition of the hyphae in time after treatment. When the drug-free control plates were substantially full, the diameter was measured using the crosshatch method and the data was processed, and the inhibition rate was calculated and photographed. The inhibition ratio (%) (diameter of control newly grown hyphae-diameter of treated newly grown hyphae)/diameter of control newly grown hyphae × 100. Each treatment was repeated 3 times.

The bacteriostatic effect of benzydamine hydrochloride on the growth of pathogenic bacteria hyphae is shown in figures 1 and 2. As can be seen from FIGS. 1 and 2, the inhibitory effect on two phytopathogens was gradually increased with the increase of benzydamine hydrochloride concentration, i.e., the inhibitory rate on the growth of tomato obligate hyphae of Botrytis cinerea and Fusarium oxysporum was dose-dependent. When benzydamine hydrochloride is at 1.5 mM, hyphae of the two phytopathogens do not grow substantially.

Table 1 shows the inhibition rate of benzydamine hydrochloride on the growth of tomato dedicated hyphae of Botrytis cinerea and Fusarium oxysporum. As is clear from Table 1, when the concentration of benzydamine hydrochloride is 1.0 mM, the inhibition rate of benzydamine hydrochloride against both phytopathogens is 60% or more. When the concentration of the benzydamine hydrochloride is 1.5 mM, the inhibition rate on botrytis cinerea reaches 97.5%, and the inhibition rate on fusarium oxysporum tomato specialization type reaches 94.1%, which indicates that the benzydamine hydrochloride has obvious inhibition effect on the hypha growth of two pathogenic bacteria.

TABLE 1 inhibition rate of benzydamine hydrochloride on the growth of pathogenic bacteria hyphae

2. Inhibition effect of benzydamine hydrochloride on spore germination of plant pathogenic bacteria

The experimental method comprises the following steps: inoculating the two pathogenic bacteria on a PDA plate respectively to culture until spore is produced; collecting spores and adjusting the concentration of spores to about 6.0X 10 using YPD liquid medium⁵Per ml; adding benzydamine hydrochloride with different masses (concentration 0, 0.1, 0.2, 0.3 and 0.4 mM) into conidium suspension respectively; the inhibition effect of the benzydamine hydrochloride on the germination of 2 pathogenic bacteria spores is detected by adopting a concave slide method (50 ul of spore suspension is respectively added into a sterilized concave slide, the spore suspension is subjected to moisturizing culture at 26 ℃, when the germination rate of control conidia reaches 90%, the germination rate of the conidia treated by the benzydamine hydrochloride with different concentrations is detected, and the inhibition rate of the medicament on the germination of the conidia is calculated).

The experimental result is shown in figure 3, and the inhibition rate of the germination of conidium of 2 pathogenic bacteria is obviously improved along with the increase of the content of benzydamine hydrochloride. When the content exceeds 0.2 mM, the germination of two conidia can be basically inhibited, which shows that the benzydamine hydrochloride has stronger inhibiting effect on the germination of botrytis cinerea and fusarium oxysporum tomato specialized spores.

3. Influence of benzydamine hydrochloride on elongation of botrytis cinerea germ tubes

The experimental method comprises the following steps: and (3) detecting by adopting the same concave slide method, culturing for 6-10 h, observing and measuring the elongation condition of the bud tube by using a microscope, and calculating the inhibition rate of the medicament on the elongation of the bud tube. Inhibition (%) (control sprout tube length-treated sprout tube length)/control sprout tube length × 100. Each treatment was repeated 3 times.

As shown in fig. 4, the inhibition rate of the growth of the germ tube was gradually increased with the increase in the benzydamine hydrochloride concentration at the same time point. When the benzydamine hydrochloride is 0.2 mM, the elongation of the botrytis cinerea germ tube is basically inhibited.

Table 2 summarizes the inhibition rate of benzydamine hydrochloride on Botrytis cinerea germ tube elongation. As can be seen from Table 2, the inhibition rate of the growth of the germ tubes of Botrytis cinerea can reach 97.6% when the concentration of the drug is 0.2 mM.

TABLE 2 inhibition rate of benzydamine hydrochloride (0.2 mM) on Botrytis cinerea germ tube elongation

4. Influence of benzydamine hydrochloride on elongation of fusarium oxysporum tomato specialized germ tube

The experimental method comprises the following steps: and (3) detecting by adopting the same concave slide method, measuring the elongation condition of the germ tube after culturing for 3-9 h, and calculating the inhibition rate of the benzydamine hydrochloride on the elongation of the germ tube by adopting the same method.

As shown in FIG. 5, at the same time point, the inhibition rate of the growth of the germ tube was higher as the concentration of benzydamine hydrochloride was increased, i.e., the inhibition rate of the growth of the germ tube was dose-dependent, and when benzydamine hydrochloride was 0.2 mM, the growth of the germ tube was substantially inhibited.

Table 3 summarizes the inhibition rate of benzydamine hydrochloride on the elongation of fusarium oxysporum tomato specialized germ tubes. As can be seen from Table 3, when the benzydamine hydrochloride concentration is 0.2 mM, the elongation inhibition rate of the tomato obligate germ tube of Fusarium oxysporum can reach 98.0%.

TABLE 3 inhibition rate of benzydamine hydrochloride (0.2 mM) on elongation of fusarium oxysporum tomato specialized germ cells

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.