Application of 4-Propylphenol in the Control of Soil-borne Diseases

Abstract

The present invention discloses the application of 4-propylphenol in the control of soil-borne diseases, belonging to the technical field of plant-derived fungicides. The present invention has found through research that 4-propylphenol has significant inhibitory effects on various soil-borne pathogens such as Fusarium graminearum, Pseudograminearum, Phytophthora sojae, and Phytophthora capsici; moreover, 4-propylphenol is a plant-derived volatile substance with high safety and low cost, and has broad application prospects in the control of soil-borne diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of plant-derived fungicides, and particularly to the application of 4-propylphenol in the control of soil-borne diseases. Background Art

[0002] Soil health is closely related to the development of sustainable agriculture. In agricultural production, soil health plays a crucial role. As a dynamic ecosystem, a healthy soil environment can maintain water balance, store organic and inorganic carbon, control the cycling and decomposition of soil nutrients, and improve plant productivity (Doran, 2000). However, the frequent occurrence of soil-borne diseases seriously disrupts the soil health balance. Soil-borne diseases are a relatively serious type of disease in agriculture. After the disease occurs, it leads to a reduction in crop yield and causes serious economic losses. Their disease characteristics are numerous types, great harm, and difficulty in prediction and control.

[0003] Soil-borne diseases refer to the general term for diseases in which pathogens (such as fungi, bacteria, oomycetes, viruses, and nematodes) infect plants from the roots or stems through the soil as a transmission medium under suitable environmental conditions (Li Xinglong, 2015). Since the pathogens of soil-borne diseases parasitize in the soil and have strong concealment, it is difficult to judge the disease type through specific symptoms, which increases the difficulty of controlling soil-borne diseases (Panth et al., 2020; Yang Zhen, 2019). Among them, the pathogens causing the diseases are mainly fungi. The main types of fungal pathogens are: Rhizoctonia solani, Fusarium moniliforme, Fusarium graminearum, Pythium aphanidermatum, etc. These pathogens will cause the prevalence of diseases such as damping-off, Rhizoctonia blight, root rot, fusarium wilt, etc., resulting in a reduction in crop yield and damage to quality.

[0004] In recent years, the intensive agricultural production model has developed rapidly. People blindly pursue high yields and high efficiency of crops, and unreasonable land use has led to frequent occurrence of soil-borne diseases (Wei Yong, et al., 2022). This not only reduces crop yields and causes economic losses but also disrupts the soil ecological balance. The reasons for the prevalence of soil-borne diseases are as follows: The single-crop planting pattern causes continuous cropping effects, making the reproduction of pathogenic bacteria more rampant (Cai Zucong, Huang Xinqi, 2016); Straw returning to the field, poor land management by farmers, and inappropriate farming operations provide a more suitable growth environment for pathogenic bacteria; The excessive use of chemical fertilizers reduces soil fertility (Wu and Li, 2022); The unscientific use of pesticides increases the drug resistance of soil-borne pathogenic bacteria (Chu Bingyao, et al., 2020; Ma Huiling, Fang Yuanyuan, 2014). Therefore, paying attention to the interaction relationship among the soil ecological environment, soil microorganisms, and soil-borne pathogenic bacteria is beneficial to the prevention and control of soil-borne diseases (Garibaldi et al., 2014; Raviv and M, 2009).

[0005] Regarding the current situation of soil-borne diseases and combining with the infection mode of soil-borne pathogenic bacteria, among the current research methods such as disease-resistant varieties, chemical control, and biological control, chemical control is the most widely used and the most rapid and efficient. Therefore, the development of new green and safe pesticides is an urgent requirement for the sustainable development of agriculture.

[0006] Plant-derived fungicides come from nature and degrade in nature, are not prone to produce residues, and are beneficial to avoiding environmental pollution, water pollution, and soil ecological imbalance (Chuanwan et al., 2004; Sundin et al., 2016; Yi Yongfeng, Zhou Jiechen, 2022). Currently, the application of plant-derived fungicides is relatively extensive. For example, in the medical field, in inhibiting and killing bacteria and pests of plant diseases, in food storage, and in the preservation and anti-corrosion of fruits, vegetables, etc. (Wang et al., 2015).

[0007] In terms of disease prevention, compared with traditional chemical pesticides, plant-derived fungicides have the following advantages: (1) Wide sources. There are numerous plant resources in China and even the world, which also means the abundance of plant resources that can be developed into plant-derived fungicides (Chuanwan et al., 2004). (2) Many plant-derived fungicides can not only inhibit and kill bacteria, but also play a certain role in promoting growth (Liang Changhui, 2021; Ge Ting, 2021). (3) Plant-derived fungicides have strong targeting, low toxicity to non-target organisms, are easy to volatilize and degrade, with less residue, which is beneficial to human food safety (Balogh et al., 2010; Louws et al., 2001). (4) The simultaneous action of multiple active ingredients in plant-derived fungicides on pathogenic bacteria has stronger antibacterial activity, which also provides more possibilities for later compounding (Kim and Rhee, 2016; Medina et al., 2006).

[0008] 4-Propylphenol, also known as p-propylphenol and tetra-n-propylphenol, with the English name 4-Propylphenol and the molecular formula C9H 12 O and a molecular weight of 136.19. It is a light yellow liquid. 4-Propylphenol naturally exists in oriental tobacco leaves and cigarette smoke. As a kind of phenolic substance, it can be released from plant tissues in gaseous form and is an important plant secondary metabolite. In daily production applications, 4-Propylphenol is commonly used as a liquid crystal raw material and intermediate. In addition, 4-Propylphenol is also a phenolic flavor allowed to be used in the national food additive use standard (GB2760-2014) of China and is widely used in food production: for example, in the production of baked products, soft candies, jellies, puddings, non-alcoholic beverages, fruit ice products, preserved fruits, frostings, jellies, jams, hard candies, chewing gums, etc. Currently, in the research related to 4-Propylphenol, there are very few reports on the inhibition of plant pathogenic bacteria. Only the literature reports that 4-Propylphenol has an inhibitory effect on four bacteria, namely Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, and Staphylococcus aureus (Sun Jiewen et al., 2017). Summary of the Invention

[0009] Aiming at the above-mentioned existing technologies, the purpose of the present invention is to provide the application of 4-propylphenol in the prevention and control of soil-borne diseases. The present invention has found through research that 4-propylphenol has varying degrees of inhibitory effects on various soil-borne pathogenic bacteria such as Fusarium graminearum, Pseudograminearum, Phytophthora sojae, and Phytophthora capsici; and 4-propylphenol is a plant-derived volatile substance with high safety and low cost, and has broad application prospects in the prevention and control of soil-borne diseases.

[0010] To achieve the above purpose, the present invention adopts the following technical solutions:

[0011] In the first aspect of the present invention, there is provided the use of 4-propylphenol in the preparation of an antibacterial agent against soil-borne pathogens.

[0012] In the above application, preferably, the soil-borne pathogen is one or more of Fusarium graminearum, Fusarium pseudograminearum, Fusarium oxysporum, Phytophthora sojae, Phytophthora nicotianae and Phytophthora capsici.

[0013] In the second aspect of the present invention, there is provided the use of 4-propylphenol in the preparation of a drug for preventing and treating soil-borne diseases.

[0014] In the above application, the soil-borne diseases include: wheat scab, wheat basal stalk rot, Phytophthora sojae, tobacco black shank and / or Phytophthora capsici.

[0015] In the third aspect of the present invention, there is provided a method for preventing and treating wheat scab, comprising the following steps:

[0016] During the flowering stage of wheat, the ears of wheat are treated with 4-propylphenol.

[0017] Preferably, the ears of wheat are sprayed with 4-propylphenol at a concentration of 150 μg / ml - 200 μg / ml.

[0018] In the fourth aspect of the present invention, there is provided a method for preventing and treating wheat basal stalk rot, comprising the step of performing seed coating treatment on wheat seeds with 4-propylphenol before sowing.

[0019] In the fifth aspect of the present invention, there is provided the use of a composition in the preparation of a product for preventing and treating Fusarium graminearum infection;

[0020] The composition consists of 150 μg / ml 4-propylphenol and 0.6 mg / ml phenethyl alcohol.

[0021] In the sixth aspect of the present invention, there is provided a pharmaceutical preparation for preventing and treating wheat scab, and the pharmaceutical preparation contains 150 μg / ml 4-propylphenol and 0.6 mg / ml phenethyl alcohol.

[0022] Advantages of the present invention:

[0023] (1) It is found in the research of the present invention that 4-propylphenol has varying degrees of inhibitory effects on various soil-borne pathogens such as Fusarium graminearum, Fusarium pseudograminearum, Phytophthora sojae, Phytophthora capsici, etc.; and 4-propylphenol is a plant-derived volatile substance, with high safety and low cost, and has broad application prospects in the prevention and treatment of soil-borne diseases.

[0024] (2) 4-Propylphenol is both a plant-derived volatile and an edible spice in China, and its safety in use is more guaranteed. Moreover, 4-propylphenol is inexpensive and has a low cost. Therefore, 4-propylphenol has greater development potential and a broader application prospect.

[0025] (3) The combined use of 4-propylphenol and phenethyl alcohol has a synergistic effect in preventing the infection of Fusarium graminearum. Description of the Drawings

[0026] Figure 1 : Gas chromatography-mass spectrometry diagram of tetrapropylphenol; in the figure, a. is the peak diagram of the disease-resistant material treated with Fusarium graminearum; b. is the peak diagram of the disease-resistant material treated with water; c. is the peak diagram of the susceptible material treated with Fusarium graminearum; d. is the peak diagram of the disease-resistant material treated with water. The blue line represents the quantitative ion; the red and blue lines represent the quantitative ion.

[0027] Figure 2 : Effect of 4-propylphenol on the colony formation of Fusarium graminearum.

[0028] Figure 3 : Effect of 4-propylphenol on the radial growth of Fusarium graminearum hyphae; A. Different concentrations of tetrapropylphenol were added to the PDA medium; B. The colony diameter of Fusarium graminearum was measured after 7 days; C. Inhibition rate of tetrapropylphenol on the hyphal diameter of Gibberella zeae.

[0029] Figure 4 : Effect of 4-propylphenol on the dry weight of Fusarium graminearum hyphae.

[0030] Figure 5 : Effect of 4-propylphenol on the hyphal morphology of Fusarium graminearum.

[0031] Figure 6 : Effect of 4-propylphenol on the leakage amount of intracellular substances of Fusarium graminearum; in the figure, A: Effect of different concentrations of 4-propylphenol on the DNA leakage amount of Fusarium graminearum; B: Effect of different concentrations of 4-propylphenol on the protein leakage amount of Fusarium graminearum.

[0032] Figure 7 : Effect of 4-propylphenol on the conductivity of Fusarium graminearum solution.

[0033] Figure 8 : Antibacterial activity test of 4-propylphenol against other Fusarium oxysporum; in the figure, a. Fusarium oxysporum f. sp. lycopersici b. Fusarium oxysporum f. sp. cucumerinum.

[0034] Figure 9 : Antibacterial activity test of 4-propylphenol against Phytophthora; in the figure, a. Phytophthora sojae b. Phytophthora capsici c. Phytophthora nicotianae.

[0035] Figure 10 : Safety detection of 4-propylphenol on wheat seeds.

[0036] Figure 11 : Safety detection of 4-propylphenol on wheat plants; in the figure, A. Treating soil with 4-propylphenol at different concentrations; B. Growth chart of wheat plant height measured after 14 days.

[0037] Figure 12 : Field control effect of tetrapropylphenol on Fusarium head blight of wheat; in the figure, A. Control effect of tetrapropylphenol at different concentrations on wild-type wheat Fielder; B. Diseased spikelet rate after inoculation for 14 days. Detailed implementation manners

[0038] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0039] As mentioned above, in recent years, soil-borne diseases have occurred frequently, posing a huge challenge to the sustainable development of agriculture. At present, the main prevention and control methods for soil-borne diseases are the extensive use of pesticides. For a long time, this will not only enhance the drug resistance of pathogenic bacteria, but also cause environmental pollution and soil ecological imbalance. As an important leading source of green fungicides, plant-derived antibacterial substances are widely present in nature. When plants are stressed by the outside world, they can quickly synthesize and release a large number of volatile organic compounds. Some of these volatiles can help plants resist the invasion of pathogens. Therefore, it is an urgent requirement in agricultural production to develop eco-friendly new pesticides for soil-borne pathogens by using volatiles.

[0040] Based on this, the present invention optimizes and innovates in the screening method of volatile compounds, and uses near-isogenic line materials for treatment, which can not only reduce the difference in volatile components caused by different genetic backgrounds of materials, but also accurately screen the range. After different treatments on wheat resistant and susceptible materials, the present invention uses a gas chromatography-mass spectrometry (GC-MS) to determine the components in the volatile compound database established in this laboratory according to the peak shape and peak area, and finds that the content of some volatile compounds in resistant varieties is more than that in susceptible varieties. Therefore, it is speculated that these volatiles may play an important role in the plant's resistance to pathogen infection ( Figure 1 ).

[0041] In the preliminary antibacterial activity test, when using the drug-containing medium method, among the four selected substances, only 4-propylphenol can inhibit the radial growth of the radial mycelium of Fusarium graminearum, while phenethyl alcohol, hexyl acetate and decanal at the same concentration have no inhibitory effect on the growth of the radial mycelium of Fusarium graminearum. Under the fumigation treatment, the test results show that 4-propylphenol also has good antibacterial activity. In addition, an interesting phenomenon was found: when using the drug-containing medium method, phenethyl alcohol, which has no obvious inhibitory effect on the growth of the radial mycelium of Fusarium graminearum at a concentration of 400 μg / ml, shows a significant inhibitory effect at a concentration of only 200 μg / ml under the fumigation method.

[0042] This invention mainly investigated the antibacterial activity of 4-propylphenol. The results first proved that 4-propylphenol has antibacterial activity against fungi such as Fusarium oxysporum and oomycetes such as Phytophthora sojae, and has the application prospect of being developed into a soil-borne disease control agent. To realize its practical application, this invention further detected the use safety of 4-propylphenol and found that within a certain concentration range, 4-propylphenol does not affect the germination of wheat seeds and the growth of plants.

[0043] Based on the excellent antibacterial activity of 4-propylphenol, this invention also investigated the antibacterial activity of other phenolic compounds. The antibacterial results of different phenolic compounds against Fusarium graminearum are as follows:

[0044]

[0045] The results show that the relationship between the alkyl carbon chain of phenolic compounds and antibacterial activity has a certain regularity: the antibacterial effect of phenolic compounds increases with the increase of the length of the alkyl carbon chain, and the change of the position of the carbon chain does not affect the antibacterial activity, which provides a reference basis for the later research of phenolic substances. The field control test also verified the regularity of the carbon chain length and antibacterial effect, and the control effect on wheat scab is tetrabutylphenol > tetrapropylphenol > tetraethylphenol.

[0046] Considering the actual production application in the later stage, in terms of safety, compared with tetraethylphenol, tetrapropylphenol has a better antibacterial effect in in vitro tests and less odor irritation. On the premise that the control effects of tetrabutylphenol and tetrapropylphenol are similar, tetrapropylphenol is both a plant-derived volatile and an edible spice in China, and its safety is more guaranteed than tetrabutylphenol. In terms of production cost, the price of tetrapropylphenol is only one-tenth of that of tetrabutylphenol, and the cost is low. Therefore, tetrapropylphenol has more development potential and a broader application prospect.

[0047] To further enhance the antibacterial effect of 4-propylphenol, this invention considered combining 4-propylphenol with phenethyl alcohol, which also has antibacterial effects, and found that it has a synergistic effect in preventing the infection of Fusarium graminearum.

[0048] Thus, the present invention is proposed.

[0049] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0050] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. Among them: 4-propylphenol was purchased from Shanghai Macklin Biochemical Co., Ltd., and its chemical structural formula is shown as follows:

[0051]

[0052] Phenylethyl alcohol was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0053] Fusarium oxysporum f. sp. cucumerinum and Fusarium oxysporum f. sp. lycopersici were purchased from BNCC.

[0054] The Fusarium graminearum used in this embodiment is the wild-type strain PH-1 of Fusarium graminearum; pathogenic fungi such as Fusarium pseudograminearum, Phytophthora sojae, Phytophthora nicotianae, and Phytophthora capsici have all been disclosed in non-patent literature, and the public can obtain them from the applicant within 20 years from the application date for repeating this experiment (the above-mentioned pathogenic fungi are stored in the National Plant Protection Microbial Germplasm Resource Bank (Shandong)).

[0055] The composition of the culture medium used in the embodiments of the present invention is as follows:

[0056] (1) CMC (Carboxymethylcellulose) medium (Hou et al. 2002)

[0057]

[0058] Note: Since CMC is difficult to dissolve at low temperatures, if it needs to be dispensed and sterilized, first dispense it, then separately weigh it into a bottle, and add 1.5 g of CMC powder to every 100 mL. After autoclaving at 121 °C for 25 min, store it in a 4 °C refrigerator.

[0059] (2) PDA medium (1 L):

[0060]

[0061] Wash the potatoes, peel them, weigh 200 g and cut them into small pieces. Add an appropriate amount of distilled water and heat for about 20 min until the potatoes are cooked to the extent that they can be mashed with a glass rod. Filter the potato pieces with four layers of gauze, collect the filtrate, add 15 g of glucose and 12 g of agar powder, slightly heat and stir evenly, make up the volume to 1 L with distilled water, dispense into conical flasks, sterilize at 121 °C under high-pressure steam for 25 min, and store in a 4 °C refrigerator.

[0062] (3) YEPD (Yeast extraction peptone dextroglucose) liquid medium:

[0063]

[0064] After sterilizing at 121 °C by moist heat for 25 min, store in a 4 °C refrigerator.

[0065] Example 1: Investigation on the antibacterial activity of 4-propylphenol

[0066] 1. Investigation on the antibacterial activity of 4-propylphenol against Fusarium graminearum:

[0067] (1) Effect of 4-propylphenol on the spore germination of Fusarium graminearum:

[0068] In the prepared CMC medium, add 4-propylphenol at different concentrations so that the final concentrations are 50 μg / ml, 100 μg / ml, 150 μg / ml, 200 μg / ml, and 250 μg / ml respectively, and use the treatment with the addition of the same volume of ddH2O as the blank control. Inoculate 6 Fusarium graminearum discs with a diameter of 6 mm in each bottle of the medium, and culture them on a shaker at 25 °C and 200 rpm in the dark for 3 - 4 days. Each treatment has 3 replicates. Filter the CMC culture solution with four layers of sterile gauze, collect the filtrate in a 50 ml sterile centrifuge tube, centrifuge at 2000 rpm for 5 - 10 minutes, discard the supernatant, and collect the conidial precipitate. Add the same volume of ddH2O to resuspend the conidia, and use a hemocytometer to observe and count the amount of conidia under a microscope.

[0069] 4-Propylphenol can significantly inhibit the spore germination of Fusarium graminearum. Moreover, as the concentration of added 4-propylphenol increases, the germinated conidia decrease, and the inhibitory effect is enhanced. Compared with the control group, when the concentration of added 4-propylphenol is 200 μg / ml, 4-propylphenol completely inhibits the spore germination of Fusarium graminearum (Table 1).

[0070] Table 1: Effect of 4-propylphenol on the spore germination of Fusarium graminearum

[0071]

[0072] (2) Effect of 4-propylphenol on the colony formation of Fusarium graminearum:

[0073] 0.1 ml of Fusarium graminearum spore suspension was evenly spread on PDA medium containing 4-propylphenol at a concentration of 20 μg / ml to 220 μg / ml, and the treatment with the same volume of ddH2O spread was used as the control. Each treatment had 3 replicates. Incubate at 25 °C for 3 days in the dark to observe the effect of 4-propylphenol on the colony formation of Fusarium graminearum.

[0074] The results are as Figure 2 shown. The results indicate that 4-propylphenol can inhibit the colony formation of Fusarium graminearum. The spore suspension was evenly spread on the medium containing different concentrations of 4-propylphenol. It was observed that in the medium containing 4-propylphenol, the speed and area of colony formation were significantly lower than those of the control group. Moreover, the higher the concentration of 4-propylphenol added, the stronger the inhibitory effect.

[0075] After two days of incubation, the colonies in the control group had expanded to cover the entire plate, and the colony color was bright red, mixed with some yellow. When the concentration of 4-propylphenol added was 40 μg / ml, only several sporadic small dots were formed, and the colony color was white. When the added concentration was 200 μg / ml, 4-propylphenol completely inhibited the colony formation.

[0076] (3) Effect of 4-propylphenol on the radial growth of Fusarium graminearum hyphae:

[0077] Different concentrations of 4-propylphenol were added to sterile petri dishes containing PDA medium to make the final concentrations 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, and 100 μg / ml respectively, and the treatment with the same volume of ddH2O added was used as the blank control. A 6-mm-diameter Fusarium graminearum mycelial disc was inoculated into each petri dish, sealed with a sealing film, and incubated upside down at 25 °C in the dark. Each treatment had 3 replicates. Observe and photograph the mycelial growth, measure and record the colony diameter using the cross-cross method, and calculate the inhibition rate using the formula.

[0078] Mycelial growth inhibition rate = ((colony diameter of the control group - diameter of the mycelial disc) - (colony diameter of the treatment group - diameter of the mycelial disc)) / (colony diameter of the control group - diameter of the mycelial disc) × 100%

[0079] The results showed that 4-propylphenol had a significant inhibitory effect on the radial growth of Fusarium graminearum hyphae. As the concentration of added 4-propylphenol increased, the inhibitory effect enhanced. When the concentration of added 4-propylphenol was 80 μg / ml, the inhibition rate reached 97.44%. When the added concentration was 100 μg / ml, the inhibition rate reached 100%, and 4-propylphenol completely inhibited the radial growth of Fusarium graminearum hyphae. By observing the petri dishes after 7 days of cultivation, it was found that the entire colony in the control group presented bright red, and the surface was completely spread and covered by yellowish-white villous hyphae. When the added concentration was 20 μg / ml, only the center of the colony surface showed red, the external hyphae were white, and some hyphae collapsed, with the hyphal growth being weak ( Figure 3 ).

[0080] (4) Effects of 4-propylphenol on the mycelium of Fusarium graminearum:

[0081] Different concentrations of 4-propylphenol were added to the YEPD medium to make the final concentrations 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, and 100 μg / ml respectively, and the treatment with the addition of the same volume of ddH2O was used as the control. A spore suspension of Fusarium graminearum with a spore concentration of 5×10 5 spores / mL was added to each bottle of the medium, and it was cultured on a shaker at 25°C and 200 rpm for 4 days in the dark. Each treatment had 3 replicates. The mycelium was collected using a funnel, filter paper, etc., placed in an oven at 60°C for drying for 24 hours, and weighed after cooling to calculate the inhibition rate of 4-propylphenol on the mycelial growth of Fusarium graminearum.

[0082] Inhibition rate of mycelial growth (%) = (dry weight of control mycelium - dry weight of treated mycelium) / dry weight of control mycelium × 100%

[0083] The results showed that 4-propylphenol significantly inhibited the growth of Fusarium graminearum mycelium. Moreover, the higher the added concentration of 4-propylphenol, the stronger the inhibitory effect ( Figure 4 ). When the concentration of added 4-propylphenol was 60 μg / ml, the mycelial inhibition rate reached 63.8%. The dry weight of the mycelium was only 0.094 g, which was less than half of the dry weight of the mycelium in the control group. When the concentration of added 4-propylphenol was 100 μg / ml, the mycelium in the medium no longer grew, and the inhibition rate of 4-propylphenol on the mycelium of Fusarium graminearum reached 100% (Table 2).

[0084] Table 2: Effects of 4-propylphenol on the dry weight of Fusarium graminearum mycelium

[0085]

[0086] (5) Effects of 4-propylphenol on the mycelial morphology of Fusarium graminearum:

[0087] Collect the mycelia of Fusarium graminearum, and treat the obtained mycelia with 4-propylphenol (100 μg / ml) for 24 hours, using the treatment with the same volume of ddH2O added as the control. There are 3 replicates for each treatment. Observe the morphological characteristics of the mycelia of Fusarium graminearum after different treatments under an optical microscope.

[0088] Observation under the optical microscope of the control group and the 4-propylphenol treatment group found that, as Figure 5 shown, the mycelia in the control group were plump, smooth, naturally extended, with more mycelial branches, thick and strong branch growth, and obvious mycelial septum separation. The mycelia after treatment with tetrapropylphenol grew slender, were relatively disordered and twisted, had fewer mycelial branches, and the mycelial septum separation was not obvious. We speculate that 4-propylphenol may have caused damage to the cells of Fusarium graminearum.

[0089] (6) Effect of 4-propylphenol on the cell membrane of Fusarium graminearum:

[0090] To investigate whether 4-propylphenol causes damage to the cell membrane of Fusarium graminearum, detect the extracellular leakage of DNA, extracellular leakage of protein, and solution conductivity of Fusarium graminearum. The specific methods are as follows:

[0091] Mycelia preparation: In a laminar flow hood, use a sterile pipette tip with a diameter of 6 mm to punch out mycelial cakes (5 - 8 pieces) from the edge of the activated Fusarium graminearum colony and inoculate them into CMC medium. Culture in a shaker at 25°C and 200 rpm in the dark for 4 days to obtain a spore suspension of Fusarium graminearum. Inoculate the suspension into YEPD medium and culture in a shaker in the dark for 4 days to obtain the mycelia of Fusarium graminearum.

[0092] Detection of extracellular DNA leakage: Place a certain amount of mycelia in a centrifuge tube containing medium, and add 4-propylphenol respectively to make its final concentrations 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, using the treatment with the same volume of ddH2O added as the control. There are 3 replicates for each treatment. Absorb a fixed amount of the culture solution every 2 hours and measure the absorbance at 260 nm for different treatments using a micro-spectrophotometer.

[0093] Detection of extracellular protein leakage: Place a certain amount of mycelia in a centrifuge tube containing medium, and add 4-propylphenol respectively to make its final concentrations 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, using the treatment with the same volume of ddH2O added as the control. There are 3 replicates for each treatment. Absorb a fixed amount of the culture solution every 2 hours and measure the absorbance at 280 nm for different treatments using a micro-spectrophotometer.

[0094] Conductivity detection: A certain amount of mycelium was placed in a centrifuge tube containing culture medium, and 4-propylphenol was added respectively to make its final concentrations 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, and 100 μg / ml. The treatment with the addition of the same volume of ddH2O was used as the control. There were 3 replicates for each treatment. The conductivity of the solution under different treatments was detected with a conductivity detector every 2 hours.

[0095] The reason why cells can perform normal physiological functions is that the cell contents play a crucial role. To verify whether 4-propylphenol causes the leakage of cell contents by destroying the cell membrane structure of Fusarium graminearum, we next detected the leakage amount of intracellular substances of Fusarium graminearum by 4-propylphenol. The test results showed ( Figure 6 ), within the same time, the higher the concentration of tetrapropylphenol in the treatment group, the more the DNA leakage amount and protein leakage amount of Fusarium graminearum. And at the same concentration, there was a certain positive correlation between the treatment time and the DNA leakage amount and protein leakage amount of Fusarium graminearum. Generally speaking, the DNA leakage amount and protein leakage amount of Fusarium graminearum treated with 4-propylphenol were significantly higher than those of the control group. This also proved that 4-propylphenol destroyed the cell membrane of Fusarium graminearum, resulting in the leakage of cell contents.

[0096] Relative conductivity is an important index to measure cell membrane permeability. The larger its value, the more the leakage amount of electrolytes, indicating that the degree of damage to the cell membrane integrity is greater. To further study the effect of 4-propylphenol on the cell membrane permeability of Fusarium graminearum, the determination of the conductivity of the solution of Fusarium graminearum by 4-propylphenol was carried out. From Figure 7 it can be seen that compared with the control group, the conductivity of the mycelium solution of Fusarium graminearum treated with 4-propylphenol increased significantly. And as the concentration of 4-propylphenol added increased, the conductivity also increased. This shows that 4-propylphenol changed the permeability of the cell membrane of Fusarium graminearum, resulting in the damage of the cell membrane. And within the concentration range of 0 μg / ml to 100 μg / ml, there was a positive correlation between the concentration of 4-propylphenol and the degree of cell membrane damage.

[0097] 2. Investigation on the antibacterial activity of 4-propylphenol against other soil-borne pathogens:

[0098] Two other Fusarium oxysporum: Fusarium oxysporum f. sp. cucumerinum and Fusarium oxysporum f. sp. lycopersici were selected for the antibacterial activity test of 4-propylphenol. At the same time, three oomycetes: Phytophthora sojae, Phytophthora nicotianae, and Phytophthora capsici were selected for the antibacterial activity test. The method was the same as the above-mentioned "(3) Effect of 4-propylphenol on the radial growth of Fusarium graminearum mycelium".

[0099] Mycelial growth inhibition rate = ((colony diameter of the control group - diameter of the fungal disc) - (colony diameter of the treatment group - diameter of the fungal disc)) / (colony diameter of the control group - diameter of the fungal disc) × 100%

[0100] The results showed that 4-propylphenol had inhibitory effects on other soil-borne diseases to varying degrees, and the inhibitory intensity increased with the increase in the concentration of added 4-propylphenol. 4-Propylphenol had the strongest inhibitory effect on Phytophthora sojae. When the added concentration was 60 μg / ml, the inhibition rate against Phytophthora sojae reached 100%. When the added concentration was 80 μg / ml, 4-propylphenol could completely inhibit the radial mycelial growth of Fusarium graminearum, Fusarium oxysporum f. sp. cucumerinum, Phytophthora sojae, and Phytophthora nicotianae( Figure 8 , Figure 9 ).

[0101] Example 2: Effect of 4-propylphenol on wheat seed germination

[0102] Disinfection and germination acceleration treatment of wheat seeds: Take out the wheat seeds required for the experiment from the -20°C refrigerator, soak them in 0.1% sodium hypochlorite solution for 10 minutes, and rinse them once with distilled water. Then soak them in distilled water for ten minutes, rinse, and repeat twice. Place the disinfected wheat seeds in a petri dish with two layers of sterile filter paper at the bottom, pour in an appropriate amount of distilled water, and incubate at 4°C for 24 hours for germination acceleration.

[0103] Add different concentrations of 4-propylphenol to a petri dish with two layers of sterile filter paper at the bottom, so that the final concentrations are 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, and 100 μg / ml respectively. Use the treatment with the addition of the same volume of ddH2O as the blank control. Arrange 9 disinfected wheat seeds with the ventral groove facing down in the petri dish, culture at room temperature, and observe and record the germination of wheat seeds.

[0104] The results of the previous in vitro experiments showed that 4-propylphenol had a significant inhibitory effect on Fusarium graminearum. Next, an actual application experiment was carried out. We treated wheat seeds with different concentrations of 4-propylphenol and then conducted a germination experiment. After moisturizing culture, it was found through observation that within the concentration range of 0 μg / ml to 100 μg / ml, 4-propylphenol did not affect the germination of wheat seeds, and the germination rate was 100% (Table 3, Figure 10 ).

[0105] Table 3: Safety detection of 4-propylphenol on wheat seeds

[0106]

[0107] Example 3: Effect of 4-propylphenol on wheat plant growth

[0108] Using the soil treatment method, 10 ml of 4-propylphenol solutions with final concentrations of 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, and 100 μg / ml were added to each pot of soil, respectively, and the treatment with the addition of the same volume of ddH2O was used as the control. The disinfected and germinated wheat seeds were planted in the pots. There were 3 replicates for each treatment. Observations and photographs were taken on the 7th and 14th days after sowing, and the germination situation and plant height were recorded.

[0109] The test results showed that within the concentration range of 0 μg / ml to 100 μg / ml, the wheat seeds could all emerge from the soil, and the germination rate was 100%. After measurement and statistics, the average plant height of wheat under each treatment had little difference ( Figure 11 , Table 4).

[0110] Table 4: Safety detection of 4-propylphenol on wheat plants

[0111]

[0112] Example 4: Control test of 4-propylphenol on Fusarium graminearum infecting wheat in the field

[0113] During the full bloom stage of wheat flowering, the wheat ears were treated with 4-propylphenol. 4-propylphenol was formulated into medicated solutions with final concentrations of 50 μg / ml, 100 μg / ml, 150 μg / ml, and 200 μg / ml. The treatment with a final concentration of 0 μg / ml of 4-propylphenol was used as the negative control, denoted as CK0; the treatment with the common pesticide tebuconazole was used as the positive control (the tebuconazole treatment was carried out according to the instructions: 15 ml of 43% tebuconazole suspension was mixed with 30 L of water), denoted as CK1.

[0114] Spraying treatment: At the flowering stage, the prepared medicated solutions of 4-propylphenol with different concentrations were evenly and quantitatively sprayed (30 L per mu) on the ears of the marked wheat.

[0115] The spore concentration of the Fusarium graminearum bacterial solution was adjusted to 5×10 5 CFU / mL, and three days after the treatment, the ears were inoculated by the single-flower dropping method. There were 10 replicates for each treatment. The disease index was statistically analyzed after 7 days and 14 days, and the disease phenotype was photographed and recorded. The percentage of scabbed spiklets was calculated to judge the severity of scab disease.

[0116] Percentage of scabbed spiklet (PSS) = (number of scabbed spiklets / total number of spiklets) × 100%.

[0117] The results showed that: under spraying conditions, 4-propylphenol had a good control effect on Fusarium head blight of wheat. When the concentration of 4-propylphenol sprayed was 200 μg / ml, the diseased spikelet rate was only 32.5%. It could be seen from the phenotypic diagram at 14 days after inoculation that the inoculated part of the negative control group CK0 was significantly diseased, and the degree of lesion expansion was severe, resulting in the withering and death of the entire wheat ear. After treatment with 4-propylphenol, the wheat ears were slightly diseased, the lesion expansion was slower, the number of diseased spikelets and diseased grains decreased significantly, and the degree of reduction in the diseased spikelet rate was positively correlated with the concentration of 4-propylphenol applied. This indicated that under spraying treatment, 4-propylphenol had a good control effect on Fusarium head blight of wheat( Figure 12 ).

[0118] Example 5: Effect of 4-propylphenol coating on wheat seed germination

[0119] Using Jimai 22 as the experimental object for coating test, 20 μL of 4-propylphenol and an appropriate amount of ddH2O were used to coat 100 g of seeds by mixing, and the treatment with the addition of the same volume of ddH2O was used as the control. The coated wheat seeds were neatly placed with the ventral groove facing down in a petri dish with a diameter of 15 cm lined with two layers of sterilized and moist filter paper. Each treatment had 3 replicates. After 2 days, observations and photos were taken, and the germination situation was recorded.

[0120] The test results are shown in Table 5.

[0121] Table 5: Germination rate of coated seeds

[0122]

[0123] The test results showed that the germination rate of the seeds in the 4-propylphenol coating treatment group was 97%, and the germination rate of the ddH2O control group was only 96%. The 4-propylphenol coating treatment did not affect the germination of wheat seeds.

[0124] Example 6: Investigation on the control effect of 4-propylphenol coating on wheat basal stalk rot

[0125] Using Jimai 22 as the experimental object for coating test, the control effect of 4-propylphenol on wheat basal stalk rot caused by infection with Fusarium pseudograminearum was investigated. The specific treatments were as follows:

[0126] The pathogen was propagated using millet medium. The millet was washed, put into a pot, boiled with water for 2 min, cooled to the surface without water, then dispensed, sealed, and the Erlenmeyer flask filled with millet was sterilized in a moist heat sterilizer (121 °C, 30 min), and cooled for later use. Fusarium graminearum was inoculated into the sterilized millet medium, and placed in an incubator at 25 °C for 7 days, and shaken regularly to ensure that the colonies were evenly distributed on the surface of the millet.

[0127] The millet culture medium and sterile soil were mixed evenly at a ratio of 0.75:100 (weight ratio). The inoculated soil was transferred into nutrient pots (the nutrient pots were 6 cm in height and 9 cm in diameter). 8 seeds coated with 4-propylphenol were sown in each pot, and seeds inoculated with the treatment without the same volume of ddH2O were set as the control (CK). 3 replicates were set for each treatment. They were cultured at 25 °C, watered to keep moist, and after 21 days, the plant height, disease incidence rate, and disease index of each treatment were calculated.

[0128] Grading standard:

[0129] Grade 0: The whole plant had no browning symptoms;

[0130] Grade 1: Only the first leaf sheath of the plant turned brown, and the browning area did not exceed 1 / 2;

[0131] Grade 2: Only the first leaf sheath of the plant turned brown, and the browning area exceeded 1 / 2;

[0132] Grade 3: Only the first leaf sheath of the plant turned brown, the browning area exceeded 1 / 2, and the brown color deepened;

[0133] Grade 4: The second leaf sheath of the plant turned brown;

[0134] Grade 5: The third leaf sheath of the plant turned brown or the plant died.

[0135] Calculate the disease incidence rate and disease index of each treatment, and measure the plant height of each treatment.

[0136] Disease index = 100×Σ (number of diseased plants at each level × disease level at each level) / (total number of plants surveyed × highest disease level)

[0137] Disease incidence rate (%) = number of diseased plants / total number of plants surveyed * 100

[0138] Control effect (%) = [control average disease incidence rate (disease index) - treatment disease incidence rate (disease index)] / control average disease incidence rate (disease index) * 100

[0139] The results are shown in Table 6 and Table 7

[0140] Table 6: Plant height of each treatment

[0141]

[0142]

[0143] Table 7: Disease index and control effect of stem base rot treated with 4-propylphenol coating

[0144]

[0145] The results showed that the plant heights of the control group were 23.63±2.55a, 22.56±2.15ab, and 22.13±3.50ab respectively, and those of the 4-propylphenol coating treatment group were 21.13±1.48ab, 21.44±2.11ab, and 20.56±1.80b respectively. This indicates that coating treatment with 4-propylphenol before sowing wheat has an impact on the plant height of wheat, but the difference is not significant. The disease plant rate of the control group reached 75%, and those of the 4-propylphenol coating treatment group were 25.00%, 25.00%, and 37.50%, with an average disease plant rate of 29.17%; the control effects on diseased plants were 66.67%, 66.67%, and 50.00%, with an average control effect reaching 61.11%; the control effects on disease index were 88.24%, 88.24%, and 82.35%, with an average control effect reaching 86.28%. This indicates that coating treatment with 4-propylphenol before sowing wheat has a good control effect on wheat basal rot disease.

[0146] Example 7: Pot experiment

[0147] A pot experiment was conducted using the wild-type wheat Fielder as the experimental object to investigate the control effect of the combined use of 4-propylphenol and phenethyl alcohol on the infection of Fusarium graminearum. The specific treatments were as follows:

[0148] Treatment 1: During the flowering stage, a liquid medicine containing 150 μg / ml of 4-propylphenol was evenly and quantitatively sprayed on the ears of the marked wheat, 1 ml per ear. The spore concentration of the Fusarium graminearum liquid was adjusted to 5×10 5 CFU / mL, and three days after treatment, the ears were inoculated using the single-flower drip method.

[0149] Treatment 2: During the flowering stage, a liquid medicine containing 0.6 mg / ml of phenethyl alcohol was evenly and quantitatively sprayed on the ears of the marked wheat, 1 ml per ear. The spore concentration of the Fusarium graminearum liquid was adjusted to 5×10 5 CFU / mL, and three days after treatment, the ears were inoculated using the single-flower drip method.

[0150] Treatment 3: During the flowering stage, a liquid medicine containing 150 μg / ml of 4-propylphenol and 0.6 mg / ml of phenethyl alcohol was evenly and quantitatively sprayed on the ears of the marked wheat, 1 ml per ear. The spore concentration of the Fusarium graminearum liquid was adjusted to 5×10 5 CFU / mL, and three days after treatment, the ears were inoculated using the single-flower drip method.

[0151] Treatment with an equal amount of clear water was used as the control (CK).

[0152] There were 10 replicates for each treatment. After 10 days, the disease phenotypes were counted and photographed. The diseased spikelet rate was calculated to judge the severity of scab.

[0153] Percentage of scabbed spiklet (PSS) = (Number of scabbed spiklets / Total number of spiklets) × 100%.

[0154] The results are shown in Table 8.

[0155] Table 8: Percentage of scabbed spiklets under different treatments

[0156]

[0157] The results show that the combined use of 4-propylphenol and phenethyl alcohol has a synergistic effect in controlling the infection of Fusarium graminearum.

[0158] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a composition in the preparation of a product for preventing and controlling infection by Fusarium graminearum; characterized in that, The composition consists of 150 μg / ml of 4-propylphenol and 0.6 mg / ml of phenethyl alcohol.

2. A pharmaceutical preparation for preventing and treating wheat scab caused by infection of Fusarium graminearum, characterized in that, The pharmaceutical preparation consists of 150 μg / ml of 4-propylphenol and 0.6 mg / ml of phenethyl alcohol.

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