Application of turmeric rhizome extract missible oil in prevention and treatment of corn leaf spot disease and tomato gray mold

Abstract

The invention relates to application of turmeric rhizome extract emulsifiable concentrate in preventing and treating corn leaf spot and tomato gray mold. Compared with the prior art, the turmeric rhizome extract missible oil disclosed by the invention contains an effective substance aromatic turmerone, and the aromatic turmerone can reduce the spore germination rate and sporulation quantity of pathogenic bacteria, increase the permeability of hypha cell membranes and cause overflow of contents, so that normal growth of hyphae is inhibited, and an antibacterial effect is achieved. Aromatic turmerone acts on plants, so that plant cells generate more polyphenol and flavonoid substances, and the immunity of the plants is improved. Pharmacodynamic test results in fields in two fields show that the 6.5% turmeric rhizome extract emulsifiable concentrate can be used for stably and effectively preventing and treating corn northern leaf blight and tomato gray mold after being diluted by 150-300 times, and also has a yield increasing effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant disease control, and in particular relates to the application of turmeric rhizome extract emulsifiable concentrate in controlling maize leaf spot and tomato gray mold Background Art

[0002] Maize is an important crop in China for both food and industrial raw materials. Due to the development needs of the green industry and renewable energy industry in recent years, maize has become an important strategic resource in the 21st century [2] 。

[0003] Maize southern leaf blight is one of the most destructive leaf diseases of maize caused by Bipolaris maydis, which seriously endangers maize yield and quality globally, especially in tropical and subtropical regions [3] 。 Usually, at the initial stage of the disease, irregularly scattered water-soaked lesions appear on the lower leaves of maize, and continue to expand and spread to the entire leaf, and then spread to the upper leaves as the disease progresses [4] 。 When the disease is severe, it will cause the maize leaves to wither and even the stems to lodge [5] 。 In recent years, serious outbreaks of maize southern leaf blight have occurred in China, and maize southern leaf blight has become an important factor restricting maize yield in maize-producing areas. During the epidemic period, it can cause a yield loss of 10%-68% [6] 。 In the research of biological control, scholars such as Ye found a cyclic lipopeptide antibiotic iturin A in Bacillus subtilis B47 2 which has a control effect on Bipolaris maydis, and the field control effect reaches 64.2%, significantly better than chlorothalonil [7] ; Cui et al. found that Bacillus amyloliquefaciens B9601-Y2 has the effect of controlling maize southern leaf blight while promoting crop growth, and the control effect can reach 61.38% in pot experiments [8] 。 Currently, the pesticides registered for maize southern leaf blight in China include flusilazole, propiconazole·azoxystrobin, trifloxystrobin·tebuconazole, azoxystrobin·tebuconazole, tebuconazole·azoxystrobin, jinggangmycin, etc [9] 。 However, the control effect of microbial live bodies is generally weak, and it is easily affected by environmental factors and its stability is relatively poor. For example, the common biological control agent for maize southern leaf blight, jinggangmycin, has been used for a long time in the market. The long-term use of a single agent for control will cause the pathogen to develop drug resistance

[0004] Northern corn leaf blight is a fungal disease caused by Exserohilum turcicum, which mainly damages the leaves, leaf sheaths and husks of corn. At the initial stage of the disease, dark brown, nearly circular lesions appear on the corn leaves. As the disease develops, the lesions gradually expand and connect into large, irregular lesions, and in severe cases, the leaves will wither and die. In the research on biological control, scholars such as Ma found that the Trichoderma strain 576 has the potential to control northern corn leaf blight, and the disease can be controlled by spraying the conidial suspension of Trichoderma 576 on the leaf surface and by root irrigation.

[10] ; Scholars such as Scapin conducted in vitro antibacterial tests on the northern corn leaf blight pathogen using a variety of plant extracts, and found that the extracts of rosemary and camphor had the strongest inhibitory effect on the mycelial growth of the pathogen.

[11] ; Scholars such as Sartori found that the Bacillus isolated and identified from wilted corn leaves could reduce the disease severity of northern corn leaf blight.

[12] .

[0005] Tomato gray mold is a disease caused by Botrytis cinerea. Botrytis cinerea can infect plant parts such as leaves, stems, and fruits. When Botrytis cinerea infects tomato leaves, the lesions are initially water-soaked, often starting from the leaf tip and spreading outwards, then turning into brown ring patterns and producing a gray mold layer. When the disease is severe, it can cause the entire leaf to rot; when infecting tomato fruits, the lesions on the fruit epidermis develop from grayish-white water-soaked lesions at the initial stage of the disease to a grayish-brown mold layer. When the disease is severe, the fruit will lose moisture and become a mummified fruit, seriously endangering the yield and quality of tomatoes. Botrytis cinerea has a wide range of hosts and can infect more than 200 kinds of plants. The chemical pesticides registered for tomato gray mold in China include Chlorothalonil, Pyrimethanil, Iprodione, Procymidone, Boscalid, etc.; the biological pesticides include Carvacrol, β-Lupoglobulin polypeptide (BLAD), Trichoderma harzianum, Berberine, Eugenol, Trichoderma sp, etc. Chemical synthesis pesticide control is the main means of controlling gray mold at home and abroad. Although chemical pesticides are effective quickly and have low costs, a single active ingredient is likely to cause the pathogen to develop drug resistance and will cause certain pollution to the environment. The demand for more efficient and greener biological control in gray mold control is increasing. In the research of biological control, the natural compound thymol can inhibit Botrytis cinerea by regulating polyamine oxidase in Botrytis cinerea to produce hydrogen peroxide. The control effect of the single strain solution of the biocontrol yeast Cryptococcus albidus 64 on tomato gray mold can reach 95.83%, and it has a good storage and anti-corrosion effect. The compost tea prepared with forest compost, sea compost, sheep compost, shrimp compost, and vermiculite compost can reduce gray mold on strawberry leaves by 38 - 62% within 12 days. Lemongrass essential oil can achieve the effect of resisting Botrytis cinerea by changing membrane activity and membrane permeability.

[0006] Chinese Patent CN116725012A discloses the application of a plant-derived pesticide of ar-turmerone in controlling sheath blight. This solution uses the plant-derived pesticide of ar-turmerone to control sheath blight, which can reduce the usage amount of chemical pesticides in farmland and alleviate the problem of target drug resistance. However, the turmeric rhizome extract emulsifiable concentrate disclosed in this solution is mainly used to control sheath blight. The pathogen of rice sheath blight is Rhizoctonia solani Kühn, while the pathogen of southern leaf blight of maize is Bipolaris maydis (Nishik.) Shoemaker., the pathogen of northern leaf blight of maize is Exserohilum turcicum (Pass.) Leonay et Suggs, and the pathogen of tomato gray mold is Botrytis cinerea. Although they all belong to Deuteromycotina, it can be seen from their Latin names that the three pathogens belong to different genera respectively. There are significant differences between southern leaf blight, northern leaf blight and sheath blight of maize. According to the content disclosed in this patent, it is not possible to determine how to control leaf spot of maize.

[0007] References:

[0008] [1] Ma Wenfeng. Moderately develop the deep processing industry of maize and promote the high-quality development of the maize industrial chain [J]. Grain Processing, 2023, 48(1): 1-6.

[0009] [2] YANG Q, BALINT-KURTIP, XU M l. Quantitative disease resistance: dissection and adoption in maize [J]. Molecular Plant, 2017, 10(3): 402-413.

[0010] [3] Li Zhiqiang, Zhang Xianghui, Liu Wende. Research progress on southern leaf blight of maize [J]. Plant Protection, 2023, 49(05): 80-88.

[0011] [4] HOU Y P, CHEN Y L, WU L Y et al. Baseline sensitivity of Bipolaris maydis to the novel succinate dehydrogenase inhibitor benzovindiflupyr and its efficacy. Pestic [J]. Biochem. Physiol, 2018(149): 81-88.

[0012] [5] LOPEZ-ZUNIGAL O, WOLTERS P, DAVIS S et al. Using maize chromosome segment substitution line populations for the identification of loci associated with multiple disease resistance[J]. G3-Genes Genom. Genet, 2019(9):189-201.

[0013] [6] CHEN Y L, MAO X W, WANG J X et al. Activity of the dinitroaniline fungicide fluazinam against Bipolaris maydis[J]. Pestic Biochem and Physiol, 2018(148):8-15.

[0014] [7] YE Y F, LIQ Q, FU G et al. Identification of Antifungal Substance (Iturin A2) Produced by Bacillus subtilis B47 and Its Effect on Southern Corn Leaf Blight[J]. Journal of Integrative Agriculture, 2012(11.1):95-99.

[0015] [8] CUI W Y, HE P J, MUNIR S et al. Efficacy of plant growth promoting bacteria Bacillus amyloliquefaciens B9601-Y2 for biocontrol of southern corn leaf blight[J]. Biological Control, 2019(139):104080.

[0016] [9] China Pesticide Information Network. Data Center[DB]. http: / / www.chinapesticide.org.cn / zwb / dataCenter

[0017]

[10] MA Y,LI Y,YANG S et al.Biocontrol Potential of Trichodermaasperellum Strain 576against Exserohilum turcicumin Zea mays.Journal of Fungi(Basel),2023,9(9),936.

[0018]

[11] SCAPIN C R,CARNELOSSIP R,VIEIRA R A,et al.Fungitoxidade in vitrode extratos vegetais sobre Exserohilum turcicum(Pass)Leonard Suggs In vitrofungitoxicity of plant extracts on Exserohilum turcicum(Pass)LeonardSuggs.Revista Brasileira de Plantas Medicinais,2010,12(1),57-61.

[0019]

[12] FESSIA A,SARTORI M, D et al.In vitro studies of biofilm-forming Bacillus strains,biocontrol agents isolated from the maizephyllosphere.Biofilm,2022,4,100097. Summary of the Invention

[0020] Turcicum leaf spot is a common disease in maize, seriously endangering the yield and quality of crops. Based on the current situation that there is a lack of effective technologies for controlling turcicum leaf spot in the existing technology, the present invention provides the application of turmeric rhizome extract emulsifiable concentrate in controlling turcicum leaf spot and tomato gray mold.

[0021] The object of the present invention can be achieved by the following technical solutions:

[0022] The present invention first provides the application of turmeric rhizome extract emulsifiable concentrate in controlling turcicum leaf spot.

[0023] In one embodiment of the present invention, the turcicum leaf spot is southern leaf blight of maize.

[0024] In one embodiment of the present invention, the turcicum leaf spot is northern leaf blight of maize.

[0025] In one embodiment of the present invention, the turmeric rhizome extract emulsifiable concentrate is a 6.5% (by mass concentration) turmeric rhizome extract emulsifiable concentrate.

[0026] In one embodiment of the present invention, the formulation composition of the turmeric rhizome extract emulsifiable concentrate is as follows: 50% turmeric rhizome extract, 30% methyl oleate, and 20% compound emulsifier T-3016 (composed of 30% triphenylethylene phenol polyoxyethylene ether, 38% sorbitan trioleate polyoxyethylene ether, and 32% calcium dodecylbenzenesulfonate).

[0027] The preparation method is as follows: First, mix the compound emulsifier T-3016 with methyl oleate and carry out emulsification and homogenization at 5000 rpm for 5 min, then mix with the turmeric rhizome extract and carry out emulsification and homogenization at 5000 rpm for 5 min to obtain the product, which is a yellow, flowable, homogeneous, and transparent liquid. To detect the mass fraction (%) of ar-turmerone in the emulsifiable concentrate preparation, the method is as follows: Dissolve the sample in anhydrous methanol, use methanol and water as the mobile phase, use a C18 (4.6 mm × 250 mm, 5 μm) chromatographic column, and perform high-performance liquid chromatography separation on the sample. Quantify the content of ar-turmerone in the sample by the external standard method at a UV wavelength of 242 nm. The result is that the mass fraction (%) of ar-turmerone is 6.5%. And the sample is diluted 200 times with standard hard water and tested according to GB / T 1603-2001. There is no floating oil on the upper layer, no sunken oil and precipitation on the lower layer in the measuring cylinder, and it is determined that the emulsion stability is qualified.

[0028] The present invention further provides the application of the turmeric rhizome extract emulsifiable concentrate in controlling corn leaf spot disease and promoting yield increase.

[0029] In one embodiment of the present invention, the turmeric rhizome extract emulsifiable concentrate is diluted 100 - 600 times during use.

[0030] In one embodiment of the present invention, when the turmeric rhizome extract emulsifiable concentrate is applied, the effective dose of ar-turmerone after dilution is 100 - 500 mg / kg.

[0031] In one embodiment of the present invention, the application method of the turmeric rhizome extract emulsifiable concentrate is foliar spray application.

[0032] In one embodiment of the present invention, the application period of the turmeric rhizome extract emulsifiable concentrate is as follows: For corn, the growth period is the tasseling stage, the large trumpet mouth stage, or at the 3 - 5 leaf stage of corn, that is, the initial stage of disease occurrence.

[0033] The present invention also provides the application of the turmeric rhizome extract emulsifiable concentrate in controlling tomato gray mold.

[0034] In one embodiment of the present invention, the application of turmeric rhizome extract emulsifiable concentrate in controlling tomato gray mold and promoting tomato yield increase.

[0035] In one embodiment of the present invention, the turmeric rhizome extract emulsifiable concentrate is diluted 100 - 600 times when in use.

[0036] In one embodiment of the present invention, when the turmeric rhizome extract emulsifiable concentrate is applied to tomatoes, the effective dose of ar-turmerone after dilution is 100 - 500 mg / kg.

[0037] In one embodiment of the present invention, when the turmeric rhizome extract emulsifiable concentrate is applied to tomatoes, the application method is spraying on tomato leaves or fruits.

[0038] The main innovations of the present invention are reflected in the following aspects:

[0039] The present invention first proposes the application of turmeric rhizome extract emulsifiable concentrate in controlling maize leaf spot and tomato gray mold.

[0040] Compared with the prior art, the advantages and beneficial effects of the present invention are reflected in the following aspects:

[0041] First of all, the turmeric rhizome extract emulsifiable concentrate contains the active substance ar-turmerone. Ar-turmerone can reduce the spore germination rate of pathogenic bacteria and rupture the cell walls of germinated spores and hyphae, achieving an antibacterial effect. Ar-turmerone acts on plants, causing plant cells to produce more phenolic and flavonoid substances, improving the immune ability of plants. Through experiments in two places, it is concluded that compared with the control agent, the treatment of diluting 6.5% turmeric rhizome extract emulsifiable concentrate 150 times has no significant difference or is significantly higher in controlling maize northern leaf blight and southern leaf blight. Ar-turmerone has a good and stable control effect on maize northern leaf blight and southern leaf blight.

[0042] Secondly, the turmeric rhizome extract contains compounds such as ar-turmerone, curcumin, and gingerols, which can alleviate the generation of drug resistance of the target; at the same time, the acute and sub-chronic toxicity of the turmeric rhizome extract emulsifiable concentrate is low toxicity, and it is low toxicity to terrestrial organisms in the environment, including bees, silkworms, and birds. The main component ar-turmerone and other components in 6.5% turmeric rhizome extract emulsifiable concentrate are all from the natural plant turmeric. Turmeric has been allowed to be used as a food additive in both China and the United States and does not contain toxic and harmful substances. At the same time, it is easy to hydrolyze in the environment and is easily degraded in the soil. Therefore, it will not accumulate in the environment for a long time and damage the environment, but will be degraded in the environment relatively quickly. Experiments prove that each dosage treatment of 6.5% turmeric rhizome extract emulsifiable concentrate has no phytotoxicity to maize, the crop grows normally, and it has an effect of increasing maize yield; and it has no adverse effects on non-target organisms. 6.5% turmeric rhizome extract emulsifiable concentrate has a good control effect on tomato gray mold. It is proved that the effect of 6.5% ar-turmerone emulsifiable concentrate in controlling tomato gray mold has stability and is applicable to different production situations. Brief Description of the Drawings

[0043] Figure 1 Effect diagram of art-turmerone at different concentrations inhibiting the mycelial growth of Exserohilum turcicum;

[0044] Figure 2 Effect diagram of art-turmerone at different concentrations inhibiting the mycelial growth of Bipolaris maydis;

[0045] Figure 3 Effect of art-turmerone on the mycelial morphology of Bipolaris maydis;

[0046] CK: Blank control group; EC 50 : Art-turmerone inhibits the mycelial growth of Bipolaris maydis EC 50 value concentration treatment group;

[0047] Figure 4 Art-turmerone treatment of the germination rate of Bipolaris maydis spores (p < 0.05);

[0048] Figure 5 Relative electrical conductivity of Bipolaris maydis mycelia at each measurement time;

[0049] CK-1 is the water control group, and EC50 is the art-turmerone EC 50 concentration treatment group.

[0050] Figure 6 Effect diagram of art-turmerone at different concentrations inhibiting the mycelial growth of Botrytis cinerea. Detailed Description of the Invention

[0051] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0052] Example 1

[0053] In vitro antibacterial test of art-turmerone against maize leaf spot

[0054] 1. Test materials and equipment

[0055] 1.1 Test materials

[0056] (1) Test agents:

[0057] Test compound: Art-turmerone standard (ar-turmerone), purity > 98%, provided by Sichuan Victy Biotech Co., Ltd.;

[0058] Test preparation: 6.5% turmeric rhizome extract emulsifiable concentrate;

[0059] Among them, 6.5% turmeric rhizome extract emulsifiable concentrate is a turmeric rhizome extract emulsifiable concentrate with a mass concentration of 6.5%, which is disclosed in patent CN116746589B.

[0060] The formulation composition of the turmeric rhizome extract emulsifiable concentrate is as follows: 50% of turmeric rhizome extract, 30% of methyl oleate, and 20% of compound emulsifier T-3016 (composed of 30% of triphenylethylene phenol polyoxyethylene ether, 38% of sorbitan trioleate polyoxyethylene ether, and 32% of calcium dodecylbenzenesulfonate).

[0061] Among them, for the turmeric rhizome extract, its preparation method is as follows: (1) After drying, pulverizing, and sieving the turmeric rhizome raw material, it is extracted with 75% ethanol aqueous solution (the solid-liquid ratio is 50 g / L), the extraction temperature is 20 °C, and the extraction time is 48 h to obtain the extract; (2) The extract is concentrated and then extracted with n-hexane, and the oil-phase product is subjected to molecular distillation. The pressure condition of molecular distillation is 3 Pa, and the temperature is 120 °C to obtain the turmeric rhizome extract.

[0062] The preparation method of the turmeric rhizome extract emulsifiable concentrate is: First, mix the compound emulsifier T-3016 with methyl oleate and carry out emulsification and homogenization at 5000 rpm for 5 min, then mix with the turmeric rhizome extract and carry out emulsification and homogenization at 5000 rpm for 5 min to obtain it. The product is a yellow flowable homogeneous transparent liquid.

[0063] To detect the mass fraction (%) of ar-turmerone in the emulsifiable concentrate preparation, the method is: The sample is dissolved in anhydrous methanol, using methanol and water as the mobile phase, and using a C18 (4.6 mm × 250 mm, 5 μm) chromatographic column to carry out high-performance liquid chromatography separation of the sample. The content of ar-turmerone in the sample is quantitatively determined by the external standard method at a UV wavelength of 242 nm. The result is that the mass fraction (%) of ar-turmerone is 6.5%. And the sample is diluted 200 times with standard hard water and tested according to GB / T 1603-2001. There is no floating oil on the upper layer, no sunken oil and precipitation on the lower layer in the measuring cylinder, and it is determined that the emulsion stability is qualified.

[0064] The turmeric rhizome extract emulsifiable concentrates of the following other examples are all prepared by the same method.

[0065] (2) Test strains: Bipolaris maydis, Exserohilum turcicum.

[0066] 1.2 Test instruments

[0067] Clean bench (SW-CJ-1FD), Suzhou Antai Air Technology Co., Ltd. of Sujing Group; high-pressure steam autoclave (SANYO MLS 3750); mold incubator (MJ-150F-I), Shanghai Huitai Instrument Manufacturing Co., Ltd.; pipette (Dalong Xingchuang Experimental Instrument (Beijing) Co., Ltd., TopPette manual single-channel adjustable pipette, 1000-5000μL); conical single nozzle sprayer (3WBD-20 model), etc.

[0068] 2. Test methods

[0069] 2.1 In vitro antibacterial test of turmeric flavonoids against corn leaf spot pathogen

[0070] 2.1.1 Preparation of drug-containing culture medium and pathogen inoculation

[0071] Use DMSO to dissolve the standard of turmeric flavonoids and dilute to 26 mg / mL for use. Under sterile conditions, the PDA medium heated to a liquid state was cooled to about 50°C, and the turmeric flavonoids solution was added to adjust the concentration of the drug-containing medium to 2.6, 0.52, 0.26, 0.13, 0.065, 0.0325, and 0.01625 mg / mL (i.e., the concentration of turmeric flavonoids in the medium). After sufficient mixing, the drug-containing medium was prepared and poured into a culture dish with a diameter of 90 mm, and it was cooled and solidified. The PDA medium without the test agent was used as the control group, and each treatment was repeated 3 times. The edge of the test strain grown on the PDA medium was beaten with an 8mm sterilized pipette tip to obtain the bacterial cake, and the bacterial cake was picked up with a toothpick and placed in the center of the treatment group (i.e., drug-containing medium) and the control group medium, and placed in a 28°C incubator for culture, and the antibacterial test results were observed after 5 days.

[0072] 2.1.2 Data Analysis

[0073] After 5-7 days of cultivation, the diameter of each treated colony was measured by the cross method, the average value was taken, and the inhibition rate was calculated according to formula (1).

[0074]

[0075] Among them, IR (inhibition radio) is the inhibition rate, D c is the diameter of the control colony, D t is the diameter of the treated colony, and d is the diameter of the bacterial cake. The experimental data were analyzed using the DPS toxicity regression equation and EC 50 value.

[0076] 3. Test results

[0077] The results showed that turmeric flavonoids had an inhibitory effect on the mycelial growth of Pathogenes of Southern Blight and Southern Blight of Corn under in vitro conditions. Figure 1 ,Figure 2 , calculate the toxicity regression equation and EC 50 The results are shown in Table 1.

[0078] Table 1 EC of turmeric flavonoids in inhibiting the mycelial growth of corn leaf spot pathogen 50 value

[0079]

[0080] In this Example 1, it can be confirmed that the main active compound turmeric flavonoids of 6.5% turmeric rhizome extract emulsifiable concentrate has inhibitory activity against the mycelial growth of corn leaf blight fungus (EC50 value is 0.0878 mg / mL).

[0081] Example 2

[0082] Field test on the efficacy of turmeric flavonoids in controlling corn leaf spot

[0083] 1. Materials and Instruments

[0084] (1) Test materials

[0085] Test agent (test agent): 6.5% turmeric rhizome extract emulsifiable concentrate.

[0086] Control drugs: 24% Jinggangmycin aqueous solution, Wuhan Kono Biotechnology Co., Ltd.; 400 g / L flusilazole emulsifiable concentrate, Xingnong Pharmaceutical (China) Co., Ltd.

[0087] The test time, location and corn varieties are shown in Table 2:

[0088] Table 2 Field test time, location and test corn varieties

[0089]

[0090]

[0091] (2) Test equipment

[0092] TopPette manual single-channel adjustable pipette [1 000-5000 μL, Dalong Xingchuang Laboratory Instrument (Beijing) Co., Ltd.]; 136113 manual sprayer (capacity 5 L, Shanghai Haiyang Business Consulting Co., Ltd.); 3WJD-18 electrostatic sprayer with conical nozzle (nozzle diameter 0.5 mm) (Shandong Weishi).

[0093] 2. Test methods

[0094] 2.1 Experimental design

[0095] (1) Yunnan Province pilot site

[0096] The experimental field has a gleyed soil type, with a pH value of 6.1, an organic matter content of 3.17%, good soil moisture, and medium fertility. The experimental field has good drainage and irrigation conditions, and all experimental plots have the same cultivation, water, and fertilizer management conditions. The experiment was set with 5 treatments (see Table 3), replicated 4 times, for a total of 20 plots; each plot was 40m 2 , and the plots were randomly arranged in blocks. The first application was on July 22, 2024, during the tasseling stage of the corn growth period, with sporadic occurrence of southern leaf blight. The second application was on August 1, 2024. 100 ml / acre of 32% nicosulfuron - atrazine dispersible oil suspension was used 10 days before the start of the experiment, and foliar spraying was carried out once during the 3 - 5 leaf stage of corn and the 2 - 5 leaf stage of weeds to control annual weeds in the corn field.

[0097] Table 3 Experimental design of tested pesticides at the Yunnan experimental site

[0098]

[0099] (2) Anhui experimental site

[0100] The experimental field is flat, with good irrigation and drainage conditions, and the corn is growing well. All experimental plots have the same cultivation, water, and fertilizer management conditions. The experiment was set with 5 treatments (Table 4), replicated 4 times, for a total of 20 plots; each plot was 30m 2 , and the plots were randomly arranged in blocks. The first application was on August 2, 2024, during the large - trumpet - mouth stage of the corn growth period, with sporadic occurrence of southern leaf blight. The second application was on August 9, 2024. 100 ml / acre of 32% nicosulfuron - atrazine dispersible oil suspension was used 10 days before the start of the experiment, and foliar spraying was carried out once during the 3 - 5 leaf stage of corn and the 2 - 5 leaf stage of weeds to control annual weeds in the corn field.

[0101] Table 4 Experimental design of tested pesticides at the Anhui experimental site

[0102]

[0103] 2.2 Investigation method

[0104] At both experimental sites, the pre - treatment population density was investigated on the day of the first pesticide application, and the efficacy was investigated 10 days after each of the two pesticide applications.

[0105] Five points were randomly selected from each plot, and five plants were selected from each point to investigate the disease incidence and disease grade of all leaves. The disease grading standard and the calculation method of the disease index refer to the national standard "GB / T 17980.107 - 2004 Pesticide field efficacy test guidelines (part 2): Fungicides for controlling southern leaf blight and northern leaf blight of maize";

[0106] 2.3 Efficacy calculation method

[0107] The disease index and control effect were calculated according to formulas (2) and (3).

[0108]

[0109] If the disease base number before applying the medicine was 0, the control effect was calculated according to formula (4):

[0110]

[0111] For the comparison of the efficacy differences between each treatment area, the new multiple range method (DMRT) was used to statistically analyze the experimental data, and a significance test (SAS 9.4) was carried out at the p = 0.01 level.

[0112] 2.4 Safety investigation

[0113] Observe whether there is phytotoxicity of the medicine to the crops. If there is phytotoxicity, record the type and degree of phytotoxicity, as well as the missing seedlings caused by phytotoxicity.

[0114] 2.5 Effects on the yield and quality of corn

[0115] All the corn in each plot was harvested at the Yunnan test site, and all the corn in the middle 10 square meters of each plot was harvested at the Anhui test site. After sun drying, the corn was mixed and threshed for yield measurement, and the yield per hectare was converted according to formula (5).

[0116]

[0117] The yield increase rate was calculated according to formula (6).

[0118]

[0119] 3 Test results

[0120] (1) Results of the field efficacy test of the tested medicine against Bipolaris maydis on corn

[0121] The results 10 days after the first application of the field efficacy test at the Yunnan test site showed that the three concentration treatments of the tested medicine had inhibitory effects on Bipolaris maydis on corn (Table 5). The results of the disease investigation 10 days after the second application showed that the control effects of the three concentration treatments of the tested medicine on Bipolaris maydis were all above 75%, and the effect was positively correlated with the medicine concentration. When the effective concentrations of the tested medicine were 216.67 and 433.33 mg / kg, the control effects were significantly better than those of the control medicine (p < 0.01).

[0122] Table 5 Results of the field efficacy test of the tested medicine against Bipolaris maydis on corn (Yunnan test site)

[0123]

[0124]

[0125] Different capital letters after the data in the same column indicate significant differences at p < 0.05, and different lowercase letters indicate significant differences at p < 0.01.

[0126] The results of the disease investigation 10 days after the first application at the Anhui test site (Table 6) showed that the control effects of the three concentrations of the tested agents on Bipolaris maydis were all above 60%, and the effect was positively correlated with the agent concentration. When the effective concentrations of the tested agents were 216.67 and 433.33 mg / kg, the control effects were not significantly different from those of the control agent. The results of the disease investigation 10 days after the second application showed that when the effective concentration of the tested agent was 433.33 mg / kg, the control effect was 73.66%, which was significantly better than that of the control agent (p < 0.05).

[0127] Table 6 Field efficacy test results of the tested agents against Bipolaris maydis (Anhui test site)

[0128]

[0129]

[0130] Different capital letters after the data in the same column indicate significant differences at p < 0.05, and different lowercase letters indicate significant differences at p < 0.01.

[0131] (2) Effects of the tested agents on maize yield

[0132] The results of the field trial yield investigation at the Yunnan test site (Table 7) showed that the treatment with the tested agents could increase the maize yield. The yield increase rates of the maize treated with the effective components of the tested agents at 216.7 and 433.33 mg / kg were 15.82 - 19.68%, and the yield increase effects were significantly better than those of the control agent.

[0133] Table 7 Investigation results of the tested agents on maize yield (Yunnan test site)

[0134]

[0135] The results of the field trial yield investigation at the Anhui test site (Table 8) showed that the treatment with the tested agents could increase the maize yield. The yield increase rate of the maize treated with the effective component of the tested agent at 433.33 mg / kg was 8.85%, and the yield increase effect was significantly better than that of the control agent (p < 0.01).

[0136] Table 8 Investigation results of the tested agents on maize yield (Anhui test site)

[0137]

[0138] (3) Effects of the tested agents on plants and other organisms

[0139] The treatments of each concentration of the tested agents in the two locations had no phytotoxicity to maize plants, and the crops grew normally without adverse effects on non-target organisms.

[0140] On the basis of confirming in Example 1 that the main active compound, ar-turmerone, in the 6.5% turmeric rhizome extract emulsifiable concentrate had inhibitory activity against the mycelial growth of Bipolaris maydis (the EC50 value was 0.0878 mg / mL), field efficacy trials were carried out in Anhui and Yunnan respectively in this example. The test results showed that the 6.5% turmeric rhizome extract emulsifiable concentrate had control effect on Bipolaris maydis under field conditions and had an increasing production effect on maize. When the tested agents were treated at high concentrations in the two trials, the control effect was significantly better than that of the control chemical agents. This result laid a foundation for replacing chemical synthetic pesticides with the botanical new pesticide, 6.5% turmeric rhizome extract emulsifiable concentrate, to control Bipolaris maydis.

[0141] The environmental differences between the two locations were large, and different maize varieties and different control agents were used. The results of the two groups of trials both showed that the tested agents had good control effects on Bipolaris maydis. It was proved that the control effect of the 6.5% turmeric rhizome extract emulsifiable concentrate against Bipolaris maydis was stable and applicable to different production situations.

[0142] Example 3 Field Efficacy Trial of Ar-Turmerone Against Exserohilum turcicum

[0143] 1 Materials and Instruments

[0144] (1) Test Materials

[0145] Test agent: 6.5% turmeric rhizome extract emulsifiable concentrate.

[0146] Control agents: Bacillus subtilis dispersible oil suspension with 20 billion spores / mL, Deqiang Biological Co., Ltd.; 25% pyraclostrobin suspension, Shandong Shibang Agrochemical Co., Ltd.

[0147] Test time, location and maize variety (Table 9):

[0148] Table 9 Field Test Time, Location and Test Maize Variety

[0149]

[0150] (2) Test Instruments

[0151] TopPette type manual single-channel adjustable pipette [1000 - 5000 μL, DLAB Scientific Co., Ltd.]; 136113 type manual sprayer (capacity 5L, Shanghai Haiyang Business Consulting Co., Ltd.); 3WJD-18 type electrostatic sprayer equipped with a conical nozzle (nozzle diameter 0.5 mm) (Shandong Weishi).

[0152] 2 Test Methods

[0153] 2.1 Experimental Design

[0154] (1) Xinjiang Test Site

[0155] The soil of the experimental field is loam, with a pH value of 6.82, an organic matter content of 1.48%, the soil is moist, and the fertility is medium. The experimental field has good drainage and irrigation conditions, and the cultivation, water and fertilizer management and other conditions of all experimental plots are the same. The experiment was set with 5 treatments (Table 10), repeated 4 times, for a total of 20 plots; each plot was 25m 2 , and the plots were randomly arranged in blocks. The first application time was July 15, 2024, the growth period of maize was the trumpet stage, and large leaf spot occurred sporadically. The second application time was July 22, 2024. On May 10, 2024, 150 ml / acre of 52% butachlor + atrazine suspension concentrate was used and mixed with 15 liters of water to control annual weeds; on June 28, 2024, 20 ml / acre of 2.5% deltamethrin emulsifiable concentrate was used and mixed with 45 liters of water to control maize aphids and corn borers.

[0156] Table 10 Experimental Design of Test Agents at the Xinjiang Test Site

[0157]

[0158] (2) Anhui Test Site

[0159] The soil type of the experimental field is clay, with an organic matter content of 26.58 g / kg, a pH value of 7.0, medium fertility, slightly moist soil, flat terrain, and good irrigation and drainage conditions. The maize growth is good. The cultivation, water and fertilizer management and other conditions of all experimental plots are the same. The experiment was set with 5 treatments (Table 11), repeated 4 times, for a total of 20 plots; each plot was 30m 2 , and the plots were randomly arranged in blocks. The first application time was August 2, 2024, the growth period of maize was the large trumpet stage, and large leaf spot occurred sporadically. The second application time was August 9, 2024. 100 ml / acre of 32% nicosulfuron + atrazine oil-based suspension concentrate was used 10 days before the start of the experiment, and it was sprayed once on the stems and leaves at the 3-5 leaf stage of maize and the 2-5 leaf stage of weeds to control annual weeds in maize fields.

[0160] Table 11 Experimental Design of Test Agents at the Anhui Test Site

[0161]

[0162] For both test sites, the pre-treatment base number was investigated on the day of the first application, and the efficacy was investigated 10 days after the two applications.

[0163] Five random survey points were selected in each plot, and five plants were selected from each point to investigate the disease status and disease level of all leaves. The disease classification standard and disease index calculation refer to the national standard "GB / T 17980.107-2004 Pesticide Field Efficacy Test Guidelines (II): Fungicide Control of Corn Large and Small Spot Disease";

[0164] 2.3 Calculation method of drug efficacy

[0165] The disease index and control effect are calculated according to the above formula (2) and (3). If the disease base before application is 0, the control effect is calculated according to the above formula (4):

[0166] The efficacy differences among the treatment areas were compared, and the experimental data were statistically analyzed using the new multiple range method (DMRT), and a significance test was performed at the p = 0.01 level (SAS 9.4).

[0167] 2.4 Safety Investigation

[0168] Observe whether the pesticide has any harm to the crops. If so, record the type and extent of the damage, as well as the missing seedlings caused by the damage.

[0169] 2.5 Impact on corn yield and quality

[0170] All corn in each plot was harvested in the Yunnan test site, and all corn in the middle 10 square meters of each plot was harvested in the Anhui test site. After drying, the corn was mixed and threshed to measure the yield, and the yield per hectare was converted according to the above formula (5). The yield increase rate was calculated according to the above formula (6).

[0171] 3. Test results

[0172] (1) Field efficacy test results of the tested agents against corn leaf blight

[0173] The results of the field efficacy test at the Xinjiang test site 7 days after the first application showed that the three concentrations of the test agent had an inhibitory effect on corn leaf blight (Table 12). The results of the disease investigation 14 days after the second application showed that the control effect of the three concentrations of the test agent on corn leaf blight was above 65%, and the effect was positively correlated with the concentration of the agent. When the effective concentration of the test agent was 433.33 mg / kg, the control effect was significantly better than that of the control agent (p < 0.05).

[0174] Table 12 Field efficacy test results of the tested agents for controlling corn leaf blight (test site in Xinjiang)

[0175]

[0176]

[0177] Different capital letters after the data in the same column indicate significant differences at p < 0.05, and different lowercase letters indicate significant differences at p < 0.01.

[0178] The results of the disease investigation 7 days after the first application at the Anhui test site (Table 13) showed that the control effects of the three concentration treatments of the tested agents on southern leaf blight of maize were all above 55%, and the effect was positively correlated with the agent concentration. When the effective concentrations of the tested agents were 216.67 and 433.33 mg / kg, there was no significant difference in the control effect compared with the control agent. The results of the disease investigation 14 days after the second application showed that when the effective concentration of the tested agent was 433.33 mg / kg, the control effect was 70.88%, which was significantly better than that of the control agent (p < 0.01).

[0179] Table 13 Field efficacy test results of the tested agents against northern leaf blight of maize (Anhui test site)

[0180]

[0181] Different capital letters after the data in the same column indicate significant differences at p < 0.05, and different lowercase letters indicate significant differences at p < 0.01.

[0182] (2) Effects of the tested agents on maize yield

[0183] The results of the field trial yield investigation at the Xinjiang test site (Table 14) showed that the treatment with the tested agents could increase the maize yield. The yield increase rates of maize treated with the effective components of the tested agents at 108.33, 216.7, and 433.33 mg / kg were between 4.78% and 6.29%, and the yield was significantly better than that of the blank control group.

[0184] Table 14 Investigation results of the tested agents on maize yield (Xinjiang test site)

[0185]

[0186]

[0187] Different capital letters after the data in the same column indicate significant differences at p < 0.05, and different lowercase letters indicate significant differences at p < 0.01.

[0188] The results of the field trial yield investigation at the Anhui test site (Table 15) showed that the treatment with the tested agents could increase the maize yield. The yield increase rate of maize treated with the effective component of the tested agent at 433.33 mg / kg was 7.71%, and the yield increase effect was significantly better than that of the control agent (p < 0.05).

[0189] Table 15 Investigation results of the tested agents on maize yield (Anhui test site)

[0190]

[0191] For the data in the same column, different capital letters indicate significant differences at p < 0.05, and different lowercase letters indicate significant differences at p < 0.01.

[0192] (3) Effects of the test agents on plants and other organisms

[0193] The test agents at each concentration in the two locations had no phytotoxicity to maize plants, the crops grew normally, and had no adverse effects on non-target organisms.

[0194] Based on the confirmation in Example 1 that the main active compound, arturmerone, in the 6.5% turmeric rhizome extract emulsifiable concentrate had an inhibitory activity against the mycelial growth of Exserohilum turcicum (EC 50 value was 0.0824 mg / mL), in this example, field efficacy trials were conducted in Anhui and Xinjiang respectively. The test results showed that the 6.5% turmeric rhizome extract emulsifiable concentrate had a control effect on Exserohilum turcicum under field conditions and at the same time had an effect of increasing maize yield. When the test agents were at high concentration treatments in the two trials, the control effect was significantly better than that of the control chemical agent. This result laid a foundation for replacing chemical synthetic pesticides with the botanical new pesticide 6.5% turmeric rhizome extract emulsifiable concentrate to control Exserohilum turcicum.

[0195] The environmental differences between the two locations were large, and different maize varieties and different control agents were used. The test results of both groups showed that the test agents had good control effects on Exserohilum turcicum. It was proved that the control effect of the 6.5% turmeric rhizome extract emulsifiable concentrate against Exserohilum turcicum was stable and applicable to different production situations.

[0196] Example 4 Study on the mode of action of arturmerone against Bipolaris maydis

[0197] 1. Materials and instruments

[0198] (1) Test strains

[0199] Bipolaris maydis, provided by the laboratory of Teacher Wang Xinhua from Shanghai Jiao Tong University.

[0200] (2) Test agents and culture media

[0201] Test compounds: arturmerone standard, purity > 98%, provided by Sichuan Vicibiotech Co., Ltd.;

[0202] Test reagents and culture media:

[0203] Water agar (WA) medium: 20 g of agar powder, made up to 1 L with pure water, sterilized at 121 °C for 20 minutes.

[0204] Potato Dextrose Agar (PDA) medium: 200 g of peeled and cut potatoes were boiled, and the juice was filtered through four layers of cheesecloth. 20 g of glucose and 16 g of agar were added, and pure water was added and made up to 1 L. It was sterilized at 121 °C for 20 minutes.

[0205] Yeast Extract Peptone Dextrose (YEPD) broth: 3 g of yeast extract, 10 g of peptone, and 20 g of glucose were added, and pure water was added and made up to 1 L. It was sterilized at 121 °C for 20 minutes.

[0206] (3) Test equipment

[0207] Laminar flow hood (SW-CJ-1FD), Suzhou Antai Air Technology Co., Ltd., Sujing Group;

[0208] High-pressure steam sterilizer (SANYO MLS-3750);

[0209] Mold incubator (MJ-150F-I), Shanghai Huibang Instrument Manufacturing Co., Ltd.;

[0210] Disposable petri dish (diameter d = 90 mm)

[0211] 2. Test method

[0212] 2.1 Preparation of Bipolaris maydis spore suspension

[0213] The Bipolaris maydis strain was cultured at 25 °C for 8 d. Sterile water was added to the petri dish and the mycelium was gently scraped. The suspension was pipetted and thoroughly pipetted, and the conidial suspension was obtained by filtering through three layers of sterilized lens paper. Centrifuged at 5000 rpm for 5 min, resuspended with sterile water and adjusted the conidial concentration to 1×10 4 / mL.

[0214] 2.2 Effect of ar-turmerone on the mycelial morphology of Bipolaris maydis

[0215] The ar-turmerone standard was dissolved with DMSO and diluted to 26 mg / mL for standby. The Bipolaris maydis was cultured on PDA medium for 7 d. A 5-mm-diameter mycelial disc was punched out and inoculated with the mycelium facing down in the middle of PDA medium containing 16.25 (the preparation was diluted 4000 times), 32.5 (the preparation was diluted 2000 times), 65 (the preparation was diluted 1000 times) μg / mL ar-turmerone. The one without adding ar-turmerone was used as the control. Each treatment was set with 3 replicates and cultured in an incubator at 25 °C for 5 d. The medium with mycelium was cut at the colony edge and prepared for biological samples according to Appendix 1. After the sample was sputter-coated for 15 s, it was observed using a low-vacuum ultra-high-resolution field emission scanning electron microscope (NOVA NanoSEM 230).

[0216] 2.3 Effects of ar-turmerone on the sporulation of Bipolaris maydis

[0217] A 5-mm mycelial disc was punched from the edge of a 7-day-old Bipolaris maydis colony and inoculated, with the mycelium facing downwards, in the middle of a PDA medium containing 16.25 (formulation diluted 4000-fold), 32.5 (formulation diluted 2000-fold), 65 (formulation diluted 1000-fold), 130 (formulation diluted 500-fold) μg / mL of ar-turmerone. A PDA medium without added ar-turmerone was used as a control. Each treatment was set up with 3 replicates and cultured at 25 °C in the dark for 8 days. 5 mL of sterile water was added to the petri dish and the mycelium was gently scraped, then thoroughly pipetted and filtered to obtain a spore suspension. The spore suspension was dropped onto a hemocytometer and the spore concentration was calculated under an optical microscope. The experimental data were statistically analyzed using the least significant difference method (lsd) of SAS 9.4, and significant analysis was performed according to p = 0.05.

[0218] 2.4 Effects of ar-turmerone on the spore germination of Bipolaris maydis

[0219] A Bipolaris maydis spore suspension was prepared using the same method as in 2.1 and coated on a WA medium with ar-turmerone concentrations of 8.125 (formulation diluted 8000-fold), 16.25 (formulation diluted 4000-fold), 32.5 (formulation diluted 2000-fold), 65 (formulation diluted 1000-fold), 130 (formulation diluted 500-fold) μg / mL. Each treatment was set up with 3 replicates. After culturing at 25 °C for 5 h, observations were made under an optical microscope. Multiple fields of view were selected and the germination of 100 spores was recorded. The criterion for judging spore germination was that when the germ tube after spore germination was greater than the short radius of the spore, it was considered germinated. The effects of ar-turmerone on the spore germination of Bipolaris maydis were analyzed according to the germination rate calculation formula (7). The experimental data were statistically analyzed using the least significant difference method (lsd) of SAS 9.4, and significant analysis was performed according to p = 0.05. The EC 50 value and the virulence regression equation of ar-turmerone inhibiting spore germination were calculated using DPS.

[0220]

[0221] Effects of ar-turmerone on the cell membrane permeability of Bipolaris maydis

[0222] Eight 5-mm mycelial discs were punched from the edge of a 5-day-old Bipolaris maydis colony using a sterilized pipette tip and placed into a 250-mL conical flask containing 100 mL of YEPD culture medium. Under the conditions of a rotation speed of 175 rpm and a temperature of 25 °C, after shaking culture for 48 h, ar-turmerone was added to the conical flask and the concentration was adjusted to the EC50 value of ar-turmerone inhibiting the mycelial growth of Bipolaris maydis. Each treatment was repeated 3 times, and a control group without added ar-turmerone was set up. After continuing to shake culture for 12 h, the mycelium was filtered and collected, washed 3 times with sterile water, and dried with sterilized filter paper for later use.

[0223] Weigh 0.3 g of mycelium and put it into a 50 mL centrifuge tube containing 20 mL of distilled water. Measure the conductivity of the distilled water after 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, and 220 min. After 220 min, boil the mycelium using a water bath. After the mycelium cools down, measure the final conductivity. Calculate the relative conductivity according to Equation (8) to analyze the effect of ar-turmerone on the cell membrane permeability of Bipolaris maydis, and use GraphPad Prism 10 to draw a trend image.

[0224]

[0225] 3. Experimental Results

[0226] 3.1 Observation on the Effect of Ar-Turmerone on the Mycelial Morphology of Bipolaris maydis

[0227] Observe the effect of ar-turmerone on the mycelial morphology of Bipolaris maydis by scanning electron microscopy. The results are as Figure 3 , the mycelial morphology of Bipolaris maydis without ar-turmerone treatment is normal and plump, and the surface is smooth; the mycelia of Bipolaris maydis treated with ar-turmerone show obvious shrinkage and even rupture.

[0228] 3.2 Effect of Ar-Turmerone on the Spore Production of Bipolaris maydis

[0229] The experimental results show that ar-turmerone has the effect of inhibiting the spore production of Bipolaris maydis (Table 16), and the inhibitory effect is positively correlated with the concentration of ar-turmerone.

[0230] Table 16 Effect of Ar-Turmerone on Inhibiting Spore Production of Bipolaris maydis

[0231]

[0232] Different letters after the data in the same column indicate significant differences at p < 0.05.

[0233] 3.3 Results of the Effect of Ar-Turmerone on the Spore Germination of Bipolaris maydis

[0234] Ar-turmerone has the effect of inhibiting the spore germination of Bipolaris maydis ( Figure 4 ). The spore germination rate of Bipolaris maydis after ar-turmerone treatment is significantly lower than that of the blank control group (p < 0.05), and the inhibitory effect is positively correlated with the concentration of ar-turmerone (Table 17).

[0235] It is calculated that ar-turmerone has the effect of inhibiting the spore germination of Bipolaris maydis (Table 17), and the virulence regression equation is Y = 0.7564lgX + 5.4132, EC 50The value is 0.2843 mg / mL, the 95% confidence interval is [0.1692, 0.4778], and the correlation coefficient is 0.9819.

[0236] Table 17 Inhibition rates of spore germination of Bipolaris maydis by arturmerone at different concentrations

[0237]

[0238] 3.4 Effect of arturmerone on the cell membrane permeability of Bipolaris maydis

[0239] The test results showed that the electrical conductivities of the mycelia in the control group with clear water and the arturmerone treatment group increased with the increase of treatment time (Table 18). Among them, the relative electrical conductivity of the arturmerone treatment group was higher than that of the control group with clear water ( Figure 5 ), indicating that the treatment with arturmerone led to an increase in the cell membrane permeability of Bipolaris maydis, causing damage to the cell membrane and leakage of the mycelial cell contents.

[0240] Table 18 Electrical conductivities of Bipolaris maydis mycelia at different measurement times

[0241]

[0242] The research results of this example showed that at the EC 50 concentration of arturmerone inhibiting the mycelial growth of Bipolaris maydis, it was observed by scanning electron microscopy that the mycelia shrank and deformed; after treatment with arturmerone, the cell membrane permeability of the mycelia increased; the yield of conidia decreased significantly, and spore germination was inhibited. It was shown that arturmerone controlled the occurrence of the disease by destroying the cell membrane permeability of the mycelia, resulting in abnormal growth of the mycelia, and also affecting the sporulation amount and germination of conidia, as well as the vegetative and reproductive bodies of the pathogen.

[0243] Example 5

[0244] In vitro antibacterial test of arturmerone against Botrytis cinerea on tomatoes

[0245] 1. Test materials and equipment

[0246] 1.1 Test materials

[0247] (1) Test agents:

[0248] Test compound: Standard arturmerone, with a purity > 98%, provided by Sichuan Victy Biotech Co., Ltd.;

[0249] Test preparation: 6.5% arturmerone emulsifiable concentrate, provided by the Laboratory of Plant Health and Natural Products, School of Agriculture and Biology, Shanghai Jiao Tong University;

[0250] (2) Test strain: Tomato gray mold (Botrytis cinerea), provided by Zou Lifang's research group at the College of Agriculture and Biology, Shanghai Jiao Tong University.

[0251] 1.2 Test equipment

[0252] Clean bench (SW-CJ-1FD), Suzhou Antai Air Technology Co., Ltd. of Sujing Group; high-pressure steam autoclave (SANYO MLS 3750); mold incubator (MJ-150F-I), Shanghai Huitai Instrument Manufacturing Co., Ltd.; pipette (Dalong Xingchuang Experimental Instrument (Beijing) Co., Ltd., TopPette manual single-channel adjustable pipette, 1000-5000μL); conical single nozzle sprayer (3WBD-20 model), etc.

[0253] 2. Test methods

[0254] 2.1 Preparation of drug-containing culture medium and pathogen inoculation

[0255] Use DMSO to dissolve the turmeric flavonoids standard and dilute to 26 mg / mL for use. Under sterile conditions, cool the PDA medium heated to a liquid state to about 50°C, add the turmeric flavonoids solution, adjust the concentration of the drug-containing medium to 2.6, 0.52, 0.26, 0.13, 0.065, 0.0325, 0.01625 mg / mL, mix thoroughly to make a drug-containing medium, pour the drug-containing medium into a culture dish with a diameter of 90 mm, and wait for it to cool and solidify. The control group was not added with the test agent, and each treatment was repeated 3 times. Use an 8mm sterilized pipette tip to take the bacterial cake from the edge of the test strain grown on the PDA medium, pick the bacterial cake with a toothpick and place it in the center of the culture medium of the treatment group and the control group, and culture it in a 28°C incubator. Observe the results of the antibacterial test after 5 days.

[0256] 2.1 Data Analysis

[0257] After 5-7 days of cultivation, the diameter of each treated colony was measured by the cross method, the average value was taken, and the inhibition rate was calculated according to the above formula (1).

[0258] 3. Test results

[0259] The results showed that turmeric flavonoids had an inhibitory effect on the mycelial growth of tomato gray mold under in vitro conditions ( Figure 6 ), calculate the toxicity regression equation and EC 50 The value results are shown in Table 19.

[0260] Table 19 EC of turmeric flavonoids inhibiting the mycelial growth of corn leaf spot pathogen 50 value

[0261]

[0262] Example 6

[0263] Field Efficacy Test of [(-)-Ar-turmerone] against Botrytis cinerea Pers. on Tomato

[0264] 1. Materials and Instruments

[0265] 1.1 Test Materials

[0266] Test agents: 6.5% [(-)-Ar-turmerone] EC

[0267] Control agents: 400 g / L isofetamid SC provided by Ishihara Sangyo Kaisha, Ltd., Japan; 50% boscalid WG provided by BASF SE; 500 g / L fluopyram + pyrimethanil SC provided by Bayer CropScience (China) Co., Ltd.

[0268] Test time, location and tomato variety (Table 20):

[0269] Table 20 Field Test Time, Location and Test Tomato Variety

[0270]

[0271] 1.2 Test Instruments

[0272] TopPette manual single-channel adjustable pipette [1000 - 5000 μL, DLAB Scientific (Beijing) Co., Ltd.]; 136113 manual sprayer (capacity 5 L, Shanghai Haiyang Business Consulting Co., Ltd.); 3WJD-18 electrostatic sprayer equipped with a conical nozzle (nozzle diameter 0.5 mm) (Shandong Weishi).

[0273] 2. Test Methods

[0274] (1) Test Design

[0275] Field trials in two cities and three locations were managed according to local production methods, and the tomato plants had the same growth vigor. Five treatments were set in each location test (Table 21), with each treatment replicated 4 times, for a total of 20 plots, and each plot was 12 - 16 m 2 . The plots were randomly arranged in blocks. At the first application, Botrytis cinerea had not occurred or had just started to occur on tomatoes in Fengxian, Shanghai and Urumqi, while it had already occurred on tomatoes in Songjiang, Shanghai. The agents were evenly sprayed on the whole plant by the spraying method, and the application was repeated every 5 - 7 days. The field trials in Fengxian, Shanghai and Songjiang, Shanghai were continuously applied 3 times, and the trial in Urumqi was continuously applied 2 times.

[0276] Table 21 Field Test Design of Test Agents against Botrytis cinerea on Tomato

[0277]

[0278] (2) Investigation Method

[0279] Botrytis cinerea had occurred before the first application in Songjiang, Shanghai, so the base number before application was investigated. In Fengxian, Shanghai, the disease condition was investigated on tomato leaves 7 days after the third application; in the Songjiang, Shanghai experiment, the disease condition was investigated on tomato fruits 7 days after the second and third applications. In the Urumqi experiment, the disease condition was investigated on tomato leaves and fruits 7 days after the first application and 10 days after the second application. Five-point sampling was used in each plot, 3 plants were investigated at each point, and all leaves of each plant were investigated. The disease grading standard and the calculation of disease index refer to the national standard "GB / T 17980.28-2000 Pesticide Field Efficacy Test Guidelines (I) Fungicides for Controlling Botrytis Cinerea on Vegetables".

[0280] (3) Method for calculating drug efficacy

[0281] 2.3 Method for calculating drug efficacy

[0282] The disease index and control effect were calculated according to the above formulas (2) and (3). If the disease base number before application was 0, the control effect was calculated according to the above formula (4).

[0283] To compare the differences in drug efficacy among different treatment areas, the new multiple range method (DMRT) was used to statistically analyze the experimental data, and a significant test (SAS 9.4) was carried out at the p = 0.01 level.

[0284] (4) Safety investigation

[0285] During each disease investigation, the safety of the test agents on crop growth was observed, and whether there were obvious effects of each treatment on the growth of tomatoes was compared, such as leaf discoloration, deformity, fruit drop, etc. At the same time, the effects of the test agents on other non-target organisms were observed.

[0286] (5) Effects on tomato yield and quality

[0287] Yield investigations were carried out on tomatoes during the harvest period in Urumqi. The tomato yields of each plot were measured on June 20, June 23, June 26, June 29, and July 2, 2024, respectively. The yields of 5 plots were cumulatively calculated and converted to hectare yields (kg / hm 2 ). The yield increase rate was calculated according to the above formula (6).

[0288] 3. Experimental results

[0289] 3.1 Results of the field efficacy test of the test agents for controlling Botrytis cinerea on tomato leaves (Fengxian District, Shanghai)

[0290] (1) Control effect of the test agents on Botrytis cinerea on tomato leaves

[0291] The results of the disease investigation 7 days after the third application showed that the control effects of the three concentrations of the test agents on Botrytis cinerea on tomato leaves were between 81.07% and 84.37%, and there were no significant differences in the control effects of the three concentrations compared with the treatment group of the control agent, 400 g / L isopropylthianil suspension (Table 22).

[0292] Table 22 Field efficacy test results of the test agents against Botrytis cinerea on tomato leaves (Fengxian District, Shanghai)

[0293]

[0294] Different capital letters after the data in the same column indicate significant differences at p < 0.05, and different lowercase letters indicate significant differences at p < 0.01.

[0295] (2) Effects of the test agents on plants and other organisms

[0296] All concentrations of the test agents had no phytotoxicity to tomato plants, the crops grew normally, and there were no adverse effects on non-target organisms.

[0297] 3.2 Field efficacy test results of the test agents against Botrytis cinerea on tomato fruits (Songjiang District, Shanghai)

[0298] (1) Control effects of the test agents on Botrytis cinerea on tomato fruits

[0299] The results of the disease investigation 7 days after the second and third applications showed that the test agents had an inhibitory effect on Botrytis cinerea on tomato fruits, and the inhibitory effect was positively correlated with the treatment dose (Table 23). The control effects of the active ingredients of the test agents at 216.67 - 433.33 mg / kg after three applications were between 64.91% and 75.43%, and there were no significant differences in the drug efficacy compared with the control agent.

[0300] Table 23 Field efficacy test results of the test agents against Botrytis cinerea on tomato fruits (Songjiang District, Shanghai)

[0301]

[0302] Different capital letters after the data in the same column indicate significant differences at p < 0.05, and different lowercase letters indicate significant differences at p < 0.01.

[0303] (2) Effects of the test agents on plants and other organisms

[0304] All concentrations of the test agents had no phytotoxicity to tomato plants, the crops grew normally, and there were no adverse effects on non-target organisms.

[0305] 3.3 Field efficacy test results of the test agents against Botrytis cinerea on tomatoes (Urumqi)

[0306] (1) Control effect of the test agents on Botrytis cinerea of tomatoes

[0307] The results of the disease investigation 7 days after the first application in the field efficacy test showed that the three concentration treatments of the test agents had an inhibitory effect on the disease of Botrytis cinerea on tomato leaves and fruits (Table 24). The results of the disease investigation 10 days after the second application showed that the control effects of the three concentration treatments of the test agents on Botrytis cinerea of tomatoes were all above 60% in both leaf and fruit parts, and the effect was positively correlated with the agent concentration. When the active ingredient of the test agent was treated at 433.33 mg / kg, the control effect of Botrytis cinerea on tomato leaves was above 79%, and the control effect on fruits was above 80%, with no significant difference from the control agent.

[0308] Table 24 Investigation results of the field efficacy test of the test agents against Botrytis cinerea of tomatoes (Urumqi)

[0309]

[0310] Different capital letters after the data in the same column indicate significant differences at p < 0.05, and different lowercase letters indicate significant differences at p < 0.01.

[0311] (2) Yield increase effect of the test agents on tomatoes

[0312] The results of the yield investigation in the field test (Table 25) showed that the tomato yields of the test agent treatment groups and the control agent treatment group were all significantly higher than that of the blank control group. The yield increase rates of tomatoes treated with the active ingredient of the test agent at 216.7 - 433.33 mg / kg were 6.59 - 7.74%, and there was no significant difference between the two treatment groups and the control agent.

[0313] Table 25 Investigation results of the yield of the field efficacy test of the test agents against Botrytis cinerea of tomatoes (Urumqi)

[0314]

[0315]

[0316] Different capital letters after the data in the same column indicate significant differences at p < 0.05, and different lowercase letters indicate significant differences at p < 0.01.

[0317] (3) Effects of the test agents on plants and other organisms

[0318] Each concentration treatment of the test agent had no phytotoxicity to tomato plants, the crops grew normally, and there was no adverse effect on non - target organisms.

[0319] The field efficacy trials conducted in Fengxian and Songjiang of Shanghai and Urumqi of Xinjiang in this embodiment show that the agent has inhibitory effects on the symptoms of the leaves and fruits of tomato gray mold, and also has an increasing production effect. The control effect of the high concentration of this agent is at the same level as that of the conventionally used chemically synthesized agents, and the increasing production effects of the medium and high concentrations of the tested agent reach the same level as that of the control agent. This result lays a foundation for replacing chemically synthesized pesticides with the plant-derived new pesticide 6.5% artemisiaflone emulsifiable concentrate in the control of tomato gray mold.

[0320] The results of the field efficacy trials in Shanghai and Xinjiang show that the tested agent has a similar trend in the control effect of tomato gray mold. The test results in the two places in Shanghai show that the effect of applying the drug before the onset of the disease is better than that after the onset of the disease.

[0321] In this embodiment, field trials are carried out in two places with large environmental differences, different tomato varieties and different control agents are used, and two disease grading standards for leaf parts and fruit parts are adopted. The results of the three groups of experiments all show that the tested agent has a good control effect on tomato gray mold. It is proved that the effect of 6.5% artemisiaflone emulsifiable concentrate in controlling tomato gray mold is stable and applicable to different production situations.

[0322] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those who are familiar with the technology in this field can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. Application of turmeric rhizome extract emulsifiable concentrate in the prevention and treatment of corn leaf spot disease.

2. The use of a turmeric rhizome extract emulsifiable concentrate in preventing and treating corn leaf spot according to claim 1, characterized in that: Application of turmeric rhizome extract emulsifiable concentrate in preventing and controlling corn leaf spot and promoting corn yield.

3. The use of a turmeric rhizome extract emulsifiable concentrate in preventing and treating corn leaf spot according to claim 1, characterized in that: The corn leaf spot disease is small corn leaf spot disease or large corn leaf spot disease.

4. The use of a turmeric rhizome extract emulsifiable concentrate in preventing and treating corn leaf spot according to claim 1, characterized in that: The turmeric rhizome extract emulsifiable concentrate has a mass concentration of 6.5%.

5. The use of a turmeric rhizome extract emulsifiable concentrate in preventing and treating corn leaf spot according to claim 1, characterized in that: The turmeric rhizome extract emulsifiable concentrate is diluted 100-600 times when used.

6. The use of a turmeric rhizome extract emulsifiable concentrate in preventing and treating corn leaf spot according to claim 5, characterized in that: When the turmeric rhizome extract emulsifiable concentrate is administered, the effective dosage of aromatic turmeric flavonoids after dilution is 100-500 mg / kg.

7. The use of a turmeric rhizome extract emulsifiable concentrate in preventing and treating corn leaf spot according to claim 1, characterized in that: The turmeric rhizome extract emulsifiable concentrate is used by spraying the stems and leaves.

8. The use of a turmeric rhizome extract emulsifiable concentrate in preventing and treating corn leaf spot according to claim 1, characterized in that: The turmeric rhizome extract emulsifiable concentrate is applied during the tasseling stage and the trumpet stage of the corn growth period, or during the 3-5 leaf stage of the corn, i.e., the early stage of the disease.

9. Application of turmeric rhizome extract emulsifiable concentrate in the prevention and control of tomato gray mold.

10. The use of the turmeric rhizome extract emulsifiable concentrate according to claim 9 in preventing and treating tomato gray mold, characterized in that: Application of turmeric rhizome extract emulsifiable concentrate in preventing and controlling tomato gray mold and promoting tomato yield increase.

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