Application of garlic E element combined with organic fertilizer in tobacco root knot nematode disease control

Abstract

The application belongs to the technical field of tobacco root-knot nematode prevention and treatment, and particularly relates to application of garlic E element combined with organic fertilizer in tobacco root-knot nematode disease prevention and treatment, and specifically provides application of an effective dose of garlic E element in tobacco root-knot nematode disease prevention and treatment. The garlic E element (ALE) is determined for killing activity on southern root-knot nematodes. The ALE has significant repellent and killing activity on the southern root-knot nematodes. No matter through direct contact or fumigation treatment, the ALE can effectively inhibit egg hatching and kill J2s. When the concentration is 10 muL / mL, direct contact treatment for 12 hours can achieve 100% J2s mortality and 98.69% egg hatching inhibition rate. The prevention and treatment effect of combined application of the ALE and the organic fertilizer is verified through a potting test. Greenhouse test data show that the garlic E element or the organic fertilizer can promote tobacco growth and reduce root-knot nematode disease harm in a certain extent when applied alone, but the effect of combined application of the two is more significant.

Description

Technical Field

[0001] This invention belongs to the field of tobacco root-knot nematode control technology, specifically involving the application of garlic E-in combined with organic fertilizer in the control of tobacco root-knot nematode disease. Background Technology

[0002] Tobacco root-knot nematode disease is a soil-borne disease caused by root-knot nematodes (Meloidogyne spp.). It infects the root system of tobacco plants at various stages of growth. The southern root-knot nematode (Meloidogyne incognita) is the most destructive species causing tobacco root-knot nematode disease, being one of the most widespread and destructive plant parasitic nematodes. Furthermore, this nematode is the primary pathogen of tobacco root-knot disease, severely damaging the yield and quality of tobacco crops. Root-knot nematodes penetrate the root tip and move to the vascular cylinder, inducing the formation of specialized feeding sites called "giant cells." As the tissues around the nematode feeding site proliferate, root system function is hindered, and overall metabolic functions, such as the absorption and transport of water and minerals, are impaired, leading to wilting and shrinkage, inward curling of leaf tips and margins, and ultimately, slowed plant growth. Root-knot nematodes also accumulate large amounts of toxins within the host plant, causing plant cell damage and, in severe cases, plant death. Furthermore, because root-knot nematodes parasitize tobacco roots for a long period and cause mechanical damage to the root system, they facilitate the invasion of other pathogenic microorganisms, leading to various fungal and bacterial diseases and resulting in more severe complex infections, such as Fusarium wilt and root rot. These diseases severely affect the normal growth of tobacco leaves, reducing their yield and quality. Currently, the control of root-knot nematodes relies excessively on chemical nematicides, which pose serious threats to ecology and human health due to phytotoxicity, environmental pollution, and drug resistance. Therefore, it is urgent to develop economically feasible and environmentally friendly control strategies. Summary of the Invention

[0003] This invention aims to provide a green and environmentally friendly method for controlling tobacco root-knot nematodes, thereby offering a new technical solution for the control of root-knot nematodes. Specifically, this invention provides the following technical solution:

[0004] On the one hand, the present invention provides the application of garlic E in the prevention and control of tobacco root-knot nematode disease.

[0005] On the other hand, the present invention provides the application of a combination of garlic E and organic fertilizer in the prevention and control of tobacco root-knot nematode disease.

[0006] Preferably, the mass ratio of garlic E to organic fertilizer is 0.5-1.5:20.

[0007] Furthermore, the pathogen used to control tobacco root-knot nematode disease is the southern root-knot nematode.

[0008] On the other hand, the present invention also provides a method for preventing and controlling tobacco root-knot nematode disease, comprising applying an effective dose of garlic E to the soil in which tobacco is grown.

[0009] Furthermore, application methods include spraying or fumigation.

[0010] On the other hand, the present invention also provides a method for preventing and controlling tobacco root-knot nematode disease, comprising applying an effective dose of garlic E and organic fertilizer to the soil in which tobacco is grown.

[0011] Preferably, the mass ratio of garlic E to organic fertilizer is 0.5-1.5:20.

[0012] Preferably, the main components of the organic fertilizer are as follows: total nutrients ≥ 4.0%, organic matter ≥ 30%, nitrogen ≥ 2%, phosphorus pentoxide ≥ 1%, and potassium oxide ≥ 1%.

[0013] Furthermore, the method provided by the present invention induces the production of reactive oxygen species in tobacco root tips and / or increases the content of lignin, total phenols, and the activity of resistance-related enzymes in tobacco.

[0014] The technical effects achieved by this invention are as follows:

[0015] This invention determined the nematicidal activity of garlic E-in (ALE) against the southern root-knot nematode (Meloidogyne incognita). A pot experiment verified the control effect of ALE combined with organic fertilizer, as detailed below:

[0016] 1. ALE exhibits significant repellent and nematicidal activity against the southern root-knot nematode. Experimental data show that ALE not only effectively repels its second-instar larvae (J2s) but also significantly inhibits egg hatching and shows a nematicidal effect on J2s. Whether through direct contact or fumigation, ALE effectively inhibits egg hatching and kills J2s. At a concentration of 10 μL / mL, direct contact treatment for 12 hours achieved 100% J2s mortality and a 98.69% egg hatching inhibition rate. Under fumigation conditions, after 48 hours of treatment with 10 μL / mL ALE, the J2s mortality rate was 56.27%, and the egg hatching inhibition rate reached 76.95% after 7 days. This indicates that ALE can inhibit egg hatching and kill second-instar larvae through fumigation or direct contact; it also repels second-instar larvae from infecting host plants, thus helping plants resist root-knot nematode infection.

[0017] 2. The combined application of ALE and organic fertilizer can promote tobacco growth and development while effectively controlling root-knot nematode disease. Greenhouse experiment data show that the application of garlic E alone or organic fertilizer can promote tobacco growth and reduce the damage of root-knot nematode disease to a certain extent, but the combined application of the two has a more significant effect. Under the treatment condition of adding 1.0 g of ALE and 20 g of organic fertilizer per kilogram of soil, compared with the control group, plant height, root length, above-ground fresh weight, below-ground fresh weight, above-ground dry weight, and below-ground dry weight increased by 45.14%, 9.74%, 105%, 55.36%, 74.18%, and 88.89%, respectively, and the number of root knots, number of eggs, root knot index, egg index, and disease index decreased by 78.89%, 93.91%, 86.38%, 96.06%, and 61.01%, respectively. This indicates that the combined treatment condition is more conducive to promoting tobacco growth and effectively inhibiting the occurrence of root-knot nematode disease.

[0018] 3. Combined treatment with ALE and organic fertilizer can increase the lignin and total phenol content in tobacco plants and induce a surge of reactive oxygen species (ROS) at the root tips under root-knot nematode infestation conditions. Experimental data showed that, regardless of whether root-knot nematodes were inoculated, combined treatment with ALE and organic fertilizer significantly increased the lignin and total phenol content in tobacco plants. Under root-knot nematode inoculation conditions, the lignin and total phenol content increased by 30.99% and 19.61%, respectively, compared to the uninoculated group. Furthermore, the combined application of ALE and organic fertilizer in the early stage of inoculation induced a strong surge of ROS at the root tips, thereby enhancing the plant's resistance to root-knot nematode infestation. This indicates that combined treatment with ALE and organic fertilizer can improve tobacco's resistance to root-knot nematode disease by affecting lignin, total phenols, and ROS.

[0019] 4. Combined treatment with ALE and organic fertilizer can increase the activity of tobacco resistance-related enzymes and upregulate the expression of key genes in defense-related pathways. Experimental data showed that combined treatment with ALE and organic fertilizer significantly increased the activity of antioxidant enzymes in tobacco. POD, SOD, and CAT enzyme activities peaked at 72 hpi, increasing by 2.54 times, 1.62 times, and 2.42 times, respectively, compared to the inoculated control group; while PPO activity peaked at 24 hpi, with an increase of 2.53 times. Attached Figure Description

[0020] Figure 1 shows the contact toxicity of ALE against second-instar larvae (A) and eggs (B) of Southern Root-knot Nematode.

[0021] Figure 2. Statistical chart of the fumigation effect of ALE on second-instar larvae (A) and eggs (B) of Southern Root-knot Nematode.

[0022] Figure 3. Statistical chart of tobacco growth indicators under different treatment conditions. Where A: plant height; B: root length; C: aboveground fresh weight; D: underground fresh weight; E: aboveground dry weight; F: underground dry weight.

[0023] Figure 4. Statistical chart of disease indicators in tobacco under different treatments. Where A: number of root knots; B: number of eggs; C: root knot index; D: egg index; E: reproductive factor; F: disease index.

[0024] Figure 5. Growth (A1-M1) and disease occurrence (A2-M2) of tobacco under different treatments.

[0025] Figure 6. Effects of combined application of ALE and organic fertilizer on total phenolic content (A) and lignin content (B) in tobacco at different growth stages; Note: CK: sterile distilled water treatment; MI: inoculation with 1000 J2s of southern root-knot nematodes; AF: 1.0 g ALE and 20 g organic fertilizer per kilogram of soil at transplanting; AFMI: 1.0 g ALE and 20 g organic fertilizer per kilogram of soil at transplanting, and inoculation with 1000 J2s of southern root-knot nematodes three days after transplanting.

[0026] Figure 7. Effect of combined application of ALE and organic fertilizer on reactive oxygen species (ROS) content in tobacco. Note: Scale bar = 100 μm.

[0027] Figure 8. Effects of ALE combined with organic fertilizer on reactive oxygen species (A) and hydrogen peroxide content (B) in tobacco at different growth stages.

[0028] Figure 9. Effects of combined application of ALE and organic fertilizer on the activities of peroxidase (A), superoxide dismutase (B), catalase (C), and polyphenol oxidase (D) in tobacco plants. Detailed Implementation

[0029] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.

[0030] Example 1

[0031] 1.1 Test Materials

[0032] Nematode material: Purebred Southern Root-Knot Nematodes were provided by the Nematode Laboratory of Yunnan Agricultural University. Second-instar larvae were infected with tomatoes and then propagated and preserved in a greenhouse. The isolated and purified Southern Root-Knot Nematodes were inoculated onto infected tomatoes with 3-4 true leaves. On day 45 after inoculation, the tomato roots were collected, and the Southern Root-Knot Nematodes were collected using the shallow tray method.

[0033] Plant material: *Nicotiana tabacum* L., with tobacco seeds provided by the Yunnan Academy of Tobacco Agriculture, Kunming. Tobacco seeds were sown in plastic trays and transplanted to 32-cell trays (54×28 cm) when they reached the four true leaf stage. After further growth to a suitable developmental stage, individual plants were transplanted into seedling pots (14 cm in diameter, 11 cm in height), with the soil in the pots sterilized at 121℃ for 30 minutes under high pressure. The entire growth process was conducted under controlled greenhouse conditions (day / night temperature difference 28℃ / 22℃, relative humidity 60%-70%, 14-hour light / 10-hour dark cycle).

[0034] Reagents and Fertilizers: Garlic E-type compound (ALE) was provided by Shanghai Laisen Biotechnology Co., Ltd. Avermectin (CAS No. 71751-41-2; 95.0%) was provided by Shanghai Yuanye Biotechnology Co., Ltd. The organic fertilizer substrate was provided by Yunnan Yuantian Biotechnology Group Fertilizer Co., Ltd., and its main components are as follows: total nutrients ≥ 4.0%, organic matter ≥ 30%, nitrogen ≥ 2%, phosphorus pentoxide ≥ 1%, potassium oxide ≥ 1%.

[0035] 1.2 Verification of the nematicidal activity of garlic E-in against southern root-knot nematodes

[0036] 1.2.1 Chemotactic activity of allicin E on southern root-knot nematodes

[0037] The experiment was conducted in square petri dishes, each containing 2% water agar, at room temperature. ALE standards were dissolved in sterile water to prepare solutions of four concentrations: 0.1 μL / mL, 1 μL / mL, 2 μL / mL, and 10 μL / mL. Sterile filter paper discs (5 mm in diameter) were then placed on either side of the petri dish. 5 μL of diluted ALE solution was added to the left filter paper disc (experimental group), and 5 μL of sterile distilled water was added to the right filter paper disc (control group). Sterile distilled water was used as a negative control (independent petri dish). Approximately 200 second-instar larvae suspended in 10 μL of sterile water were added to the center of each petri dish. After incubating the dishes at 25°C in the dark for 12 hours, the number of second-instar larvae in the treatment and control areas was recorded under a microscope. The chemotactic index (CI) was calculated using the following formula. All experiments were performed in triplicate.

[0038] CI = (Number of nematodes in the test area - Number of nematodes in the control area) / (Number of nematodes in the test area + Number of nematodes in the control area).

[0039] If 0 < CI < 1, it indicates that the test sample has an attractive effect on J2s; if −1 < CI < 0, it indicates that the test sample has an avoidance effect on J2s; if CI = 0, it indicates that the sample has no effect on J2s.

[0040] 1.2.2 Contact toxicity of allicin E against southern root-knot nematodes

[0041] The direct contact toxicity assay of ALE against second-instar larvae of the southern root-knot nematode was conducted using a 12-well plate. ALE standards were diluted to different concentrations as in the repulsion assay, with sterile water as a negative control and 1 μg / mL abamectin as a positive control. 1 mL of each solution of different concentrations was added to each well, followed by approximately 200 second-instar larvae. The plate was sealed with sealing film to prevent evaporation and interference, and incubated at 25°C in the dark. Nematode mortality was observed under a stereomicroscope after 12, 24, and 48 hours of treatment, with immobile and stiff individuals considered dead. Furthermore, to assess the effect of ALE on egg hatching, the same method as the second-instar larvae assay was used. 1 mL of each ALE solution of different concentrations was added to each well, followed by approximately 200 eggs. The plates were incubated at a constant temperature of 25°C, and the number of hatched second-instar larvae (J2) was recorded daily for 7 consecutive days. All treatments were performed in triplicate.

[0042] Mortality rate (%) = (Number of dead second-instar larvae / Total number of second-instar larvae) × 100%.

[0043] Hatching rate (%) = (Number of second-instar larvae hatched / Total number of eggs) × 100%.

[0044] 1.2.3 Fumigation activity of garlic E-in against southern root-knot nematodes

[0045] In the fumigation experiment, 1 mL of ALE solution of different concentrations was added to the center of a 12-well plate, and 1 mL of sterile water containing either 200 J2s or 200 eggs was added to the four surrounding wells. The culture plate was sealed with sealing film to prevent moisture evaporation and interference, and cultured at 25°C in the dark. The mortality rate of nematodes was observed using a stereomicroscope after 12, 24, and 48 hours of treatment. For eggs, the hatching rate was recorded for 7 consecutive days, and all experiments were performed in triplicate.

[0046] 1.3 Greenhouse Experiment

[0047] The greenhouse experiment included the following treatment groups: garlic emulsifier (ALE) alone (0.5, 1.0, and 1.5 g / kg soil); organic fertilizer alone (20 g / kg soil); and ALE combined with organic fertilizer (0.5 g ALE + 20 g organic fertilizer / kg soil, 1.0 g ALE + 20 g organic fertilizer / kg soil, and 1.5 g ALE + 20 g organic fertilizer / kg soil). Irrigation with sterilized distilled water served as a negative control, and 0.5 g / kg soil abamectin served as a positive control. Each treatment group consisted of 3 tobacco plants, independently replicated 3 times, for a total of 9 tobacco plants per treatment.

[0048] The experimental procedure was as follows: First, loess and humus were mixed in a 5:1 ratio and sterilized by high-temperature steam (121℃, 30 minutes). Then, different concentrations of ALE were added (e.g., 0.5 g / kg ALE + 20 g / kg organic fertilizer: 20 g organic fertilizer and 0.5 g ALE were added per kilogram of soil). After thorough mixing, approximately 300 g of the mixed soil was placed into each experimental pot, and tobacco seedlings were then transplanted. Three days after transplanting, 1000 freshly hatched second-instar larvae were evenly inoculated into the soil around the roots to a depth of approximately 5 cm using the soil inoculation method. During the growth period, water was provided as needed to ensure normal plant development.

[0049] Forty-five days after inoculation, growth indicators (plant height, root length, aboveground fresh weight, belowground fresh weight, aboveground dry weight, and belowground dry weight) and disease indicators (number of root knots, number of eggs, root knot index, egg index, reproductive factor, and disease index) were measured, and disease classification was performed according to Table 1. The root knot index, egg index, disease index, and reproductive factor were calculated using the following formulas.

[0050] Root knot index (GI) = Number of root knots per plant / Fresh weight of roots per plant

[0051] Egg Index (EI) = Number of eggs per plant / Fresh weight of roots per plant

[0052] Disease Index (DI) = [∑(Disease grade × Number of plants at that disease grade) / (Total number of plants surveyed × Highest disease grade value)] × 100%

[0053] Reproduction factor (RF) = Final population density of nematodes in grafted plants / Initial population density of nematodes in grafted plants.

[0054] Table 1 Grading Criteria for Tobacco Root-Knot Nematode Disease

[0055]

[0056] 1.4 Determination of lignin and total phenol content

[0057] Pot experiments were conducted to screen the optimal concentration of garlic E-in combined with organic fertilizer for subsequent physiological index determination. Four different treatments were set up, all inoculated using the soil inoculation method: Treatment 1 (control group, CK): irrigation with sterile distilled water served as a negative control; Treatment 2 (MI): each tobacco plant was inoculated with 1000 second-instar larvae of the southern root-knot nematode; Treatment 3 (AF): 1.0 g ALE and 20 g organic fertilizer were added per kilogram of soil at transplanting; Treatment 4 (AFMI): 1.0 g ALE and 20 g organic fertilizer were added per kilogram of soil at transplanting, and 1000 second-instar larvae of the southern root-knot nematode were inoculated three days after transplanting. Each treatment had 3 plants, independently replicated 3 times, for a total of 9 tobacco plants per group. All experiments were conducted under greenhouse conditions. Physiological indexes (lignin and total phenol content) were measured on days 0, 1, 3, 5, and 7 after inoculation. The lignin content detection kit (catalog number: ADS-W-TR014) and total phenol content detection kit (catalog number: ADS-W-KY008-48) manufactured by Jiangsu Edison Biotechnology Co., Ltd. were used for the determination, and the operation was carried out in accordance with the instructions.

[0058] 1.5 Reactive oxygen species staining and determination of H2O2 content

[0059] Tobacco root tips (1-2 cm) were collected from each treatment group at 0, 1, 3, 5, and 7 days post-inoculation for DCF (2,7-dichlorodihydrofluorescein diacetate) staining. Each treatment was repeated in triplicate, with three tobacco seedlings per replicate. Five root tips were collected from each seedling for reactive oxygen species (ROS) staining. 1-2 cm root tips were placed in 2 mL centrifuge tubes and 25 μM H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) working solution was added for staining. The H2DCFDA stock solution (10 mM) was prepared by dissolving in DMSO and stored at –20°C protected from light. It was diluted to a final concentration of 25 μM with pH 7.4 PBS buffer before use and prepared fresh each time. The root tips were incubated in the staining solution in the dark for 10-15 minutes, then rinsed with distilled water 3-5 times to remove residual dye. Finally, the samples were placed on a glass slide, covered with a coverslip, and the fluorescence intensity was observed and images were captured under blue light excitation using a Leica fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm, 10x objective). Quantitative analysis was performed using ImageJ software to calculate the relative content of reactive oxygen species (ROS) in tobacco roots at different stages.

[0060] The content of hydrogen peroxide (H2O2) was determined using a hydrogen peroxide content detection kit (product number: ADS-W-YH015) produced by Jiangsu Addison Biotechnology Co., Ltd., and the operation was carried out according to the instructions. Tobacco roots under each treatment were taken for determination at 0, 1, 3, 5, and 7 days after inoculation. Three biological replicates were set for each treatment, each replicate included 3 seedlings, and a total of 9 tobacco plants were included in each treatment.

[0061] 1.6 Detection of resistance-related enzyme activities

[0062] Root samples of each treatment group were collected at 0, 24, 48, and 72 hours after inoculation. After thoroughly rinsing with distilled water, the root samples were immediately frozen in liquid nitrogen and stored at -80 °C for subsequent analysis of the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and polyphenol oxidase (PPO). Three plants were set for each treatment group, with 3 independent repetitions, and a total of 9 tobacco plants were included in each group. All experiments were carried out under greenhouse conditions. The determination methods were all carried out using kits produced by Jiangsu Addison Biotechnology Co., Ltd. (product numbers: G0107W, G0101W, G0105W, and ADS-W-YH005-96).

[0063] 2. Results

[0064] 2.1 Toxic effect of allicin E on Meloidogyne incognita

[0065] 2.1.1 Chemotactic activity of allicin E on Meloidogyne incognita

[0066] The chemotactic effect of allicin E on Meloidogyne incognita is presented in Table 2. Allicin E has strong volatility, and volatile substances have characteristics such as strong diffusivity and permeability. Therefore, it can attract or repel nematodes, showing chemotactic activity. In this experiment, the chemotactic activity of ALE on Meloidogyne incognita at four concentrations was detected. The results showed that: the sterile water control group (CK) had no obvious chemotactic or attractive effect on nematodes; the chemotactic index of nematodes at different ALE concentrations was -1 < C.I. < 0, indicating that ALE at four concentrations showed chemotactic activity against Meloidogyne incognita. It should be noted that the repellent effect of ALE is concentration-dependent, and the higher the concentration, the stronger the repellent effect.

[0067] Table 2 Chemotactic effect of different concentrations of allicin E on Meloidogyne incognita

[0068]

[0069] Note: a, b, c indicate that at the 0.05 significance level, different letters represent significant differences between means.

[0070] 2.1.2 Contact killing activity of allicin against southern root-knot nematodes

[0071] To investigate the contact toxicity of garlic E (ALE) against southern root-knot nematodes, this experiment determined the direct contact inhibitory effects of four concentrations of ALE on second-instar larvae (J2s) and eggs of the southern root-knot nematode. The results showed that all concentrations of ALE exhibited nematicidal effects on J2s, and this effect was concentration-dependent; the higher the concentration, the more significant the effect. When the ALE concentration was 1 μL / mL or higher, the mortality rate after 48 hours of direct contact reached 100%, significantly higher than the 19.39% in the control group (CK). Figure 1 (A). Meanwhile, during continuous observation of egg hatching, all tested concentrations of ALE significantly inhibited egg hatching. When the ALE concentrations were 1 μL / mL, 2 μL / mL, and 10 μL / mL, the hatching rates after 7 days were 2.07%, 1.09%, and 0.78%, respectively, which were 57.28%, 58.26%, and 58.57% lower than the control group. Figure 1 (B). These results indicate that ALE has certain contact-killing activity against second-instar larvae of the southern root-knot nematode and also exhibits strong egg hatching inhibition ability.

[0072] 2.1.3 Fumigation activity of garlic E-in against southern root-knot nematodes

[0073] This experiment used a 12-well plate to detect the fumigation activity of garlic extract (ALE) at four concentrations against second-instar larvae (J2s) and eggs of the southern root-knot nematode. The results showed that ALE had significant fumigation activity against J2s, and within the tested concentration range, its activity increased with increasing concentration, exhibiting a good concentration-dependent effect. When the ALE concentrations were 1 μL / mL, 2 μL / mL, and 10 μL / mL, the mortality rates of J2 larvae after 48 hours were 29.91%, 35.47%, and 56.27%, respectively, significantly higher than the 19.68% in the control group (CK). Figure 2 (A). Furthermore, ALE treatment under fumigation conditions continuously inhibited egg hatching. After a 7-day observation period, the hatching rates at ALE concentrations of 1 μL / mL, 2 μL / mL, and 10 μL / mL were 17.42%, 14.11%, and 8.89%, respectively, representing decreases of 21.15%, 24.46%, and 29.68% compared to the sterile water control group. Figure 2 (B). These results indicate that ALE has a certain degree of volatility and diffusion, can generally inhibit the hatching of southern root-knot nematode eggs, and has a fumigation effect on second-instar larvae.

[0074] 2.2 Effects of different concentrations of garlic E combined with organic fertilizer on tobacco resistance to root-knot nematode disease

[0075] 2.2.1 Effects of different concentrations of garlic E combined with organic fertilizer on tobacco growth and development

[0076] To investigate the effects of combined application of ALE and organic fertilizer on tobacco growth indicators, plant height, root length, aboveground fresh weight, belowground fresh weight, aboveground dry weight, and belowground dry weight were recorded 45 days after inoculation with southern root-knot nematodes. Specific results can be found [link to relevant data]. Figure 3 45 days after inoculation, the overall growth of the potted plants is as follows: Figure 5 As shown in the data, 45 days after inoculation, all treatment groups showed better growth indicators than the sterile water control group and the abamectin treatment group, indicating differences in the promoting effects of different treatments on plant growth. When ALE was applied alone, the promoting effects on plant height, above-ground fresh weight, above-ground dry weight, and underground dry weight decreased with increasing ALE concentration, but root length and underground fresh weight did not show significant changes with increasing concentration. In the ALE-only treatment, when the ALE concentration was 1.0 g / kg, compared with the sterile water control group, plant height, root length, above-ground fresh weight, above-ground dry weight, underground fresh weight, and underground dry weight increased by 38.09%, 2.45%, 61.62%, 43.82%, 91.39%, and 57.78%, respectively. Applying organic fertilizer alone can significantly promote plant growth. When the organic fertilizer concentration is 40 g / kg, compared with the sterile water control group, plant height, above-ground fresh weight, above-ground dry weight, underground fresh weight, and underground dry weight increased by 64.48%, 124.33%, 53.58%, 102.9%, and 85.56%, respectively. When ALE is applied in combination with organic fertilizer, the growth-promoting effect is more significant than that of applying ALE or organic fertilizer alone. When the concentration is 1.0 g / kg ALE + 20 g / kg organic fertilizer, compared with the sterile water control group, plant height, root length, above-ground fresh weight, underground fresh weight, above-ground dry weight, and underground dry weight increased by 45.14%, 9.74%, 105%, 55.36%, 74.18%, and 88.89%, respectively. The combination of high-concentration ALE and organic fertilizer has a more prominent promoting effect on the growth of the underground parts of the plant. In summary, the above results indicate that under controlled greenhouse conditions, the combined application of ALE and organic fertilizer can effectively promote the growth of tobacco.

[0077] 2.2.2 Effects of different concentrations of garlic E combined with organic fertilizer on the control of tobacco root-knot nematode disease

[0078] The combined application of different concentrations of ALE with organic fertilizer has the following effects on the control of tobacco root-knot nematode disease: Figure 4 and Figure 5As shown, compared with the sterile water control group, all treatment groups significantly reduced nematode infection indicators, including root knot number, egg number, root knot index, egg index, reproductive factor, and disease index. However, the control effects varied among different treatment groups. Both ALE and organic fertilizer effectively reduced disease indicators, whether applied alone or in combination. The combined application of ALE and organic fertilizer exhibited a clear dose-dependent characteristic, showing better control of root-knot nematodes than applying ALE or organic fertilizer alone, achieving a stronger nematode suppression effect. Specifically, under the combined treatment of 1.0 g / kg ALE + 20 g / kg organic fertilizer, compared with the control group, the number of root knots, egg number, root knot index, egg index, and disease index decreased by 78.89%, 93.91%, 86.38%, 96.06%, and 61.01%, respectively. Compared with the sterile water control group, the abamectin treatment group showed reductions of 86.73%, 96.74%, 90.33%, 97.63%, and 71.18% in root knot number, egg number, root knot index, egg index, and disease index, respectively. Although the abamectin treatment was more effective than ALE or organic fertilizer alone, the difference was not significant. The results also showed that the reproduction factor (Rf) value in the sterile water control group was greater than 1, indicating an increase in the population of southern root-knot nematodes, while the Rf values ​​in all other treatment groups were less than 1, indicating a decrease in the population. These results collectively indicate that under controlled greenhouse conditions, the combined application of ALE and organic fertilizer can effectively control the occurrence of tobacco root-knot nematode disease.

[0079] 2.3 Effects of combined application of garlic E-co-organic fertilizer on the lignin and total phenol content of tobacco

[0080] To investigate the effects of combined application of garlic emulsion (ALE) and organic fertilizer on the lignin and total phenol content of tobacco, this experiment measured the lignin and total phenol content of tobacco under four treatment conditions. The results showed that combined application of garlic emulsion (ALE) and organic fertilizer significantly increased the total phenol and lignin content in tobacco. Figure 6Regardless of whether or not the plant was inoculated with the southern root-knot nematode, this combined treatment effectively induced the accumulation of lignin and total phenols in tobacco, with the induction effect being more pronounced under nematode inoculation conditions. Lignin content reached its highest level on day 3 post-inoculation. When inoculated with root-knot nematodes, the lignin content significantly increased by 30.99% after application of ALE and organic fertilizer compared to the unfertilized treatment; and when ALE and organic fertilizer were applied together, the lignin content after southern root-knot nematode infection was 37.87% higher than the unfertilized treatment. Total phenol content peaked on day 7 post-inoculation. When inoculated with root-knot nematodes, the total phenol content under the combined ALE and organic fertilizer treatment was 19.61% higher than the unfertilized treatment; and when ALE and organic fertilizer were applied together, the total phenol content increased by 9.58% compared to the unfertilized treatment. These results collectively indicate that the combined application of garlic E and organic fertilizer can significantly promote the synthesis and accumulation of total phenols and lignin in tobacco, thereby enhancing the plant's resistance to the southern root-knot nematode.

[0081] 2.4 Effects of combined application of garlic E-co-organic fertilizer on reactive oxygen species and hydrogen peroxide content in tobacco.

[0082] DCF staining of tobacco root tips at different time points under four treatments revealed that the combined application of ALE and organic fertilizer (i.e., 1.0 g ALE and 20 g organic fertilizer per kilogram of soil) under inoculation conditions could induce a burst of reactive oxygen species in tobacco root tips. Figure 7 and Figure 8 (A). The combined application of ALE and organic fertilizer did not induce significant reactive oxygen species (ROS) accumulation. However, in the case of root-knot nematode inoculation, the nematode invasion caused a violent ROS burst at the root tip. Significant ROS accumulation was observed at the root tip as early as 1 day post-inoculation (dpi), and the ROS level increased further with prolonged inoculation time, reaching a stronger burst at 5 dpi. When ALE and organic fertilizer were applied together, the ROS burst at the root tip was more intense in the initial inoculation stage than with single inoculation, thus enhancing the plant's resistance to root-knot nematode infection. Consistent with the results of fluorescent staining, under conditions of inoculation with southern root-knot nematodes, the combined application of ALE and organic fertilizer significantly affected the hydrogen peroxide content in tobacco plants. Figure 8 (B) Under inoculation conditions, compared with the untreated control group (MI), the combined application of ALE and organic fertilizer significantly increased the hydrogen peroxide content in tobacco at the initial stage of inoculation, reaching a peak at 3 days post-inoculation, which was 23.53% higher than the treatment group inoculated with root-knot nematodes alone. Subsequently, the hydrogen peroxide content gradually decreased in the later stages of inoculation. These results indicate that the combined application of ALE and organic fertilizer can induce a significant surge of reactive oxygen species in tobacco root tips at the initial stage of inoculation, thereby enhancing the plant's defense against southern root-knot nematode infection.

[0083] 2.5 Effects of combined application of garlic E and organic fertilizer on the activity of tobacco resistance-related enzymes

[0084] The combined application of ALE and organic fertilizer significantly increased the activity of antioxidant enzymes in tobacco plants. On the third day after transplanting, compared with the untreated control group, the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO) were all significantly increased. Figure 9 Regardless of whether root-knot nematode inoculation was performed, the combined application of ALE and organic fertilizer effectively enhanced the activity of these four enzymes. Furthermore, under root-knot nematode inoculation conditions, the increase in enzyme activity was more significant. The activities of POD, SOD, and CAT reached their highest values ​​72 hours post-inoculation (hpi), increasing by 2.54 times, 1.62 times, and 2.42 times, respectively, compared to the control group. PPO activity peaked at 24 hpi, with an increase of 2.53 times. These results collectively indicate that the combined application of ALE and organic fertilizer can effectively upregulate the expression of key resistance-related enzymes in tobacco, significantly enhancing the plant's ability to resist root-knot nematode infection.

[0085] In summary, this invention determined the nematicidal activity of garlic E-in (ALE) against southern root-knot nematode (M. incognita), and investigated the control effect of ALE combined with organic fertilizer on tobacco root-knot nematode disease using pot experiments. The main conclusions are as follows:

[0086] (1) ALE has a significant repellent effect on southern root-knot nematodes, and also has significant nematicidal activity and hatch-inhibiting activity against second-instar larvae and eggs of root-knot nematodes. Experimental data show that ALE can effectively kill J2s and inhibit egg hatching through direct contact or fumigation, and can also significantly hinder J2s infection of host plants, thereby helping plants resist root-knot nematode infection. The main effects are repellency and contact killing.

[0087] (2) The combined application of ALE and organic fertilizer can significantly promote the growth and development of tobacco and effectively control the occurrence of root-knot nematode disease. Greenhouse experiment data show that the application of ALE or organic fertilizer alone can promote tobacco growth and reduce root-knot nematode disease to a certain extent, but the combined application of the two (1.0 g of ALE and 20 g of organic fertilizer per kilogram of soil) has the most significant effect. This indicates that the combined treatment of ALE and organic fertilizer can effectively inhibit the occurrence of root-knot nematode disease while promoting normal plant growth and development.

[0088] (3) Combined treatment with ALE and organic fertilizer can increase the lignin and total phenol content in tobacco plants, induce reactive oxygen species bursts at the root tips under root-knot nematode infection conditions, and enhance resistance-related enzyme activity. Regardless of whether root-knot nematodes are inoculated, combined treatment with ALE and organic fertilizer can significantly increase the lignin, total phenol content, and resistance enzyme activity in tobacco plants, with even greater increases under root-knot nematode inoculation conditions. In addition, the combined application of ALE and organic fertilizer in the early stage of inoculation can induce a strong reactive oxygen species burst at the root tips, enhancing the plant's resistance to root-knot nematode infection. This indicates that combined treatment with ALE and organic fertilizer can improve tobacco's resistance to root-knot nematode disease by affecting tobacco lignin, total phenol, reactive oxygen species, and resistance enzyme activity. The experimental results can provide a theoretical basis and practical reference for the sustainable control of root-knot nematode disease.

Claims

1. Application of garlic E in the prevention and control of tobacco root-knot nematode disease.

2. Application of a combination of garlic E-in and organic fertilizer in the control of tobacco root-knot nematode disease.

3. The application according to claim 2, characterized in that, The mass ratio of garlic E to organic fertilizer is 0.5-1.5:

20.

4. The application according to any one of claims 1-3, characterized in that, The pathogen used to control tobacco root-knot nematode disease is the southern root-knot nematode.

5. A method for controlling tobacco root-knot nematode disease, characterized in that, This includes applying an effective dose of garlic E to the soil where tobacco is grown.

6. The method according to claim 5, characterized in that, Application methods include spraying or fumigation.

7. A method for controlling tobacco root-knot nematode disease, characterized in that, This includes applying an effective dose of garlic E and organic fertilizer together to the soil where tobacco is grown.

8. The method as described in claim 8, characterized in that, The mass ratio of garlic E to organic fertilizer is 0.5-1.5:

20.

9. The method according to claim 7 or 8, characterized in that, The main components of the organic fertilizer are as follows: total nutrients ≥ 4.0%, organic matter ≥ 30%, nitrogen ≥ 2%, phosphorus pentoxide ≥ 1%, potassium oxide ≥ 1%.

10. The method of claim 9 induces the production of reactive oxygen species in tobacco root tips and / or increases the content of lignin, total phenols, and the activity of resistance-related enzymes in tobacco.

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