Application of exogenous selenium in improving resistance of tomato to late blight

Abstract

The application discloses application of exogenous selenium in improving resistance of tomatoes to late blight, and belongs to the field of plant disease control. The exogenous selenium comprises Na2SeO3. It is found that, compared with a control group, the exogenous selenium can inhibit the occurrence of diseases of tomato plants. Specifically, the exogenous selenium can inhibit the expansion of late blight spots on tomato leaves, alleviate the accumulation of H2O2 and O2 ‑ in the leaves, reduce the ROS content in the leaves of tomatoes, increase the activities of antioxidant enzymes SOD, POD and CAT, and the content of chlorophyll, and reduce the generation of MDA. In addition, spraying the exogenous selenium can increase the contents of SA and JA in the tomato plants, and the selenium content in the leaves and fruits of tomatoes. Furthermore, it is detected that the exogenous selenium can improve the resistance of tomatoes to the late blight by inducing the expression of SIPR5 genes. In conclusion, it is found that the exogenous selenium can improve the resistance of tomatoes to the late blight, and a new method for preventing and treating the late blight of tomatoes is provided.

Description

Technical Field

[0001] This invention relates to the field of plant disease control, and in particular to the application of exogenous selenium in improving resistance to late blight in tomatoes. Background Technology

[0002] Tomato (Solanum lycopersicum L.) belongs to the Solanaceae family and the Solanum genus. Also known as tomato, it was historically called "June persimmon" and is an annual or perennial herbaceous plant. Tomatoes are rich in nutrients, including carotene and vitamin C, which are very beneficial to human health.

[0003] Tomato late blight (Phytophthorain festans (Mont.) De Bary), also known as black stem blight, is caused by infection with the pathogenic fungus Phytophthora. In modern agricultural production, greenhouse cultivation techniques are widely used in tomato production. However, due to the stability of factors such as temperature and humidity inside greenhouses, these environmental conditions are often very suitable for the growth and reproduction of pathogens, providing a good breeding ground for the fungus to overwinter. Coupled with excessive continuous cropping, the yield and quality of tomatoes are severely damaged.

[0004] The pathogen primarily infects tomato leaves and petioles, causing disease. In severe cases, it leads to stem rot, plant wilting, and browning of the fruit. These symptoms not only result in significant economic losses but also have adverse environmental impacts. The outbreak and prevalence of tomato late blight have seriously threatened tomato production in my country, significantly reducing tomato yields.

[0005] Biological control is a method of controlling harmful organisms by using beneficial organisms and their biological products. It is environmentally friendly and less likely to lead to pesticide resistance. Scholars both domestically and internationally are increasingly interested in this method, and its application is constantly expanding. The long-term reliance on chemical control for tomato late blight has many problems, including fostering pesticide resistance in pathogens, excessive pesticide residues, and pollution of the agricultural environment. Therefore, biological control, as an alternative, is gradually becoming a new strategy for controlling tomato late blight.

[0006] Currently, biological agents have become an effective means of controlling tomato late blight. Among them, plant extracts and microorganisms are the two most commonly used biological agents. Hou Haili et al.'s research showed that Artemisia capillaris extract has a strong inhibitory effect on tomato late blight pathogens, demonstrating protective and therapeutic effects of 66.21% and 60.01% respectively using in vivo tissue methods. Zhang Peixin et al.'s research found that extracts from Solanum nigrum and Solanum kaxiense had good inhibitory effects on the growth of tomato late blight pathogens, with inhibition rates of 54.00% and 51.58% respectively. Wu Qiong et al.'s research showed that Pseudomonas fluorescens strain YJ27 had a significant inhibitory effect on tomato late blight pathogens, with colony diameters significantly lower than the control, and an inhibition rate of 68.00%.

[0007] Swedish scientist Berzelius first discovered and named selenium (Se) during a sulfuric acid production experiment in 1817. However, as a byproduct of the experiment, it was considered a harmful substance for a long time. Research by Rofruek (1973) and Awosthi (1975) showed that selenium is not only a nutrient element involved in the synthesis of some metabolic processes in humans and animals, but also an important component of glutathione peroxidase (GSH-Px). Since then, people have changed their views on it and developed great interest, thus the importance of selenium has received increasing attention. However, whether the application of exogenous selenium affects the resistance of tomatoes to tomato late blight is not reported in current technology. Summary of the Invention

[0008] The purpose of this invention is to provide the application of exogenous selenium in improving resistance to late blight in tomatoes, in order to solve the problems existing in the prior art. Exogenous selenium can improve the resistance of tomatoes to late blight, and this invention provides a new method for the prevention and control of late blight in tomatoes.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] This invention provides the application of exogenous selenium in improving resistance to late blight in tomatoes, wherein the exogenous selenium includes Na2SeO3.

[0011] Optionally, the concentration of the exogenous selenium includes 0.025 mM.

[0012] Optionally, the exogenous selenium can improve the resistance of tomatoes to late blight by reducing the ROS content in tomato leaves.

[0013] Optionally, the exogenous selenium enhances the resistance of tomatoes to late blight by increasing the activity of SOD, POD, and CAT.

[0014] Optionally, the exogenous selenium can enhance the resistance of tomatoes to late blight by increasing chlorophyll content.

[0015] Optionally, the exogenous selenium can enhance the resistance of tomatoes to late blight by increasing the levels of the defense hormones SA and JA.

[0016] This invention also provides a product for improving resistance to late blight in tomatoes, wherein the active ingredient of the product is exogenous selenium;

[0017] The exogenous selenium includes Na2SeO3.

[0018] The present invention also provides a method for improving resistance to late blight in tomatoes, including the step of treating tomato plants with exogenous selenium.

[0019] Optionally, the exogenous selenium includes Na₂SeO₂. 3。

[0020] Optionally, the method for treating tomato plants includes: uniformly spraying the tomato plants with exogenous selenium at a concentration of 0.025 mM.

[0021] The present invention discloses the following technical effects:

[0022] Experimental verification revealed that, compared with the control group, exogenous selenium can inhibit disease development in tomato plants. Specifically, exogenous selenium can inhibit the expansion of late blight lesions on tomato leaves and alleviate H2O2 and O2 levels in the leaves. - The accumulation of exogenous selenium reduces the ROS content in tomato leaves; simultaneously, it increases the activity of antioxidant enzymes SOD, POD, and CAT, as well as chlorophyll content, and reduces MDA production, thereby improving plant growth vitality and disease resistance. Furthermore, spraying exogenous selenium can also increase the SA and JA content in tomato plants, as well as the selenium content in tomato leaves and fruits. In addition, tests have shown that exogenous selenium may enhance tomato resistance to late blight by inducing SIPR5 gene expression. In summary, this invention discovers that exogenous selenium can improve tomato resistance to late blight, providing a new method for the prevention and control of tomato late blight. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 A comparison of plant phenotypes after infection with Late Blight;

[0025] Figure 2 Results of NBT staining of tomato leaves;

[0026] Figure 3 Results of DAB staining of tomato leaves;

[0027] Figure 4 The effects of exogenous selenium treatment on the antioxidant system of tomatoes; A: SOD activity; B: POD activity; C: CAT activity; D: MDA content;

[0028] Figure 5 The effect of exogenous selenium on chlorophyll content in tomato leaves;

[0029] Figure 6 The effect of exogenous selenium treatment on the content of the tomato defense hormones SA (left panel) and JA (right panel);

[0030] Figure 7 The effect of exogenous selenium treatment on the relative expression levels of PR series genes in tomatoes; A: SIPR1; B: SIPR2; C: SIPR3; D: SIPR5. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Example 1

[0037] 1. Test materials

[0038] The tomato material used in the test was Moneymaker (LA2706, provided by TomatoGenetics Resource Center), a susceptible material that did not contain any disease-resistant genes. The pathogenic blight fungus was Phytophthora, provided by the Tomato Research Group of Northeast Agricultural University.

[0039] 2. Grouping and Inoculation with Pathogens

[0040] The experiment included a control group (CK, inoculated with only *Phytophthora blight*) and a treatment group (Se, inoculated with *Phytophthora blight* after applying exogenous 0.025 mM Na₂SeO₃). After uniform spraying with exogenous Na₂SeO₃, the seedlings were left to stand for 24 hours before inoculation with *Phytophthora blight*. Each treatment consisted of 20 tomato seedlings, with 3 biological replicates.

[0041] Late blight pathogens were inoculated into PDA medium and cultured upside down for 28 days in the dark at 20°C. Aerial mycelia were scraped from the surface, washed and soaked with sterile water, and then filtered through gauze to collect the bacterial suspension, which became a spore-containing suspension. The spores were observed and counted under a microscope using a hemocytometer, and the concentration of the spore suspension was adjusted based on the counting results until a concentration of 10⁻⁶ was reached. 6 The standard is / mL.

[0042] In the inoculation experiment of tomato seedlings, seedlings with similar growth conditions and at the 7-8 true leaf stage were first selected. To ensure effective spore infection, a humid environment was created before inoculation. Then, the spore suspension was sprayed finely and evenly onto each leaf of the seedling using a spraying method. This step was continued until spore suspension began to drip from the leaves to ensure comprehensive inoculation. After inoculation, the seedlings were immediately placed under a constant temperature of 20°C and the relative humidity was maintained at 100% to promote spore germination and infection. For the first 24 hours after inoculation, a completely dark environment was maintained, which facilitated the initial infection process. Subsequently, the seedlings were placed in an environment with alternating 12-hour light and 12-hour dark cycles to simulate the day-night cycle in the natural environment, to continue cultivating and observing seedling growth and disease development. Samples were then taken according to specific indicator requirements.

[0043] 3. DAB and NBT staining

[0044] Tomato seedlings were sampled 24 hours after exogenous spraying with Na2SeO3 (before inoculation with late blight pathogen), and this sample was recorded as 0 dpi (daypost infection). Subsequently, leaves were collected at 1 dpi and 3 dpi for DAB and NBT staining to determine H2O2 and O2. - The specific steps for accumulating the chlorophyll are as follows: First, thoroughly wash the leaves with distilled water to ensure that there are no residual impurities on the leaf surface. After washing, carefully transfer these clean leaves to pre-prepared test tubes and immerse them in DAB and NBT staining solutions respectively for 12 hours each at room temperature and in the dark. Then, transfer the leaves treated with DAB and NBT staining solutions to 95% ethanol solutions and subject these solutions to a boiling water bath for 10 to 15 minutes, shaking the centrifuge tubes every 3-5 minutes to ensure complete chlorosis. After removing the leaves, place them on a glass slide, add an appropriate amount of 50% glycerol, and cover with a coverslip, taking care to avoid air bubbles.

[0045] 4. Determination of antioxidant enzymes SOD, POD, CAT activity, MDA and chlorophyll content

[0046] Leaves from the CK and Se groups at 0 dpi, 1 dpi, 2 dpi, and 3 dpi were used for analysis. Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were accurately and rapidly measured using the SOD-WST-8 activity assay kits (ADS-W-KY011, ADS-W-KY003, and ADS-W-KY002) produced by Jiangsu Enzyme Immunoassay Co., Ltd. Malondialdehyde (MDA) content was determined using the MDA content assay kit (ADS-W-YH002-196). Chlorophyll content was determined using the chlorophyll content assay kit (ADS-W-GH001). This experiment was performed in triplicate.

[0047] 5. Determination of SA and JA content

[0048] Leaves from the CK and Se groups at 0 dpi, 1 dpi, 2 dpi, and 3 dpi were collected for measurement. The contents of salicylic acid (SA) and jasmonic acid (JA) were determined using the plant salicylic acid (SA) ELISA kit and the plant jasmonic acid (JA) ELISA kit, respectively, manufactured by Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0049] 6. Determination of Se content

[0050] Leaves and fruits from the CK and Se groups were collected separately for Se content determination. Following GB5009.268-2016 National Food Safety Standard – Determination of Multiple Elements in Food, the samples were digested using a pressure vessel digestion method, and then analyzed by inductively coupled plasma mass spectrometry. 5000 ICP-MS determination.

[0051] 7. Determination of relative expression levels of PR series genes

[0052] Leaves from the CK and Se groups at 0 dpi, 1 dpi, 2 dpi, and 3 dpi were collected, and RNA was extracted using the Trizol method and analyzed according to an RNA reverse transcription kit. The reverse transcription reaction was performed using IIQRT SuperMix for qPCR according to the instructions. See Table 1 for detailed qRT-PCR primer design. The experiment utilized... qPCR Green Master Mix (Vazyme, USA) and Quantstudio TM Real-Time PCR was performed using a 3D instrument (Thermo, USA). Gene expression levels were analyzed using a 2D instrument. -ΔΔCT Methods. The reaction system (total 20 μL) consisted of 5 parts: 1 μL cDNA template, 1 μL primer-F (10 μM), 1 μL primer-R (10 μM), 10 μL of Green Master Mix and 7 μL of RNase-Free Water were used. The qRT-PCR reaction program was: 95℃ for 3 min; 40 cycles: 95℃ for 5 s, 60℃ for 20 s, and 72℃ for 40 s.

[0053] Table 1 qRT-PCR primers

[0054]

[0055] 8. Data Statistics and Analysis

[0056] The collected data was organized using Excel software, data analysis was performed in the DPS data processing system software, and graphs were plotted using Graphpad Prism 9.5 software.

[0057] 9. Results

[0058] 9.1 Phenotypic Identification

[0059] Depend on Figure 1It can be seen that there are significant differences in the phenotypes of plants in the control group (CK) and the exogenous selenium-treated group (Se). The CK group showed relatively severe disease on the leaves, with water-soaked dark green lesions on the upper surface of the leaves and a white mold layer on the lower surface. The number of diseased leaves was also relatively higher. Compared with the CK group, the leaves of the tomato plants sprayed with exogenous selenium had fewer diseased leaves, smaller lesion areas, and significantly lower disease incidence, indicating that exogenous selenium treatment significantly improved the resistance of tomatoes to late blight.

[0060] 9.2 NBT and DAB staining results

[0061] By comparison Figure 2 The NBT staining results of the control group and the selenium-treated group showed that, compared with the CK group, the leaves of the selenium-treated group exhibited fewer blue spots and lower reactive oxygen species (ROS) staining intensity, indicating lower superoxide radical production and enhanced antioxidant capacity of the leaves. Through comparison... Figure 3 DAB staining results of the control group and the Se-treated group showed that, compared with the CK group, the leaves of the Se-treated group exhibited more brown precipitate, indicating that the tomato leaves in the treated group accumulated less H2O2. These results indicate that exogenous selenium treatment can inhibit the accumulation of H2O2 and O2 in tomato leaves. - The accumulation of these substances reduces the ROS content in tomato leaves.

[0062] 9.3 Effects of exogenous selenium on SOD, POD, CAT activity and MDA content

[0063] Depend on Figure 4 It can be seen that, compared with the CK group, the SOD activity in the Se treatment group was significantly increased, reaching its highest value at 3 dpi, which was 34.86% higher than that in the CK group; compared with the CK group, the POD activity in the Se treatment group was significantly increased, reaching its highest value at 1 dpi, which was 12.26% higher than that in the CK group, and then slightly decreased, possibly because the response was more intense in the early stage of infection by Late Pulmonaria; compared with the CK group, the CAT activity in the Se treatment group was significantly increased, reaching its highest value at 3 dpi, which was 17.48% higher than that in the CK group; the changes in MDA content in both the CK group and the Se treatment group showed a trend of first increasing and then decreasing. Compared with the CK group, the MDA content in the Se treatment group was significantly decreased, reaching its lowest value at 3 dpi, which was 12.19% lower than that in the CK group.

[0064] 9.4 Effects of exogenous selenium on chlorophyll content in tomatoes

[0065] Depend on Figure 5It was found that, compared with the control group, the chlorophyll content of the Se-treated group was increased, reaching its highest value at 2 dpi, which was 14.02% higher than that of the control group. These results indicate that exogenous selenium treatment can increase the chlorophyll content of tomato plants, enhance photosynthesis, and thus improve plant growth vitality and disease resistance.

[0066] 9.5 Effects of exogenous selenium on the content of tomato defense hormones SA and JA

[0067] Depend on Figure 6 It was found that, compared with the control group (CK), the SA content in the Se treatment group was significantly increased, and significantly higher than that in the CK group at 2 dpi and 3 dpi, exceeding it by 45.65% and 56.03%, respectively. The JA content in both the CK and Se treatment groups showed a trend of first increasing and then decreasing. Compared with the CK group, the JA content in the Se treatment group was significantly increased, exceeding that in the CK group by 58.76%, 35.32%, 42.12%, and 38.78% at 0 dpi, 1 dpi, 2 dpi, and 3 dpi, respectively, reaching its highest value at 1 dpi. These results indicate that the SA and JA content in tomato plants treated with exogenous selenium was significantly increased, enhancing the plant's defense response and resistance to pathogens, thereby improving disease resistance.

[0068] 9.6PR series gene relative expression levels

[0069] PR genes are a series of key genes in the tomato disease resistance response pathway. To investigate whether the expression of key genes in the tomato disease resistance response pathway is induced by Se, the expression of four PR genes in leaves of the CK group and the Se treatment group at 0, 1, 2, and 3 dpi was analyzed using qRT-PCR. Figure 7 The results showed that the SlPR5 gene was significantly upregulated in the exogenous selenium-induced resistance to late blight in tomatoes, reaching its highest expression level at 3 dpi, which was 1.76 times that of the control (CK). Meanwhile, SIPR1, SIPR2, and SIPR3 showed downregulated expression. These results indicate that exogenous selenium treatment may enhance tomato resistance to late blight by inducing SIPR5 gene expression.

[0070] 9.7 Effects of exogenous selenium on selenium content in tomato leaves and fruits

[0071] Table 2 shows that in the leaves, the selenium content in the Se-treated group was significantly higher than that in the control group, increasing from 0.05 mg / kg to 5.11 mg / kg. This indicates that exogenous selenium treatment can significantly increase the selenium content in tomato leaves. Similarly, in the fruit, the selenium content in the Se-treated group was also higher than that in the control group, increasing from 4.00 mg / kg to 7.08 mg / kg. This also demonstrates that exogenous selenium treatment can significantly increase the selenium content in tomato fruit.

[0072] Table 2 Effects of exogenous selenium on Se content in tomato leaves and fruits

[0073]

[0074] In summary, compared with the control group, exogenous selenium can inhibit the disease occurrence in tomato plants. Specifically, exogenous selenium can inhibit the growth of H2O2 and O2 in tomato leaves. - The accumulation of selenium reduces the ROS content in tomato leaves; it can increase the activity of antioxidant enzymes SOD, POD, and CAT in tomato plants and reduce MDA production; it can increase the chlorophyll content in tomato plants, enhance photosynthesis, and thus improve plant growth vitality and disease resistance; it can increase the SA and JA content in tomato plants, enhance the plant's defense response, and thus improve the plant's resistance to pathogens and disease resistance; at the same time, exogenous selenium can increase the selenium content in tomato leaves and fruits; and this invention also found that exogenous selenium may improve the resistance of tomatoes to late blight by inducing the expression of the SIPR5 gene. In summary, this invention has discovered that exogenous selenium can improve the resistance of tomatoes to late blight, and this invention provides a new method for the prevention and control of tomato late blight.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of exogenous selenium in improving resistance to late blight in tomatoes, characterized in that, The exogenous selenium includes Na2SeO3; The exogenous selenium enhances the resistance of tomatoes to late blight by increasing the levels of the defense hormones SA and JA.

2. The application as described in claim 1, characterized in that, The concentration of the exogenous selenium includes 0.025 mM.

3. A method for improving resistance to late blight in tomatoes, characterized in that, This includes the step of treating tomato plants with exogenous selenium; The exogenous selenium includes Na2SeO3; The exogenous selenium enhances the resistance of tomatoes to late blight by increasing the levels of the defense hormones SA and JA.

4. The method as described in claim 3, characterized in that, The method for treating tomato plants includes: uniformly spraying the tomato plants with an exogenous selenium concentration of 0.025 mM.

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