Application of exogenous selenium in improvement of tomato late blight resistance

Abstract

The invention discloses application of exogenous selenium to improvement of tomato late blight resistance, and belongs to the field of plant disease control. The exogenous selenium comprises Na2SeO3 (sodium selenite). Compared with a control group, exogenous selenium can inhibit the morbidity of tomato plants. Specifically, the exogenous selenium can inhibit the expansion of tomato leaf late blight scabs, relieve the accumulation of H2O2 and O2 <-> in the leaves and reduce the ROS content in the tomato leaves; meanwhile, the activity of antioxidant enzymes SOD, POD and CAT and the content of chlorophyll can be improved, and generation of MDA is reduced; besides, the content of SA and JA of tomato plants and the content of selenium in tomato leaves and fruits can also be increased by spraying exogenous selenium; in addition, through detection, exogenous selenium can improve the resistance of tomatoes to phytophthora infestans by inducing expression of the SIPR5 gene. In conclusion, the invention finds that the exogenous selenium can improve the resistance of the tomato to the late blight, and provides a new method for preventing and treating the tomato late blight.

Description

Technical Field

[0001] The present invention relates to the field of plant disease control, and particularly to the application of exogenous selenium in enhancing the resistance of tomatoes to late blight. Background Art

[0002] Tomato (Solanum lycopersicum L.), belonging to the Solanaceae family and the Solanum genus, is also known as tomato, and was also called June persimmon in ancient times. It is an annual or perennial herbaceous plant. Tomatoes are rich in nutrients, including carotenoids, vitamin C, etc., which are very beneficial to human health.

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

[0004] The pathogenic bacteria mainly cause diseases by infecting tomato leaves and petioles. Seriously, it will cause the stems of tomatoes to start to rot, the plants to wilt, and the fruits to turn brown. These symptoms will not only cause serious economic losses, but also have an adverse impact on the environment. The outbreak and prevalence of tomato late blight have seriously threatened the production of tomatoes in China and greatly reduced the yield of tomatoes.

[0005] Biological control is a method of controlling harmful organisms by using beneficial organisms and their biological products. It is environmentally friendly and not prone to drug resistance. Scholars at home and abroad have increasing research interests in this method, and its application scope is also constantly expanding. There are many problems in the long-term reliance on chemical control for the prevention and control of tomato late blight, including promoting the generation of drug resistance of pathogenic bacteria, exceeding the standard of pesticide residues, and polluting the agricultural ecological environment. Therefore, biological control, as an alternative solution, is gradually becoming a new strategy for controlling tomato late blight.

[0006] At present, biological agents have become an effective means for the prevention and control of tomato late blight. Among them, plant extracts and microorganisms are the two most commonly used biological agents. The research by Hou Haili et al. showed that Artemisia capillaris extract has a strong inhibitory effect on Phytophthora infestans of tomato. The in vivo tissue method showed that the protective efficacy and therapeutic efficacy of Artemisia capillaris extract against late blight reached 66.21% and 60.01% respectively. The research by Zhang Peixin et al. found that the extracts of Solanum coagulans and Solanum aculeatissimum have a good inhibitory effect on the growth of Phytophthora infestans of tomato, and the inhibition rates reached 54.00% and 51.58% respectively. The research by Wu Qiong et al. showed that the Pseudomonas fluorescens strain YJ27 has an obvious inhibitory effect on Phytophthora infestans of tomato. The diameter of the colony growth was significantly lower than that of the control, and the inhibition rate reached 68.00%.

[0007] In 1817, the Swedish scientist Berzelius first discovered the trace element selenium (Se) during a sulfuric acid production experiment and named it. However, since it was a by-product of the experiment, it was considered a harmful substance for a long time. The research by Rofruek (1973) and Awosthi (1975) showed that selenium is not only a nutrient element involved in partial metabolic synthesis in humans and animals, but also an important component of glutathione peroxidase (GSH-Px). Since then, people have changed their views on it and shown great interest. Therefore, the importance of selenium has received more and more attention. However, whether the application of exogenous selenium will affect the resistance of tomatoes to tomato late blight has not been reported in the existing technology. Summary of the Invention

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

[0009] To achieve the above purpose, the present invention provides the following solutions:

[0010] The present invention provides the application of exogenous selenium in improving the resistance of tomatoes to late blight, and the exogenous selenium includes Na 2 SeO 3 .

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

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

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

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

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

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

[0017] The exogenous selenium includes Na 2 SeO 3 .

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

[0019] Optionally, the exogenous selenium includes Na 2 SeO 3。

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

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

[0022] Verified by experiments, the present invention finds that, compared with the control group, exogenous selenium can inhibit the disease incidence of tomato plants. Specifically, exogenous selenium can inhibit the expansion of late blight lesions on tomato leaves, relieve the accumulation of H 2 O 2 , O 2 - , reduce the ROS content in tomato leaves; at the same time, it can increase the activities of antioxidant enzymes SOD, POD, CAT and the chlorophyll content, reduce the production of MDA, thereby enhancing the growth vitality and disease resistance of plants; and spraying exogenous selenium can also increase the contents of SA and JA in tomato plants and the selenium content in tomato leaves and fruits; in addition, through detection, exogenous selenium may enhance the resistance of tomatoes to Phytophthora infestans by inducing the expression of the SIPR5 gene. In summary, the present invention discovers that exogenous selenium can enhance the resistance of tomatoes to late blight, and the present invention provides a new method for the prevention and control of tomato late blight. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings based on these drawings without creative efforts.

[0024] Figure 1 For comparison of plant phenotypes after infection with Phytophthora infestans;

[0025] Figure 2 For the results of NBT staining of tomato leaves;

[0026] Figure 3 For the results of DAB staining of tomato leaves;

[0027] Figure 4 For 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 For the effects of exogenous selenium on the chlorophyll content of tomato leaves;

[0029] Figure 6 For the effects of exogenous selenium treatment on the contents of defense hormones SA (left figure) and JA (right figure) in tomatoes;

[0030] Figure 7 For the effects 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 manners

[0031] Now, various exemplary implementation manners of the present invention will 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, characteristics, and implementation schemes of the present invention.

[0032] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention's specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0035] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0036] Example 1

[0037] 1. Test materials

[0038] The tomato material for testing was the susceptible material Moneymaker (LA2706, provided by the Tomato Genetics Resource Center) without any disease-resistant genes. The Phytophthora infestans causing tomato late blight was provided by the tomato research group of Northeast Agricultural University.

[0039] 2. Grouping and pathogen inoculation

[0040] The experiment included a control group CK (only inoculated with Phytophthora infestans) and a treatment group Se (exogenous 0.025 mM Na 2 SeO 3 followed by inoculation with Phytophthora infestans). After the exogenous Na 2 SeO 3 was evenly sprayed and left to stand for 24 h, it was ready for inoculation with Phytophthora infestans. There were 20 tomato seedlings in each treatment, with 3 biological replicates.

[0041] The Phytophthora infestans strain was inoculated into PDA medium and cultured in the dark at 20 °C. After 28 days of inverted culture, the aerial mycelium on the surface was scraped off, washed and soaked with sterile water, and then filtered with gauze to collect the bacterial liquid. The obtained bacterial liquid was a suspension containing spores. The spores were observed and counted using a hemocytometer under a microscope, and the concentration of the spore suspension was adjusted according to the counting results until the concentration reached 10 6 / mL standard.

[0042] When conducting the inoculation experiment on tomato seedlings, first select tomato seedlings with similar growth conditions and at the stage of 7-8 true leaves. To ensure that the spores can effectively infect the seedlings, it is necessary to create a humid environment before inoculation. Subsequently, the spore suspension is carefully and evenly sprayed on each leaf of the seedlings by the spraying method, and this step should continue until the spore suspension starts to drip from the leaves to ensure the comprehensiveness of inoculation. After inoculation, the seedlings should be immediately placed under a constant temperature of 20°C, and the relative humidity of the environment should be maintained at 100% to promote the germination and infection of the spores. Within the first 24 hours after inoculation, a completely dark environment should be maintained, which helps the initial infection process of the pathogen. Thereafter, the seedlings should be placed in an environmental cycle of 12 hours of light and 12 hours of darkness, simulating the day-night changes in the natural environment, to continue culturing and observing the growth and disease development of the seedlings. Subsequently, samples are taken according to specific index requirements.

[0043] 3. DAB and NBT staining

[0044] Exogenous spraying of Na 2 SeO 3 Samples are taken 24 hours later (before inoculating Phytophthora infestans), and this sampling is recorded as 0 dpi (day post infection). Subsequently, leaves at 1 dpi and 3 dpi are taken respectively for DAB and NBT staining to determine the accumulation amounts of H 2 O 2 and O 2 - as follows: First, thoroughly wash the leaves with distilled water to ensure that there is no residual impurity on the leaf surface; after washing, carefully transfer these clean leaves to pre-prepared test tubes, and at room temperature and in the dark, soak the leaves in DAB and NBT staining solutions respectively for 12 hours. Subsequently, transfer the leaves treated with DAB and NBT staining solutions to 95% ethanol solutions respectively, and perform a boiling water bath treatment on these solutions for 10 to 15 minutes. During this period, shake the centrifuge tube every 3-5 minutes to completely decolorize the leaves; after taking out 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 the activities of antioxidant enzymes SOD, POD, CAT, MDA and chlorophyll content

[0046] Leaves of the CK group and Se group at 0 dpi, 1 dpi, 2 dpi, and 3 dpi were taken for determination respectively. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were accurately and rapidly determined using the SOD-WST-8 method activity assay kit ADS-W-KY011, peroxidase kit ADS-W-KY003, and catalase kit ADS-W-KY002 in the enzyme immunoassay series produced by Jiangsu Enzyme Immunoassay Industry Co., Ltd. respectively. The content of malondialdehyde (MDA) was determined using the malondialdehyde content kit ADS-W-YH002-196. The chlorophyll content was determined using the chlorophyll content assay kit ADS-W-GH001. This experiment was set with 3 replicates in total.

[0047] 5. Determination of SA and JA Contents

[0048] Leaves of the CK group and Se group at 0 dpi, 1 dpi, 2 dpi, and 3 dpi were taken for determination respectively. The determination of salicylic acid (SA) and jasmonic acid (JA) contents was completed using the plant salicylic acid (SA) ELISA detection kit and plant jasmonic acid (JA) ELISA detection kit produced by Shanghai Enzyme-linked Biotechnology Co., Ltd. respectively.

[0049] 6. Determination of Se Content

[0050] Leaves and fruits of the CK group and Se group were taken for the determination of Se content respectively. Referring to GB5009.268-2016 National Food Safety Standard Determination of Multiple Elements in Foods, after the samples were digested by the pressure tank digestion method, they were determined by an inductively coupled plasma mass spectrometer 5000 ICP-MS.

[0051] 7. Determination of the Relative Expression Levels of PR Series Genes

[0052] Leaves of the CK group and Se group at 0 dpi, 1 dpi, 2 dpi, and 3 dpi were taken respectively. RNA was extracted using the Trizol method, and reverse transcription reaction was carried out according to the instruction manual of the RNA reverse transcription kit IIQRT SuperMix for qPCR. The design of qRT-PCR primers is shown in Table 1 in detail. The experiment was carried out using qPCR Green Master Mix (Vazyme, USA) and Quantstudio TM 3 Real-Time PCR Instrument (Thermo, USA). The analysis of gene expression levels was carried out using 2 -ΔΔCTMethod. The reaction system (20 μL in total) consists of five parts: 1 μL of cDNA template, 1 μL of primer-F (10 μM), 1 μL of primer-R (10 μM), 10 μL of Green Master Mix and 7 μL of RNase-Free Water. The qRT-PCR reaction procedure is as follows: 95°C for 3 min; 40 cycles of reaction: 95°C for 5 s, 60°C for 20 s, 72°C for 40 s.

[0053] Table 1 qRT-PCR primers

[0054]

[0055] 8. Data statistics and analysis

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

[0057] 9. Results

[0058] 9.1 Phenotypic identification

[0059] It can be seen from Figure 1 that there are obvious differences in the phenotypes of the control group CK and the exogenous selenium-treated group Se plants. The tomato leaves in the CK group were relatively severely diseased, with water-soaked dark green lesions on the front of the leaves and white mold layers on the back of the leaves, and the number of diseased leaves was also relatively large; compared with the CK group, the tomato leaves sprayed with exogenous selenium had fewer diseased leaves and smaller lesion areas, and the disease incidence was significantly lower than that of the control group, indicating that exogenous selenium treatment significantly improved the resistance of tomatoes to late blight.

[0060] 9.2 NBT and DAB staining results

[0061] By comparing Figure 2 the NBT staining results of the control group and the selenium-treated group in Figure 3 , compared with the CK group, the leaves of the Se-treated group showed fewer blue spots and lower staining intensity of reactive oxygen species (ROS), indicating a lower production of superoxide ion radicals and enhanced antioxidant capacity of the leaves; by comparing 2 O 2 the DAB staining results of the control group and the Se-treated group in 2 O 2 , compared with the CK group, the leaves of the Se-treated group showed more brown precipitates, indicating less accumulation of H 2 - O in the treated tomato leaves. The above results indicate that exogenous selenium treatment can inhibit the accumulation of H 2 O 2 , O 2 - in tomato leaves and reduce the ROS content in tomato leaves.

[0062] 9.3 Effects of Exogenous Selenium on the Activities of SOD, POD, CAT and the Content of MDA

[0063] As Figure 4 can be seen, compared with the CK group, the SOD activity in the Se treatment group increased significantly and reached the highest value at 3 dpi, with an increase of 34.86% compared to the CK group; compared with the CK group, the POD activity in the Se treatment group increased significantly and reached the highest value at 1 dpi, 12.26% higher than the CK group, and then decreased slightly, possibly because the response was more intense in the early stage of Phytophthora infestans infection; compared with the CK group, the CAT activity in the Se treatment group increased significantly and reached the highest value at 3 dpi, 17.48% higher than the CK group; the 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 decreased significantly and reached the lowest value at 3 dpi, 12.19% lower than the CK group.

[0064] 9.4 Effects of Exogenous Selenium on the Chlorophyll Content of Tomatoes

[0065] As Figure 5 can be seen, compared with the CK group, the chlorophyll content in the Se treatment group increased, and reached the highest value at 2 dpi, 14.02% higher than the CK group. The above results indicate that the tomato plants treated with exogenous selenium can increase the chlorophyll content, enhance the photosynthesis of plants, and thus improve the growth vitality and disease resistance of plants.

[0066] 9.5 Effects of Exogenous Selenium on the Contents of Defense Hormones SA and JA in Tomatoes

[0067] As Figure 6 can be seen, compared with the CK group, the SA content in the Se treatment group increased significantly and was significantly higher than the CK group at 2 dpi and 3 dpi, 45.65% and 56.03% higher respectively; the JA 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 JA content in the Se treatment group increased significantly, 58.76%, 35.32%, 42.12% and 38.78% higher than the CK group at 0 dpi, 1 dpi, 2 dpi and 3 dpi respectively, and reached the highest value at 1 dpi. The above results indicate that the contents of SA and JA in tomato plants treated with exogenous selenium increased significantly, enhancing the plant's defense response, improving the plant's resistance to pathogens, and thus improving disease resistance.

[0068] 9.6 Relative Expression Levels of PR Series Genes

[0069] PR genes are a series of key genes in the disease resistance response pathway of tomatoes. To explore whether the key genes in the tomato disease resistance response pathway are induced by Se, qRT-PCR was used to analyze the expression of four PR genes in the leaves of the CK group and the Se-treated group at 0, 1, 2, and 3 dpi. As Figure 7 can be seen, the SlPR5 gene was significantly up-regulated in the reaction of exogenous selenium-induced tomato resistance to late blight, and the expression level reached the highest at 3 dpi, which was 1.76 times that of CK. While SIPR1, SIPR2, and SIPR3 were down-regulated. The results showed that exogenous selenium treatment might improve the resistance of tomatoes to Phytophthora infestans by inducing the expression of the SIPR5 gene.

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

[0071] As can be seen from Table 2, in the leaves, the selenium content of the Se-treated group was significantly higher than that of the CK group, and the Se content increased from 0.05 mg / kg to 5.11 mg / kg, indicating that exogenous selenium treatment of tomato plants could significantly increase the selenium content in tomato leaves. In the fruits, the selenium content of the Se-treated group was also higher than that of the CK group, and the Se content increased from 4.00 mg / kg to 7.08 mg / kg, which also indicated that exogenous selenium treatment of tomato plants could significantly increase the selenium content in tomato fruits.

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

[0073]

[0074] Based on the above results, compared with the control group, exogenous selenium could inhibit the disease incidence of tomato plants. Specifically, exogenous selenium could inhibit the accumulation of H 2 O 2 and O 2 - in tomato leaves, reduce the ROS content in tomato leaves; could increase the activities of antioxidant enzymes SOD, POD, and CAT in tomato plants, reduce the production of MDA; could increase the chlorophyll content of tomato plants, enhance the photosynthesis of plants, thereby improving the growth vitality and disease resistance of plants; could increase the SA and JA contents of tomato plants, enhance the defense response of plants, thereby improving the resistance of plants to pathogens and enhancing disease resistance; at the same time, exogenous selenium could increase the selenium content in tomato leaves and fruits of tomato plants; and, the present invention found that exogenous selenium might improve the resistance of tomatoes to Phytophthora infestans by inducing the expression of the SIPR5 gene. In summary, the present invention found that exogenous selenium could improve the resistance of tomatoes to late blight, and the present invention provided a new method for the prevention and control of tomato late blight.

[0075] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. The application of exogenous selenium in improving resistance to tomato late blight, characterized in that: The exogenous selenium includes Na2SeO3.

2. The use according to claim 1, characterized in that The concentration of the exogenous selenium includes 0.025 mM.

3. The use according to claim 1, characterized in that The exogenous selenium improves the resistance of tomatoes to late blight by reducing the ROS content in tomato leaves.

4. The use according to claim 1, characterized in that The exogenous selenium improves the resistance of tomatoes to late blight by increasing the activities of SOD, POD and CAT.

5. The use according to claim 1, characterized in that The exogenous selenium improves the resistance of tomatoes to late blight by increasing the chlorophyll content.

6. The use according to claim 1, characterized in that The exogenous selenium improves the resistance of tomatoes to late blight by increasing the contents of defense hormones SA and JA.

7. A product for improving resistance to tomato late blight, characterized in that: The active ingredient of the product is exogenous selenium; The exogenous selenium includes Na2SeO3.

8. A method for improving resistance to tomato late blight, characterized in that: The method comprises the steps of treating tomato plants with exogenous selenium.

9. The method according to claim 8, characterized in that The exogenous selenium includes Na2SeO3.

10. The method according to claim 8, characterized in that The method for treating tomato plants comprises: uniformly spraying exogenous selenium with a concentration of 0.025 mM on the tomato plants.

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