Preparation method and application of culture medium for improving high temperature resistance of tomatoes

Abstract

The invention discloses a preparation method and application of a culture medium for improving high temperature resistance of tomatoes, and belongs to the technical field of horticultural crop protection. It is found that when a magnesium-based hydrogen storage material MgH2 with a specific concentration and the culture medium are mixed and applied to roots of the tomatoes, the tolerance of tomato seedlings under high temperature stress can be remarkably enhanced; the invention further provides a preparation method of the culture medium for improving the high temperature resistance of the tomatoes, homogeneous culture soil with the MgH2 concentration being 5 mg / kg is prepared through a step-by-step dilution and mixing technology, after the medium is applied, high-temperature damage can be effectively relieved through a synergistic mechanism, and the method is easy and convenient to operate, environmentally friendly and suitable for industrial production. And a new effective technical means is provided for prevention and control of tomato high temperature stress.

Description

Technical Field

[0001] This invention relates to the field of horticultural crop protection technology, specifically to a method for preparing and applying a cultivation substrate that improves the high-temperature resistance of tomatoes. Background Technology

[0002] With the intensification of global climate change and the continuous rise in ambient temperature, the abiotic stress caused by high temperature to plants is becoming increasingly severe. High temperature stress disrupts the balance between the production and scavenging of reactive oxygen species (ROS) in plant cells, leading to the excessive accumulation of ROS such as superoxide anions and hydrogen peroxide, and inducing lipid peroxidation, which damages the integrity of cell membrane structure. This process is usually manifested as an increase in relative conductivity and an increase in malondialdehyde content (Noctor G, Mhamdi A, Foyer C H. Theroles of reactive oxygen metabolism in drought: Not so cut and dried[J].Plant Physiology, 2014, 164(4): 1636-1648. Mhamdi A, Noctor G F. Reactiveoxygen species in plant development and stress responses[J]. Annual Review of Plant Biology, 2016, 67: 219-246.). In addition, high temperatures can significantly inhibit plant photosynthesis, damage the heat-sensitive thylakoid membrane structure, reduce the activity of photosystem II (PSII), decrease the maximum photochemical efficiency, and ultimately lead to a weakening of plant growth potential and a severe decline in yield.

[0003] High temperatures pose a serious threat to the growth, development, yield, and quality of tomatoes (Solanum lycopersicum L.). Originating in the Andes Mountains of South America, tomatoes are an important economic crop with an optimal growth temperature of 20–25°C. Under high-temperature stress, tomatoes exhibit a series of physiological abnormalities, including inhibited pollen tube development, decreased pollen viability, weakened photosynthetic capacity, and excessive ROS accumulation, leading to flower and fruit drop and severely reducing yield and quality. Therefore, exploring effective ways to improve the high-temperature resistance of tomatoes has become a research hotspot.

[0004] Hydrogen (H2), as a novel green gaseous signaling molecule, possesses the antioxidant capacity to selectively scavenge hydroxyl radicals, and its role in plant responses to abiotic stress is gradually attracting attention. Previous studies have shown that H2 can alleviate the damage to plants from various abiotic stresses such as drought, salinity, heavy metals, and low temperature by regulating plant hormone signals (such as ABA and JA), activating antioxidant defense systems, and regulating the expression of stress-related genes. (Jin Q, Zhu K, Cui W, et al. Hydrogen-modulated stomatal sensitivity to abscisic acid and drought tolerance via the regulation of apoplastic pH in Medicago sativa[J]. Journal of Plant Growth Regulation, 2016, 35(2): 565-573. Xie Y, Mao Y, Lai D, et al. H2 enhances Arabidopsis salt tolerance by manipulating ZAT10 / 12-mediated antioxidant defense and controlling sodium exclusion[J]. PLoS ONE, 2012, 7(11): e49800. Xie Y, Mao Y, Zhang W, et al. Reactive oxygen species-dependent nitric oxide production contributes to hydrogen-promoted stomatal closure.) In Arabidopsis[J]. Plant Physiology, 2014, 165(2): 759-773.). Magnesium-based hydrogen storage material MgH2, as an environmentally friendly H2 donor, does not require external energy for its hydrolysis process, and the H2 release rate can be slowed and sustained by adjusting the concentration, making it suitable for agricultural field applications.Previous studies have shown that the magnesium-based hydrogen storage material MgH2 can maintain redox balance in mung beans under osmotic stress by increasing ascorbic acid (AsA) content and enhancing the activity of key enzymes such as ascorbate peroxidase (APX) and glutathione reductase (GR), thereby promoting root growth and biomass accumulation (Zhang Y, Lu X, Yao W, et al. Magnesium Hydride Confers Osmotic Tolerance in Mung Bean Seedlings by Promoting Ascorbate–Glutathione Cycle[J]. Plants, 2024, 13(19): 2819.). However, whether the magnesium-based hydrogen storage material MgH2 participates in regulating the resistance of tomatoes to high-temperature stress has not yet been reported. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to address the shortcomings of existing technologies by providing a method for preparing and applying a cultivation substrate that improves the high-temperature resistance of tomatoes. By treating tomatoes with exogenous substances, the high-temperature resistance of tomatoes is enhanced, which is of great significance for achieving stable and high-quality tomato cultivation.

[0006] Technical solution: The present invention provides a method for preparing a cultivation substrate that improves the heat resistance of tomatoes, comprising the following steps:

[0007] S1. Mix the magnesium-based hydrogen storage material MgH2 solid powder with the first part of the dry matrix to make a primary parent material. The amount of magnesium-based hydrogen storage material MgH2 solid powder is 25mg, and the amount of the first part of the dry matrix is ​​500g.

[0008] S2. Mix the primary parent soil with the second part of the dried matrix to make the secondary parent soil. The second part of the dried matrix is ​​1500g.

[0009] S3. Mix the secondary parent soil with the third part of the dried substrate. The third part of the dried substrate is 3000g. Finally, a total of 5kg of magnesium-based hydrogen storage material MgH2 cultivation substrate with a concentration of 5mg / kg is obtained.

[0010] This invention addresses the challenge of uniform distribution when applying trace amounts of MgH2 using a "stepwise dilution method." Because the amount of MgH2 used is extremely small (only 25 mg in total), direct mixing with 5 kg of soil can easily lead to uneven mixing, resulting in locally excessively high or low concentrations, thus affecting the reproducibility of experimental or application results. This three-step method, by gradually increasing the mixing volume, ensures statistically significant uniform dispersion of the extremely low concentration of MgH2 in a large soil matrix.

[0011] Furthermore, in step S2, the primary parent soil and the second part of the dry matrix are mixed for more than 15 minutes, providing sufficient mechanical mixing time and energy.

[0012] The cultivation substrate prepared according to the above method is used to improve the high temperature resistance of tomatoes. Because the MgH2 in the cultivation substrate reacts slowly with soil moisture, it continuously and in low doses releases hydrogen (H2). As a gaseous signal molecule, H2 is absorbed by the tomato roots and triggers a series of cascade physiological and biochemical reactions in the plant, thereby systematically improving the plant's heat resistance.

[0013] Furthermore, the application involves applying the cultivation substrate to the tomato roots; the roots are the main organs for plants to absorb water and mineral nutrients, and also a key site for sensing soil environmental signals. Applying a cultivation substrate containing MgH2 to the roots ensures that the hydrogen production reaction occurs in the rhizosphere microenvironment, and the released H2 can be directly absorbed by the roots or act on root cells.

[0014] Furthermore, the cultivation substrate enhances the high-temperature resistance of tomatoes through at least one of the following mechanisms: maintaining cell membrane homeostasis, protecting the photosynthetic system, enhancing the activity of osmotic regulation and antioxidant systems, and upregulating the expression of the heat shock factor HSF gene.

[0015] Beneficial effects: Compared with the prior art, the advantages of the present invention are as follows:

[0016] (1) This invention can effectively alleviate the damage of high temperature stress to tomato seedlings, and the tomatoes grow well;

[0017] (2) Through the treatment of the present invention, heat damage and water loss of tomato seedling leaves can be reduced;

[0018] (3) Through the treatment of the present invention, the total chlorophyll content, chlorophyll a and chlorophyll b in the leaves of tomato seedlings are significantly increased; the treatment of the present invention can better alleviate the damage of heat stress to the photosynthetic system II (PSII) of tomato leaves and enhance the heat resistance of tomato seedlings.

[0019] (4) Through the treatment of the present invention, the activity of antioxidant enzymes (SOD, POD, CAT) in tomato leaves can be increased and the content of growth regulators related to stress resistance (MDA and H2O2) can be reduced.

[0020] (5) The application method of the present invention is simple and effective. Attached Figure Description

[0021] Figure 1The phenotypes of tomato plants treated with 0 mg / kg MgH2 and 5 mg / kg MgH2 at room temperature (25℃) and high temperature (42℃) for two days are shown. Higher leaf wilting indicates more severe damage from high-temperature stress. 0 mg / kg MgH2 represents soil without added MgH2; 2.5 mg / kg MgH2 represents soil with a concentration of 2.5 mg / kg MgH2; 5 mg / kg MgH2 represents soil with a concentration of 5 mg / kg MgH2; and 10 mg / kg MgH2 represents soil with a concentration of 10 mg / kg MgH2.

[0022] Figure 2 The relative moisture content of tomatoes after two days of external application of 0 mg / kg MgH2 and 5 mg / kg MgH2 at room temperature (25℃) and high temperature (42℃) is given. Figure 2 A) and thermal damage index ( Figure 2 B); where, the lower the relative water content, the more severe the damage under high temperature stress; the higher the thermal damage index, the more severe the damage under high temperature stress. Figure 2 Different letters on the bars in the bar chart indicate that the differences between treatments reached a significant level (P < 0.05); 25℃ 0mg indicates that tomatoes were treated with 0mg / kg MgH2 at room temperature for two days; 25℃ 5mg indicates that tomatoes were treated with 5mg / kg MgH2 at room temperature for two days; 42℃ 0mg indicates that tomatoes were treated with 0mg / kg MgH2 at high temperature for two days; 42℃ 5mg indicates that tomatoes were treated with 5mg / kg MgH2 at high temperature for two days.

[0023] Figure 3 The chlorophyll fluorescence parameters of tomato leaves after two days of external application of 0 mg / kg MgH2 and 5 mg / kg MgH2 at room temperature (25℃) and high temperature (42℃) are compared. Figure 3 A) and Fv / Fm ( Figure 3 B); Figure 3 In the B-bar chart, different letters on the bars indicate that the differences between treatments reached a significant level (P < 0.05); Fv / Fm represents the maximum photochemical efficiency.

[0024] Figure 4 The total chlorophyll content of tomato leaves after two days of external application of 0 mg / kg MgH2 and 5 mg / kg MgH2 at room temperature (25℃) and high temperature (42℃) is calculated. Figure 4 A), Chlorophyll a ( Figure 4 B), Chlorophyll b ( Figure 4 C) and carotenoids ( Figure 4 D) Content; different letters on the bars in the bar chart indicate that the differences between treatments reached a significant level (P < 0.05);

[0025] Figure 5The changes in the oxidation system of tomato leaves after two days of external application of 0 mg / kg MgH2 and 5 mg / kg MgH2 at room temperature (25℃) and high temperature (42℃); among them, Figure 5 BF represents the MDA content, H2O2 content, SOD activity, POD activity, and CAT activity in the leaves, respectively; different letters on the bars in the bar chart indicate that the differences between treatments reached a significant level (P < 0.05); 0d, 1d, and 2d represent 0 days, 1 day, and 2 days, respectively;

[0026] Figure 6 The relative expression levels of heat-response genes in tomatoes treated with 0 mg / kg MgH2 and 5 mg / kg MgH2 at room temperature (25℃) and high temperature (42℃) for two days are shown. Different letters on the bars in the bar chart indicate that the differences between treatments reached a significant level (P < 0.05). 0d, 1d and 2d are day 0, day 1 and day 2, respectively. Detailed Implementation

[0027] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings; however, the scope of protection of the present invention is not limited to the described embodiments. The dry substrate mentioned herein is a common planting substrate, which can be purchased commercially.

[0028] Example 1: Optimization of MgH2 Concentration

[0029] To investigate the effects of exogenous MgH2 on the heat stress tolerance of tomato seedlings and to determine its optimal concentration for alleviating heat stress, this study set up concentrations of 0, 2.5, 5, and 10 mg / kg. -1 Four concentration gradients were applied, and after 2 days of exposure to ambient temperature (25℃) and high temperature stress (42℃), the growth status of tomato seedlings was observed. Figure 1 The results showed that under normal temperature control (25℃), different concentrations of MgH2 treatment had no significant effect on the phenotype of tomato seedlings; while under high temperature stress (42℃), the control group without MgH2 (0 mg·kg⁻¹) showed no significant effect. -1 The plants showed obvious wilting and yellowing of leaves. Under high temperature stress (42℃), 5 mg·kg -1 The MgH2 treatment group showed the best plant growth and good leaf expansion; 2.5 mg·kg -1 and 10mg·kg -1 While the treatment group also alleviated heat stress, the effect was less than that of 5 mg·kg⁻¹. -1 Group, 10 mg·kg -1 The treatment group even showed slight signs of growth inhibition.

[0030] Example 2: Effect of external application of 5 mg / kg MgH2 on the growth status of tomatoes under high temperature stress.

[0031] After two days of treatment with ambient temperature (25℃) and high temperature (42℃), the growth status of tomato plants treated with 0 mg / kg MgH2 and 5 mg / kg MgH2 was observed. The results are as follows: Figure 1 As shown, under normal temperature control (25℃), there was no significant difference in phenotype between tomato plants treated with 0 mg / kg MgH2 and those treated with 5 mg / kg MgH2; however, under high temperature stress (42℃), tomato plants treated with 0 mg / kg MgH2 showed obvious wilting and yellowing of leaves, while tomato plants treated with 5 mg / kg MgH2 showed good growth.

[0032] Further investigation was conducted on the relative water content and heat damage index of tomatoes in different treatment groups. The results showed that at 25℃, the relative water content of the 5 mg / kg MgH2 treatment group was approximately 95%, significantly higher than that of the 0 mg / kg MgH2 treatment group (approximately 85%), indicating that external application of 5 mg / kg MgH2 at room temperature can improve the water retention capacity of tomato seedlings. However, at 42℃, the relative water content of the 5 mg / kg MgH2 treatment group was approximately 65%, still significantly higher than that of the 0 mg / kg MgH2 treatment group (approximately 55%). Figure 2 (A) indicates that under high-temperature stress, external application of 5 mg / kg MgH2 can effectively alleviate water loss in tomato seedlings and maintain higher tissue water content. The heat damage index showed that at 42℃, the heat damage index of the 0 mg / kg MgH2 treatment group and the 5 mg / kg MgH2 treatment group was significantly higher than that of the treatment groups at 25℃; and at 42℃, the heat damage index of the 5 mg / kg MgH2 treatment group was significantly lower than that of the 0 mg / kg MgH2 treatment group, indicating that external application of 5 mg / kg MgH2 can significantly reduce the heat damage index of tomato seedlings under high temperature (…). Figure 2 B). In summary, external application of 5 mg / kg MgH2 effectively improved the growth status of tomato seedlings under 42℃ high-temperature stress by maintaining a higher relative water content and reducing the heat damage index. Moreover, this regulatory effect has shown an improvement in water retention capacity at room temperature, laying the foundation for its high-temperature resistance.

[0033] Example 3: Effects of external application of 5 mg / kg MgH2 on the photosynthetic system of tomato under high temperature stress.

[0034] The photosynthetic system of tomato leaves at the same location was observed and analyzed using a chlorophyll fluorescence imaging system. For example... Figure 3As shown in Figure A, after 2 days of treatment at room temperature, there was no significant difference in chlorophyll fluorescence imaging results between tomato leaves treated with 0 mg / kg MgH2 and those treated with 5 mg / kg MgH2. However, under high temperature stress, the tomato leaves treated with 0 mg / kg MgH2 were severely damaged, while the leaves of the tomato group treated with 5 mg / kg MgH2 did not show significant damage. The maximum photochemical efficiency (Fv / Fm) results showed that after 2 days of high temperature stress, the Fv / Fm value of the control group decreased significantly, while the Fv / Fm value of the 5 mg / kg MgH2 treatment group did not change significantly compared to the 5 mg / kg MgH2 treatment group at 25℃. Figure 3 B).

[0035] Further analysis was conducted on the chlorophyll and carotenoid content of the treated tomato leaves. The results are as follows: Figure 4 As shown, after 2 days of high-temperature stress, the contents of chlorophyll a, b, and total chlorophyll in the leaves of tomato seedlings treated with 0 mg / kg MgH2 were significantly reduced; the contents of chlorophyll a, b, and total chlorophyll in the leaves of tomato seedlings treated with 5 mg / kg MgH2 were significantly increased compared with the control group. Figure 4 AC). The content of carotenoids showed no significant change under both room temperature and high temperature treatments. Figure 4 D). This shows that external application of 5 mg / kg MgH2 can significantly reduce the damage of high temperature stress to the photosynthetic system II (PSII) of tomato leaves and enhance the heat resistance of tomatoes.

[0036] Example 4: Effect of external application of 5 mg / kg MgH2 on the tomato oxidation system under high temperature stress.

[0037] When plants are subjected to heat stress, they produce excessive amounts of ROS. DAB staining results showed no significant difference in the leaves of tomato seedlings treated with 0 mg / kg MgH2 and 5 mg / kg MgH2 at room temperature; under high temperature stress, the leaves of the 0 mg / kg MgH2 group showed large areas of dark brown coloration, indicating severe oxidative damage; while the leaves of the 5 mg / kg MgH2 group showed only a few brown spots, with significantly less damage than the former. Figure 5 A).

[0038] Under normal temperature conditions, the MDA content in the 5 mg / kg MgH2 exogenous treatment group was significantly lower than that in the 0 mg / kg MgH2 exogenous treatment group, indicating that exogenous MgH2 could reduce membrane lipid peroxidation levels at normal temperature. Under normal temperature conditions, there was no significant difference in superoxide dismutase (SOD) and catalase (CAT) activities between the 5 mg / kg MgH2 and 0 mg / kg MgH2 exogenous treatment groups (P > 0.05) (Figures 5B-F). However, after high temperature stress, physiological indicators and antioxidant enzyme activities showed significant differential responses. Regarding membrane lipid peroxidation and reactive oxygen species accumulation, the malondialdehyde (MDA) content in the 5 mg / kg MgH2 exogenous treatment group was significantly higher than that in the control group after 1 and 2 days of high temperature stress; the hydrogen peroxide (H2O2) content in the 5 mg / kg MgH2 exogenous treatment group was also significantly higher than that in the control group after 1 day of stress (Figures 5B and 5C). This indicates that while the initial treatment with 5 mg / kg MgH2 under high-temperature stress did not directly reduce membrane lipid peroxidation and reactive oxygen species accumulation, it subsequently produced an indirect alleviating effect through the regulation of antioxidant enzymes. Regarding antioxidant enzyme activity, the SOD activity in the 5 mg / kg MgH2 treatment group under high-temperature stress was not significantly different from that in the 0 mg / kg MgH2 treatment group at both days 1 and 2 of stress; the POD activity in the 5 mg / kg MgH2 treatment group was significantly higher than that in the 0 mg / kg MgH2 treatment group at both days 1 and 2 of stress; and the CAT activity in the 5 mg / kg MgH2 treatment group was significantly lower than that in the 0 mg / kg MgH2 treatment group at day 2 of stress. Figure 5 (DF). The above results indicate that external application of 5 mg / kg MgH2 mainly improves the heat resistance of tomatoes by increasing the activity of POD in tomato plants, effectively removing excess ROS in the plants, reducing leaf membrane peroxidation damage, and thus enhancing the heat resistance of tomatoes.

[0039] Example 5: Effects of external application of 5 mg / kg MgH2 on heat response-related genes in tomatoes under high temperature stress.

[0040] HSF (heat shock factor) and HSP (heat shock protein) play important roles in plant resistance to abiotic stress. The relative expression levels of heat response-related genes (including SlHSP70, SlHSP90, SlHSFA1a, and SlHSFA2) in leaves were detected at different time points using qRT-PCR. Primer sequences are shown in Listing 1 as SEQ ID NO: 1-10.

[0041] Figure 6This diagram compares the expression levels of heat-related genes among the control group at room temperature (25℃), the group treated with 5 mg / kg MgH2 at room temperature (25℃), the control group treated with 5 mg / kg MgH2 at high temperature (42℃), and the group treated with 5 mg / kg MgH2 at high temperature (42℃) after 0h, 1h, 3h, 6h, 12h, and 24h of treatment. The results show that the expression levels of HsfA1a and HsfA1b in the drug-treated group were significantly higher than those in the water-treated group under high temperature, while the expression levels of HSP70 and HSP90 were significantly lower than those in the water-treated group. This indicates that the drug composition for improving the heat resistance of tomatoes may upregulate the expression of HSF by reducing the expression level of HSP genes under heat stress, thereby further activating other transcription factors and enhancing the heat resistance of tomato seedlings at the molecular level.

[0042] Table 1 Primers for thermal response gene detection

[0043] name sequence serial number Hsp70-F 5'-GGATCAACACGAATACCGGC-3' SEQ ID NO: 1 Hsp70-R 5'-CAGCCAAGATACCAGCCTGAA-3' SEQ ID NO: 2 Hsp90-F 5'-CAAGGAAGACCAGTTGGAGT-3' SEQ ID NO: 3 Hsp90-R 5'-TCACTGATTTCCTTCTCAGTTGTCT-3' SEQ ID NO: 4 HsfA1a-F 5'-TCAACAACAGCAGCAGCCACAT-3' SEQ ID NO: 5 HsfA1a-R 5'-TCAGCCTCTCAACCTCTTTCTTCAAG-3' SEQ ID NO: 6 HsfA1b-F 5'-ACTGACGCAGGCAGGGTA-3' SEQ ID NO: 7 HsfA1b-R 5'-CCAGTTCAGAGTATCCACCTGAC-3' SEQ ID NO: 8 HSFA2-F 5'-GAGCAGCAGAGGAAGTAGTG-3' SEQ ID NO: 9 HSFA2-R 5'-TCTGCATCTTAACCTCTAAAGGAA-3' SEQ ID NO: 10

[0044] .

[0045] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for preparing a cultivation substrate to improve the heat resistance of tomatoes, characterized in that... Includes the following steps: S1. Mix the magnesium-based hydrogen storage material MgH2 solid powder with the first part of the dry matrix to prepare the primary parent material. The magnesium-based hydrogen storage material MgH2 solid powder is 25mg and the first part of the dry matrix is ​​500g. S2. Mix the primary parent soil with the second part of the dried matrix to make secondary parent soil, wherein the second part of the dried matrix is ​​1500g; S3. Mix the secondary parent soil with the third part of the dried substrate. The third part of the dried substrate is 3000g. Finally, a total amount of 5kg and a concentration of 5mg / kg of magnesium-based hydrogen storage material MgH2 cultivation substrate are obtained.

2. The method for preparing a cultivation substrate to improve the high-temperature resistance of tomatoes according to claim 1, characterized in that: In step S2, the primary parent soil and the second part of the dry matrix are mixed for more than 15 minutes.

3. The application of the cultivation substrate obtained by the preparation method according to any one of claims 1 to 2 in improving the high temperature resistance of tomatoes.

4. The application according to claim 3, characterized in that: The application involves applying the cultivation substrate to the roots of tomatoes.

5. The application according to claim 3, characterized in that: The cultivation substrate enhances the high-temperature resistance of tomatoes through at least one of the following methods: maintaining cell membrane homeostasis, protecting the photosynthetic system, strengthening the activity of osmotic regulation and antioxidant systems, and upregulating the expression of the heat shock factor HSF gene.

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