Method for repairing tomato fruit softening inhibition caused by penthiopyrad application

Abstract

The invention provides a method for repairing inhibition of penthiopyrad stress on fleshy fruit softening. According to the method, nano-selenium is applied in the immature green fruit period of fleshy fruits, and fruit softening is positively regulated and controlled, so that inhibition of penthiopyrad stress on fleshy fruit softening is repaired. The nano-selenium and penthiopyrad are combined in the immature green fruit period, so that the loss caused by the penthiopyrad to the fruit softening of the fleshy fruit crops can be effectively repaired. And the nano-selenium has the advantages of high bioavailability, good safety, strong oxidation resistance and the like.

Description

Technical Field

[0001] The invention belongs to the field of agriculture, and specifically relates to a method for repairing the inhibition of tomato fruit softening caused by the application of penthiopyrad, and more specifically relates to applying nano-selenium during the immature green fruit stage of tomatoes to repair the inhibitory effect of penthiopyrad on tomato fruit softening. Background Art

[0002] Cherry tomatoes are herbaceous plants of the genus Tomato in the Solanaceae family, also known as cherry tomatoes, small tomatoes, etc. They are used as both fruits and vegetables, and are popular among consumers for their unique flavor, bright colors, and anti-cancer properties. Cherry tomatoes are rich in health-promoting ingredients (such as ascorbic acid, carotenoids, etc.), which are conducive to a balanced diet. In addition, it has always been a model system for plant genetics, development, physiology, pathology, and ripening of fleshy fruits. In recent years, with the improvement of living standards, more and more consumers are willing to pay for higher quality cherry tomatoes, and the demand for high-quality traits of tomatoes is also increasing.

[0003] However, a variety of pests and diseases are prone to occur during tomato growth, causing huge economic losses to agricultural production. At present, chemical control is still the main means of tomato pest and disease management. In recent years, succinate dehydrogenase inhibitor (SDHI) fungicides, such as fluopyram, pyraclostrobin and penthiopyrad, have been introduced into the market to control a variety of crop diseases. Among them, penthiopyrad can effectively control a series of pathogens such as gray mold and powdery mildew. It has been registered in China for the control of tomato, grape gray mold and cucumber powdery mildew. Although the application of pesticides can effectively control pests and diseases, as an exogenous additive, it may cause a certain degree of abiotic stress to tomato plants, thereby affecting the traits of tomatoes.

[0004] Selenium is considered one of the most important micronutrients as an antioxidant for animal and human nutrition. Selenium has been shown to promote plant growth under normal and stress conditions. In addition, selenium-enriched agricultural products are becoming increasingly popular among the public. The use of nanoparticles has attracted increasing attention from agricultural researchers due to their low area / surface ratio, aggregation state, and stability. Compared with inorganic selenium, nano-selenium has the characteristics of small particle size, good sustained release and absorption effect, and high plant utilization efficiency.

[0005] Nano-selenium has a positive effect on crop growth and development, but research on the combined application of nano-selenium and pesticides is very limited. Summary of the invention

[0006] In view of the deficiencies in the prior art, one of the objectives of the present invention is to provide an environmentally friendly, safe and efficient method for repairing the damage caused by softening of fleshy fruits due to penthiopyrad stress.

[0007] The method provided by the present invention for repairing and improving the inhibition of softening of fleshy fruits caused by penthiopyrad stress is to apply nano-selenium during the immature green fruit stage of fleshy fruits to repair the damage caused by softening of fleshy fruits caused by penthiopyrad stress.

[0008] The fleshy fruit may specifically be a tomato, and more specifically may be a cherry tomato.

[0009] Apply nano-selenium at the same time as pesticides.

[0010] In one embodiment of the present invention, the nano-selenium is chitosan-based nano-selenium, and the nano-selenium is prepared by a method comprising the following steps, referring to the method in the literature (Shang, H.; Zhang, H.; Zhao, R.; Yu, M.; Ma, Y.; Sun, Z.; Wu, X.; Xu, Y., Selenium nanoparticles are effective in penetrating pine and causing highoxidative damage to Bursaphelenchus xylophilus in pine wilt disease control. Pest Management Science 2022, 78 (8), 3704-3716): A certain amount of selenium dioxide solution was slowly added to 20 mL of 1% chitosan solution to prepare a solution containing 20 mM selenium dioxide, and the mixture was stirred continuously at 500 rpm at 25°C. Then, 4 mL of 1% ascorbic acid solution was added dropwise at 25°C, and the mixture was stirred continuously at 500 rpm for 3 h. The color of the solution gradually changed to transparent red, and nano-selenium was obtained.

[0011] In one embodiment of the present invention, the pesticide preparation is penthiopyrad emulsifiable concentrate, more specifically 20% penthiopyrad emulsifiable concentrate, and its formula is: 98.0% of the original drug, 20% of penthiopyrad, 5% of the anionic emulsifier Nongru 500, 5% of the nonionic emulsifier Nongru 601, and 70% of the solvent oil S-150.

[0012] The recommended dosage of the penthiopyrad emulsifiable concentrate is 150 g ai / hectare; The dosage of the nano-selenium is 0.45 g / hectare; The application of nano-selenium in repairing the damage caused by softening of fleshy fruits due to penthiopyrad stress also falls within the protection scope of the present invention.

[0013] The fleshy fruit may specifically be a tomato, and more specifically may be a cherry tomato.

[0014] The nano-selenium is chitosan-based nano-selenium; The pesticide preparation is penthiopyrad emulsifiable concentrate, more specifically 20% penthiopyrad emulsifiable concentrate, and its formula is: 98.0% of the original drug, 20% of penthiopyrad, 5% of the anionic emulsifier Nongru 500, 5% of the nonionic emulsifier Nongru 601, and 70% of the solvent oil S-150.

[0015] The recommended dosage of the penthiopyrad emulsifiable concentrate is 150 g ai / hectare; The dosage of the nano-selenium is 0.45 g / hectare.

[0016] In the application, nano-selenium increases the soluble pectin content in the fruit after the application of penthiopyrad; enhances the activity of polygalacturonase and pectin methylesterase in the fruit after the application of penthiopyrad; positively regulates the expression level of genes related to cell wall metabolism, promotes cell wall degradation, and thus repairs the inhibition of penthiopyrad on fruit softening.

[0017] The present invention uses nano-selenium and penthiopyrad together in the immature green fruit stage of fleshy fruit crops, which can effectively repair the loss caused by the softening of fruits of fleshy fruit crops by the application of penthiopyrad. In addition, nano-selenium has the advantages of high bioavailability, good safety, strong antioxidant properties, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The experimental flow chart of the present invention.

[0019] Figure 2 These are electronic photographs of tomato fruits in the control group and each treatment group in Example 1 of the present invention. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0021] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0022] Example 1: Application of penthiopyrad and nano-selenium in combination to repair tomato fruit softening during the immature green fruit stage The specific steps include: Step 1: Prepare chitosan-based nano-selenium with the following formula: 20 mL of 1% chitosan solution (containing 20 mM selenium dioxide); 4 mL of 1% ascorbic acid solution A certain amount of selenium dioxide solution was slowly added to 20 mL of 1% chitosan solution to prepare a solution containing 20 mM selenium dioxide, and stirred continuously at 500 rpm at 25°C. Then, 4 mL of 1% ascorbic acid solution was added dropwise at 25°C, and stirred continuously at 500 rpm for 3 h. The color of the solution gradually changed to transparent red, and nano-selenium was obtained; Step 2, prepare 20% penthiopyrad emulsifiable concentrate, the formula is: 98.0% of the original drug, 20% of penthiopyrad, 5% of the anionic emulsifier Nongru 500, 5% of the nonionic emulsifier Nongru 601, and 70% of the solvent, which is solvent oil S-150; Step 3, applying the prepared penthiopyrad emulsifiable concentrate (recommended dosage 150 g ai / hectare) and chitosan-based nano-selenium (0.45 g / hectare) twice at the immature green fruit stage of tomatoes, with an interval of 7 days, and diluting with water at a dosage of 30 L / mu during spraying; Step 4, collecting tomato fruit samples at 0, 5, 10, 15, and 20 days after application of the pesticide; Step 5, at each sampling period, measuring the hardness of the fruit samples of each treatment group; Step 6: Weigh 0.2 g of each sample and place it in a 2 ml centrifuge tube. Add 800 μL of 1× phosphate buffer and use a ball mill to prepare the sample into a 20% tissue homogenate. Centrifuge at 4°C, 10,000 rpm for 10 minutes and take the supernatant. Step 7: The supernatant obtained in step 6 is treated with a cellulose (CLL) content kit (Cat. No. QYS-235028) from Qiyi Biotechnology (Shanghai) Co., Ltd., and the absorbance at 620 nm is measured using a spectrophotometer. The cellulose content in the sample is calculated according to the instructions of the kit; Step 8: The supernatant obtained in step 6 is treated with a protopectin content kit (Cat. No. QYS-23098) from Qiyi Biotechnology (Shanghai) Co., Ltd., and then the absorbance at 530 nm is measured using an ELISA reader. The protopectin content in the sample is calculated according to the instructions of the kit; Step 9: The supernatant obtained in step 6 is treated with a soluble pectin content kit (Cat. No. QYS-23092) produced by Qiyi Biotechnology (Shanghai) Co., Ltd., and then the absorbance at 530 nm is measured using an ELISA reader to calculate the soluble pectin content in the sample; Step 10: The supernatant obtained in step 6 is treated with a polygalacturonase (PG) kit (Cat. No. QYS-231005) from Qiyi Biotechnology (Shanghai) Co., Ltd., and then the absorbance at 540 nm is measured using an ELISA reader to calculate the PG activity in the sample; Step 11: The supernatant obtained in step 6 is treated with a pectin methylesterase (PME) kit (Cat. No. QYS-231007) from Qiyi Biotechnology (Shanghai) Co., Ltd., and then the PME activity in the sample is determined using an automatic potentiometric titrator; Step 12, at each sampling period, using the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Cat. No. DP441) of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the total RNA of tomato fruits in each treatment group; Step 13, reverse transcribe the RNA obtained in step 12 into cDNA using the FastKing cDNA First Strand Synthesis Kit (Genomic Depleted) (Cat. No. KR116-02) from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Step 14: Use the SuperReal Fluorescence Quantitative Premix Enhanced Edition (SYBR Green) Kit (Cat. No. FP205-02) of Tiangen Biochemical Technology (Beijing) Co., Ltd. and CFX ConnectTM Fluorescence Quantitative PCR Instrument to perform real-time fluorescence quantitative PCR on the cDNA obtained in step 13. PCR primers were synthesized by Sangon Biotechnology Technology Service (Beijing) Co., Ltd. Actin ( Actin ) was used as a reference gene to normalize the mRNA expression levels associated with fruit firmness.

[0023] Test method: The test was conducted in a constant temperature room. When tomatoes were in the immature green fruit stage, fruits of uniform size were selected for labeling. A backpack sprayer was used to spray penthiopyrad and nano-selenium on the stems and leaves, once every 7 days, for a total of two sprayings. The control group was sprayed with an equal amount of water. Four replicates were set for each treatment, and each replicate had 60 marked tomato fruits. Samples were taken 0, 5, 10, 15, and 20 days after the last application of the pesticide (see Figure 1 ).

[0024] 1) Experimental site overview: Constant temperature room (24°C), light intensity 300 μmol photons / m 2 / s, light / dark cycle was 16 h / 8 h, and field management was conventional management; 2) Group design: This experiment has 2 treatments, 1 control, and 4 repetitions. Treatment 1 is a blank control (P1), treatment 2 is treated with penthiopyrad (P2), and treatment 3 is treated with penthiopyrad + nano-selenium (P3); 3) Application period: Apply the pesticide twice during the immature green fruit stage of tomatoes; 4) Result statistics: 0, 5, 10, 15, and 20 days after the last application of pesticide, 20 fruits with the same name in the treatment group and the control group were randomly collected to measure the hardness; 10 fruits with the same name in the treatment group and the control group were randomly collected at each sampling period and immediately placed in liquid nitrogen, frozen and placed at -80°C until the analysis of cellulose, protopectin, soluble pectin content and polygalacturonase and pectin methylesterase activities (using cellulose, protopectin, soluble pectin, polygalacturonase, and pectin methylesterase kits from Shanghai Qiyi Biotechnology Co., Ltd. for determination, respectively); 12 fruits with the same name in the treatment group and the control group were randomly collected at each sampling period to analyze the expression of genes related to cell wall metabolism (using the polysaccharide and polyphenol plant total RNA extraction kit, FastKing cDNA first-strand synthesis kit (genome-free), and SuperReal fluorescence quantitative premix enhanced version (SYBR Green) kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. for determination); 5) Cellulose determination method: Take 300µL of the supernatant obtained in step 6 from each sample, add 1mL of 80% ethanol, quickly homogenize at room temperature, place in a 95℃ water bath for 20min, cool to room temperature, centrifuge at 4000g at 25℃ for 10min, and discard the supernatant. Add 1.5mL of 80% ethanol and acetone to the precipitate and wash once (vortex oscillation for about 2min, centrifuge at 4000g at 25℃ for 10min, and discard the supernatant). The precipitate is the crude cell wall. Add 1mL of the reagent provided by the kit and soak for 15 hours. Centrifuge at 4000g at 25℃ for 10min, discard the supernatant, wash twice with 1mL of 80% ethanol, discard the supernatant, dry the precipitate, and weigh the cell wall material. Weigh about 5mg of the dried precipitate, add 0.5mL of distilled water to fully homogenize, transfer the homogenate to an EP tube, dilute to 0.5mL with distilled water, place in an ice water bath, slowly add 0.75mL of concentrated sulfuric acid, mix well, and let stand in an ice water bath for 30min. Centrifuge at 8000g for 10min at 4℃, take the supernatant, dilute it 20 times with distilled water and test it. Dilute the 10 mg / mL glucose standard solution provided by the kit by two times to obtain 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.00156 mg / mL standard solutions. Preheat the spectrophotometer for more than 30min, adjust the zero with distilled water, adjust the wavelength to 620nm, and measure the absorbance of each treatment group sample. Draw a standard curve with the concentration of the standard as the horizontal axis and the absorbance as the vertical axis, and calculate the sample concentration from the standard curve according to the absorbance of the sample.

[0025] 6) Determination of original pectin content: Take 50μL of the supernatant obtained in step 6 from each sample and add 1mL of the extract solution 1 provided by the kit, place in a 90℃ constant temperature water bath for 30min, take out and cool, centrifuge at 5000g, 25℃ for 10min, remove the supernatant, add 1mL of the extract solution 1 to the precipitate, repeat the operation once, remove the supernatant after centrifugation, add 1mL of the extract solution 2 provided by the kit to the precipitate, place in a 90℃ constant temperature water bath for hydrolysis for 1h, take out and cool, centrifuge at 8000g, 25℃ for 15min. Take 200μL of the supernatant and place it in a 96-well plate. Use the standard provided by the kit (0.25mg / mL) to set the standard wells. Zero the blank wells and sequence the absorbance of each well in turn at a wavelength of 530nm. Calculate the sample concentration based on the absorbance of the standard and sample.

[0026] 7) Determination of soluble pectin content: Take 300µL of the supernatant obtained in step 6 for each sample, add 1mL of 80% ethanol, quickly homogenize at room temperature, place in a 95℃ water bath for 20min, cool to room temperature, centrifuge at 4000g at 25℃ for 10min, and discard the supernatant. Add 1.5mL of 80% ethanol and acetone to the precipitate and wash it once (vortex oscillation for about 2min, centrifuge at 4000g at 25℃ for 10min, and discard the supernatant). The precipitate is the crude cell wall. Add 1mL of reagent 1 provided by the kit and soak for 15 hours, centrifuge at 4000g at 25℃ for 10min, discard the supernatant, wash twice with 1mL of 80% ethanol, discard the supernatant, dry the precipitate, and weigh the cell wall material. Weigh 3mg of the dried cell wall material, add 1mL of reagent 2 provided by the kit, and homogenize it thoroughly. Centrifuge at 8000g at 4℃ for 10min, take the supernatant, dilute 20 times with distilled water and then test. Take 200 μL of supernatant and place it in a 96-well plate. Use the standard provided in the kit (0.25 mg / mL) to set the standard wells. Use the blank wells to zero and sequence the absorbance of each well at 530 nm. Calculate the sample concentration based on the absorbance of the standard and sample.

[0027] 8) Polygalacturonase activity assay method: Take 25µL of the supernatant obtained in step 6 for each sample and add 50µL of extract solution 1 and 50µL of extract solution 2 provided by the kit. After accurate reaction in a 40℃ water bath for 2h, heat in a boiling water bath for 10min and cool to room temperature. Add 125µL of reagent 3 provided by the kit, boil in a boiling water bath for 5min and cool to room temperature for testing. Take 200μL of the reaction solution and place it in a 96-well plate. Use the standard provided by the kit (50µmol / mL) to dilute to obtain 6, 5, 4, 3, 2, 1.5 μmol / mL standard solutions to set standard wells. Zero the blank well and sequence the absorbance of each well in turn at a wavelength of 540nm. Calculate the sample concentration based on the absorbance of the standard and sample.

[0028] 9) Pectin methylesterase activity assay method: Crush the tissue sample, grind it in an ice bath at a ratio of 1:10 between the sample mass (g) and the volume of the extract provided by the kit (mL), centrifuge it at 8000g, 4℃ for 15min, and take the supernatant for testing. Replenish the solution with Reagent 2 provided by the kit, and after the rehydration, take 50mL of Reagent 1 provided by the kit and titrate it with Reagent 2 to a pH of 7. Add 5mL of the sample to be tested, set the titration delay time to 180s, and titrate it with Reagent 2 to a pH of 7. After the titration, record the volume of Reagent 2 consumed and the time required to titrate to the endpoint to calculate the sample concentration.

[0029] 10) Method for extracting total RNA from tomato fruit: 1. Grind 100 mg of fruit pulp into powder in liquid nitrogen, add 500 µL of the lysis solution SL provided by the kit, and vortex vigorously to mix; 2. Centrifuge at 12,000 rpm for 2 min; 3. Transfer the supernatant to the filter column CS provided by the kit, centrifuge at 12,000 rpm for 2 min, and carefully pipette the supernatant in the collection tube into a new RNase-Free centrifuge tube; 4. Slowly add 0.4 times the volume of the supernatant with anhydrous ethanol, mix well, and transfer the resulting solution and precipitate to the adsorption column CR3 provided by the kit, centrifuge at 12,000 rpm for 15 sec, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube; 5. Add 350 µL of the deproteinization solution RW1 provided by the kit to the adsorption column CR3, centrifuge at 12,000 rpm for 15 sec, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube; 6. Add 80 µL of the DNase1 working solution provided by the kit to the center of the adsorption column CR3 and let it stand at room temperature for 15 min; 7. Repeat step 5; 8. Add 500 µL of the rinse solution RW provided by the kit to the adsorption column CR3, centrifuge at 12,000 rpm for 15 sec, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube; 9. Repeat step 8; 10. Centrifuge at 12,000 rpm for 2 min, put the adsorption column CR3 into a new RNase-Free centrifuge tube, add 30-50 µL of the RNase-Free ddH2O provided by the kit to the middle part of the adsorption membrane, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min to obtain the RNA solution, and use TGem Pro spectrophotometer (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to determine the total RNA concentration.

[0030] 11) RNA reverse transcription to cDNA method: 1. Add 2µL of 5×gDNA Buffer provided by the kit to an RNase-Free centrifuge tube. Add different volumes of RNA solution according to the measured RNA concentration in each treatment and control group, so that the final RNA concentration is 1µg. Add RNase-Free ddH2O provided by the kit to make the volume up to 10µL and mix thoroughly. Centrifuge briefly and incubate at 42℃ for 3 min. Then place on ice. 2. Add the reverse transcription reaction system mixture provided by the kit to the reaction solution in step 1, mix thoroughly, incubate at 42℃ for 15 min, incubate at 95℃ for 3 min, and then place on ice to obtain cDNA.

[0031] 12) Real-time fluorescence quantitative PCR method: Add 25 µL of 2×SuperReal PreMix Plus provided by the kit, 1.5 µL of forward primer, 1.5 µL of reverse primer, 1 µL of the above cDNA into an RNase-Free centrifuge tube, add RNase-Free ddH2O provided by the kit, and make up the volume to 50 µL. Use CFX Connect Fluorescence Quantitative PCR Instrument (Bio-Rad Biomedical Products (Shanghai) Co., Ltd.) for real-time fluorescence quantitative PCR. PCR conditions are: 95°C for 15 min, then set 40 cycles, in which the cycle is kept at 95°C for 10 s, annealed at 54.5°C for 20 s, and then extended at 72°C for 32 s. Actin ( Actin ) as reference genes.

[0032] Experimental results: 1) Comparison of the effects of fruit hardness As the fruit matured, the firmness of tomatoes in both the treatment and control groups showed a trend of increasing first and then decreasing. During the experiment, the fruit firmness in the penthiopyrad-treated group was the highest. At 5-20 days after treatment, the fruit firmness in the penthiopyrad-treated group increased significantly by 19.86%, 20.95%, 42.76% and 26.24% compared with the control group, respectively. During the experiment, there was no significant difference in the firmness of the penthiopyrad-treated group + nano-selenium compared with the control group, but it was always lower than the penthiopyrad-treated group. At 15 and 20 days after treatment, the fruit firmness in the penthiopyrad-treated group + nano-selenium treated group was significantly reduced by 33.64% and 27.53% compared with the penthiopyrad-treated group, respectively. The specific values ​​are shown in Table 1.

[0033] Fruit softening is a physiological and biochemical process of fruit development and ripening. During the ripening process of tomato fruit, the cell wall composition changes, causing the fruit to gradually soften and the hardness to decrease. After the application of penthiopyrad, the fruit hardness was always higher than the control. This shows that the application of penthiopyrad inhibited the softening of tomato fruit. The combined application of nano-selenium and penthiopyrad can repair the negative impact of penthiopyrad on fruit softening.

[0034] Table 1 Fruit hardness of the control group and the treatment group at different periods

[0035] ab represent the differences between different treatment groups, different letters represent significant differences (p<0.05), and the same letters represent no significant differences; * indicates that there is a significant difference between the treated group and the blank control group, * indicates p<0.05, and ** indicates p<0.01.

[0036] 2) Comparison of the effects of cellulose and pectin Cellulose and pectin are the main components of plant cell walls, among which pectin has the highest content. The transformation and decomposition of pectin are the main factors for fruit softening. Pectin participates in intercellular adhesion through calcium cross-linking with demethylated homogalacturonic acid. During the ripening process of tomato fruit, protopectin is continuously degraded, while soluble pectin is continuously accumulated, resulting in continuous softening of the fruit. During the experiment, the cellulose and protopectin contents of the fruit in the control group and the treatment group gradually decreased, while the soluble pectin content gradually increased. However, in general, the cellulose and protopectin contents in the penthiopyrad-treated group were higher than those in the control group and other treatment groups, while the soluble pectin content was always the lowest during the experiment. At 15 and 20 days after treatment, the soluble pectin content in the penthiopyrad-treated group was only 87.56% and 80.78% of that in the control group. During the experiment, there was no significant difference in the cellulose, protopectin and soluble pectin contents in the penthiopyrad+nanoselenium treatment group compared with the control group. In general, as the fruit continues to ripen, the cellulose and protopectin contents in the tomato fruit of the penthiopyrad + nano-selenium treatment group gradually become lower than those in the penthiopyrad treatment group, while the soluble pectin content gradually becomes higher than that in the penthiopyrad treatment group, indicating that nano-selenium can repair the negative impact of penthiopyrad on the softening of tomato fruit.

[0037] Table 2 Cellulose content of blank control group and treatment group at different periods

[0038] ab represent the differences between different treatment groups, different letters represent significant differences (p<0.05), and the same letters represent no significant differences; * indicates that there is a significant difference between the treated group and the blank control group, * indicates p<0.05, and ** indicates p<0.01.

[0039] Table 3 Protopectin content in blank control group and treatment group at different periods

[0040] ab represent the differences between different treatment groups, different letters represent significant differences (p<0.05), and the same letters represent no significant differences; * indicates that there is a significant difference between the treated group and the blank control group, * indicates p<0.05, and ** indicates p<0.01.

[0041] Table 4 Soluble pectin content in blank control group and treatment group at different periods

[0042] ab represent the differences between different treatment groups, different letters represent significant differences (p<0.05), and the same letters represent no significant differences; * indicates that there is a significant difference between the treated group and the blank control group, * indicates p<0.05, and ** indicates p<0.01.

[0043] 3) Comparison of the effects of polygalacturonase and pectin methylesterase Pectin-degrading enzymes play an important role in the disintegration of tissues and directly affect the texture of tomato fruit. It is generally believed that the degradation of pectin by pectin-degrading enzymes during the ripening process of fruit is closely related to the softening and pithing of fruit. Fruits contain enzymes that degrade pectin, including pectin methylesterase that decomposes galacturonic acid carboxymethyl ester and polygalacturonase that hydrolyzes the glycosidic bonds of galacturonic acid polysaccharides. These two types of enzymes have a greater effect on the degradation of pectin. In general, the activities of polygalacturonase and pectin methylesterase gradually increased from the immature green fruit stage to the color change stage (0-15 days after treatment), and the activities of the two enzymes gradually decreased during the red ripe stage of tomato (20 days after treatment). There was no significant difference in the enzyme activity of each treatment group from that of the control group 0-5 days after treatment. At 15-20 days after treatment, the activities of polygalacturonase and pectin methylesterase in the penthiopyrad-treated group were significantly reduced by 15.74%, 19.49% and 15.39%, 18.53% respectively compared with the control group. At 10-20 days after treatment, the polygalacturonase activity in the penthiopyrad+nanoselenium-treated group was significantly increased by 10.61%, 10.91% and 8.59% respectively compared with the control group, while the pectin methylesterase activity was not significantly different from that of the control group during the experimental period. The specific values ​​are shown in Tables 5 and 6.

[0044] Therefore, the results of polygalacturonase and pectin methylesterase activities showed that penthiopyrad reduced the activity of pectin-degrading enzymes, resulting in increased firmness of tomato fruit and inhibited fruit softening. Nano-selenium can restore the polygalacturonase and pectin methylesterase activities in the penthiopyrad-treated group to the control level.

[0045] Table 5 Polygalacturonase activity in the blank control group and the treatment group at different periods

[0046] abc represent the differences between different treatment groups, different letters represent significant differences (p<0.05), and the same letters represent no significant differences; * indicates that there is a significant difference between the treated group and the blank control group, * indicates p<0.05, and ** indicates p<0.01.

[0047] Table 6 Pectin methylesterase activities in blank control group and treatment group at different periods

[0048] ab represent the differences between different treatment groups, different letters represent significant differences (p<0.05), and the same letters represent no significant differences; * indicates that there is a significant difference between the treated group and the blank control group, * indicates p<0.05, and ** indicates p<0.01.

[0049] Comparison of cell wall metabolism gene effects SlPG2a Regulates polygalacturonase, thereby affecting the softening process of fruit by changing the cell wall structure. SlEXP1 Regulation of tomato expansin, in tomato SlEXP1 Induces the accumulation of mRNA and protein of genes related to fruit ripening. Endoglucanase plays an important role in the degradation of tomato fruit cell walls. SlCEL2 and expansin-encoding genes SlEXP1 It has a synergistic effect on the softening of tomato fruit and the degradation of cell walls. -△△Ct After normalization and calibration, the expression levels of the three genes in the control group were all 1.00. SlPG2a , SlEXP1 and SlCEL2 The expression levels of SlPG2a and SlCEL2 The expression levels were significantly downregulated by 60.00% and 79.00% compared with the control group. This showed that penthiopyrad had an inhibitory effect on fruit softening, which was consistent with the results of fruit hardness. As the tomato fruit matured, the expression levels of the three genes in the penthiopyrad+nanoselenium treatment group showed an upward trend, and the expression levels were higher than those in the penthiopyrad treatment group. At 15 and 20 days after treatment, the expression levels of the three genes in the penthiopyrad+nanoselenium treatment group were significantly lower than those in the penthiopyrad+nanoselenium treatment group. SlPG2a , SlEXP1 and SlCEL2The gene expression levels were approximately 1.93 and 4.26 times, 1.65 and 2.89 times, and 1.67 and 5.10 times that of the control group.

[0050] The results showed that nano-selenium promoted cell wall degradation by positively regulating the expression levels of genes related to cell wall metabolism, thereby repairing the inhibitory effect of penthiopyrad on tomato fruit softening.

[0051] Table 7 Blank control group and treatment group at different periods SlPG2a Expression level

[0052] abc represent the differences between different treatment groups, different letters represent significant differences (p<0.05), and the same letters represent no significant differences; * indicates that there is a significant difference between the treated group and the blank control group, * indicates p<0.05, and ** indicates p<0.01.

[0053] Table 8 Blank control group and treatment group at different periods SlEXP1 Expression level

[0054] ab represent the differences between different treatment groups, different letters represent significant differences (p<0.05), and the same letters represent no significant differences; * indicates that there is a significant difference between the treated group and the blank control group, * indicates p<0.05, and ** indicates p<0.01.

[0055] Table 9 Blank control group and treatment group at different periods SlCEL2 Expression level

[0056] abc represent the differences between different treatment groups, different letters represent significant differences (p<0.05), and the same letters represent no significant differences; * indicates that there is a significant difference between the treated group and the blank control group, * indicates p<0.05, and ** indicates p<0.01.

[0057] It can be seen from the experimental data of the embodiment that the application of penthiopyrad in the immature green fruit stage of tomatoes has a negative impact on the softening of tomato fruits. The combined application of nano-selenium and penthiopyrad can restore the inhibitory effect of penthiopyrad on the softening of tomato fruits to the control level, so the combined application of penthiopyrad and nano-selenium is recommended in the immature green fruit stage.

[0058] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for repairing the inhibition of softening of fleshy fruits caused by penthiopyrad stress, which comprises applying nano-selenium during the immature green fruit stage of fleshy fruits to repair the inhibition of softening of fleshy fruits caused by penthiopyrad stress.

2. The method according to claim 1, characterized in that The fleshy fruit is a tomato.

3. The method according to claim 1 or 2, characterized in that: Nano-selenium was applied at the same time as penthiopyrad.

4. The method according to claim 1 or 2, characterized in that: The nano-selenium is chitosan-based nano-selenium; the pesticide preparation is penthiopyrad emulsifiable concentrate.

5. The method according to claim 4, characterized in that The recommended dosage of the penthiopyrad emulsifiable concentrate is 150 ga.i. / hectare; the dosage of the nano-selenium is 0.45 g / hectare.

6. Application of nano-selenium in repairing the damage caused by softening of fleshy fruits due to penthiopyrad stress.

7. The use according to claim 6, characterized in that: The fleshy fruit is a tomato.

8. The use according to claim 6, characterized in that: The nano-selenium is chitosan-based nano-selenium; the pesticide preparation is penthiopyrad emulsifiable concentrate.

9. The use according to claim 6, characterized in that: In the application, nano-selenium increases the soluble pectin content in the fruit after the application of penthiopyrad; enhances the activity of polygalacturonase and pectin methylesterase in the fruit after the application of penthiopyrad; positively regulates the expression level of genes related to cell wall metabolism, promotes cell wall degradation, and thus repairs the inhibition of penthiopyrad on fruit softening.

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