Application of dipropyl disulfide in promoting tomato growth

Abstract

The present invention discloses the application of dipropyl disulfide in promoting tomato growth, belonging to the field of biotechnology. The specific application method is: dissolving dipropyl disulfide in a surfactant, adding water to obtain a dipropyl disulfide solution, then placing the solution in a container, and allowing dipropyl disulfide to evaporate outward from the container in the form of gas, and placing the container containing the dipropyl disulfide solution on the root of a tomato plant. The present invention induces tomatoes to increase the content of phenylalanine and proline secreted by the root system through the method of exogenous addition of dipropyl disulfide, significantly increases the abundance of bacteria and fungi in the rhizosphere soil microorganisms of tomatoes, changes the bacterial and fungal community structure, induces changes in tomato metabolism, improves the rhizosphere microecological environment of tomatoes, promotes tomato growth, and improves the resistance of tomatoes to diseases.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of dipropyl disulfide in promoting tomato growth. Background Art

[0002] Tomatoes are an important vegetable. In recent years, driven by farmers' pursuit of high economic returns, continuous cropping has become the primary method of tomato cultivation. However, when the same crop or related species are continuously planted on the same soil, even with normal cultivation and management practices, soil physical and chemical properties can deteriorate, harmful microorganisms can accumulate, autotoxic substances can be produced, and pests and diseases can increase. This can lead to serious continuous cropping problems. Ultimately, this can affect tomato growth, resulting in reduced fruit quality and yield.

[0003] Plants coexist in diverse communities and can sense and identify conspecifics and heterospecifics within the community by producing and releasing secondary metabolites, such as volatile organic compounds (VOCs), and interact with other plants to create and / or maintain suitable living conditions. VOCs are important mediators of the interaction between plants and their physical and biological environment. A growing body of research has demonstrated that plant volatiles play an important signaling role in plant-to-plant communication. It is through the production, release, and transmission of these airborne signaling substances, such as ethylene, methyl jasmonate, methyl salicylate, indole, and several volatile terpenes, that they are ultimately perceived by receptor plants. This, in large part, drives plant-to-plant interactions and enables information exchange between plants. Studies have shown that this type of VOCs can also affect the defense and growth of recipient plants. When VOCs are perceived by recipient plants, the recipient plants will respond in a timely manner and adjust their own growth patterns. For example, β-caryophyllene, the main volatile substance released by spotted knapweed, promotes the seed germination and growth of neighboring plants in the same area; potatoes exposed to onion volatiles will change their physiological characteristics, thereby avoiding feeding by herbivorous insects; at the same time, orchid volatiles induce physiological changes in peanuts and increase the amount of organic acids secreted by peanut roots. The increase in organic acid content may promote the resistance of peanut rhizosphere to biotic stress.

[0004] Tomatoes are an extremely important vegetable in people's lives, with high demands for both yield and quality. Providing a VOC that can improve tomatoes' resistance to soil-borne pathogens or regulate their growth would undoubtedly be of great significance to the development of the tomato industry. Summary of the Invention

[0005] To solve the above problems in the prior art, the present invention provides the use of dipropyl disulfide in promoting tomato growth. By exogenously adding the volatile organic compound dipropyl disulfide during the growth process of tomatoes, the resistance of tomatoes to diseases is effectively improved, thereby promoting the growth of tomatoes.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides application of dipropyl disulfide in promoting tomato growth.

[0008] As a preferred embodiment of the present invention, the tomatoes are potted tomatoes; the specific application method is: dissolving dipropyl disulfide in a surfactant, adding water to obtain a dipropyl disulfide solution, then placing the solution in a container, and allowing the dipropyl disulfide to evaporate outward from the container, and placing the container containing the dipropyl disulfide solution at the roots of the potted tomato plants.

[0009] As a preferred embodiment of the present invention, the purity of the dipropyl disulfide is 99wt%, the surfactant is Tween-80, the mass ratio of the dipropyl disulfide to the surfactant is 1:(1.5-2.5), and the concentration of the dipropyl disulfide solution is 8-12mmol / L.

[0010] As a preferred embodiment of the present invention, the volatilization rate of dipropyl disulfide is 0.8 to 1.3 μmol / L.

[0011] As a preferred embodiment of the present invention, the horizontal distance between the container and the tomato plant is 4 to 5 cm.

[0012] As a preferred embodiment of the present invention, after the tomato plants are established and have grown, a container containing the dipropyl disulfide solution is placed at the roots of the tomato plants.

[0013] As a preferred embodiment of the present invention, the time for dipropyl disulfide to volatilize outwards is from the time when the seedlings are transplanted to the time when the tomatoes are harvested.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention uses a safe and efficient organic compound - dipropyl disulfide, through an exogenous addition method, to induce tomatoes to increase the content of phenylalanine and proline secreted by the root system, significantly increase the abundance of bacteria and fungi in the rhizosphere soil microorganisms of tomatoes, change the bacterial and fungal community structure, improve the rhizosphere microecological environment of tomatoes, promote tomato growth, and enhance the resistance of tomatoes to diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 Schematic diagram of the experimental device in Example 1, wherein A represents exogenous addition of dipropyl disulfide, and B represents no addition of dipropyl disulfide;

[0018] Figure 2 This is a graph showing the effect of dipropyl disulfide on tomato plant height and biomass in Example 1;

[0019] Figure 3 This is a graph showing the α-diversity test results of the rhizospheric bacterial community of tomato plants with and without exogenous addition of dipropyl disulfide in Example 1;

[0020] Figure 4 This is a principal coordinate analysis result of β-diversity of tomato rhizospheric bacterial community based on Bray-Curtis distance difference between tomato plants with and without exogenous addition of dipropyl disulfide in Example 1;

[0021] Figure 5 This is a graph showing the effect of dipropyl disulfide on the abundance of bacteria and fungi in tomato rhizosphere soil in Example 1;

[0022] Figure 6 This is the effect of dipropyl disulfide on phenylalanine and proline in tomato root exudates in Example 2;

[0023] Figure 7 This is a graph showing the effect of dipropyl disulfide on tomato growth in sterilized soil in Example 3. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0025] In addition, for numerical ranges in the present invention, it is understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] In the following examples, all the raw materials used are commercially available.

[0030] Example 1

[0031] Effects of dipropyl disulfide on the rhizosphere microbial flora of tomato

[0032] Dipropyl disulfide (purity 99wt%) was dissolved in Tween-80 at a ratio of 1:2 (w / w), and then distilled water was added to obtain a 10mmol / L dipropyl disulfide solution, which was stored at 4°C for the following experiments. The soil was collected from the upper soil (0-15cm) of the tomato continuous cropping greenhouse at the Northeast Agricultural University Experimental Station using a potted method. The collected continuous cropping soil was sieved through a 2mm sieve, and all the experimental soils were thoroughly mixed and set aside. After soaking the tomato seeds in warm water for half an hour, they were placed in sterilized soil for germination. When they had two true leaves, the seedlings were transplanted into nutrient pots. When the tomatoes have 4 true leaves, they are transplanted into a handmade 38μm nylon membrane bag filled with 1kg of soil. 5mL of dipropyl disulfide solution is placed in a small glass bottle, the mouth of the glass bottle is sealed with a rubber stopper, and a 10uL pipette tip is inserted between the rubber stoppers. The same volume of Tween-80 is used as a control. The glass bottle is placed outside the nylon bag, 5cm away from the tomato plant. The schematic diagram of the experimental device is shown below. Figure 1As shown (where A is exogenously added dipropyl disulfide; B does not add dipropyl disulfide, and the container contains the same volume of Tween-80. In A and B, the horizontal distance between the container and the tomato root is 5 cm, and the vertical distance between the container and the root is 5 cm). The experiment has two treatments: (1) exogenous addition of dipropyl disulfide (volatile); (2) no addition of dipropyl disulfide (control), and one tomato plant is transplanted in each device. In treatment (1), a bottle of dipropyl disulfide solution is replaced every two days (the dipropyl disulfide in the solution is completely volatilized in two days). After 20 days of tomato planting, the height and biomass of the tomatoes are measured. The specific method is: take the tomato plants and wash them, first fix the samples at 105°C for 30 minutes, and then adjust them to 60°C and dry them to constant weight, and finally weigh the plant biomass. The measurement results are as follows: Figure 2 shown. Figure 2 In the figure, the horizontal axis "control" represents the test results of tomato plants without adding dipropyl disulfide, and the horizontal axis "volatiles" represents the test results of tomato plants with exogenous addition of dipropyl disulfide. Figure 2 As can be seen, treatment with dipropyl disulfide significantly increased tomato biomass and promoted tomato growth. Rhizosphere soil samples were collected using the shaking root method. The soil was passed through a 2mm soil sieve. Five plants from the same treatment were mixed together as a biological replicate, with three replicates per treatment. The rhizosphere soil was stored at -80°C, and DNA was extracted for soil microbial analysis.

[0033] Determination of soil microbial flora abundance

[0034] Soil DNA extraction

[0035] Weigh 0.25 g of tomato rhizosphere soil. Extract total soil DNA using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, CA, USA). Store at -20°C for subsequent experiments.

[0036] Drawing of the standard curve

[0037] Standard curve preparation: Amplify the corresponding gene fragments of soil bacteria and fungi, using them as templates. This involves PCR amplification of a clear, single target band, followed by gel excision and recovery under a UV imager, purification and ligation using a kit, plating on plates for blue-white screening, and plasmid extraction from positive clones. Plasmids of appropriate concentration and purity are diluted 10-fold to create a series of standard samples, which are then stored in a -20°C refrigerator. A standard curve consisting of at least five consecutive points is used, and quantitative fluorescence PCR is performed simultaneously with soil samples.

[0038] Formula for converting plasmid concentration (ng / μL) to template copy number concentration (CN)

[75]

[0039]

[0040] Real-time PCR analysis

[0041] Bacteria: A 20 μl reaction system contains 2 μl DNA, 9 μl SYBR Mixture, 0.2 / 0.2 μl (10 mM) upstream / downstream primers, and the remaining volume is made up with deionized water. Reaction conditions: 95°C initial denaturation for 5 min, 95°C denaturation for 50 s, 62°C annealing for 30 s, 72°C extension for 1 min, 30 cycles, and a final extension of 10 min at 72°C. Fungi: A 20 μl reaction system contains 2 μl DNA, 9 μl SYBR Mixture, 0.25 / 0.25 μl (10 mM) upstream / downstream primers, and the remaining volume is made up with deionized water. Reaction conditions: 94°C initial denaturation for 5 min, 94°C denaturation for 1 min, 58°C annealing for 1 min, 72°C extension for 1 min, 31 cycles, and a final extension of 10 min at 72°C.

[0042] Miseq high-throughput sequencing analysis

[0043] The bacterial 16S rRNA V3-V4 region was PCR amplified using the 338F / 806R sequence: 338F (5'-ACTCCTACGGGAGGCAGCAG-3'), 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The PCR amplification products were identified and separated using 2% agarose gel electrophoresis, and then purified using a DNA gel purification kit (Agarose Gel DNA Purification Kit, TakaRa). The purified products were then subjected to high-throughput sequencing, and the data results were quality controlled and analyzed using QIIME (Quantitative Insights Into Microbial Ecology, Version 1.9.0) software.

[0044] The fungal ITS1F / ITS2R sequences (ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2R (5'-GCTGCGTTCTTCATCGATGC-3')) were used for PCR amplification of the fungal ITS1 region. The PCR amplification products were identified and separated using 2% agarose gel electrophoresis, and then purified using a DNA gel purification kit (Agarose Gel DNA Purification Kit, TakaRa). The purified products were then subjected to high-throughput sequencing, and the data were quality-controlled and analyzed using QIIME (Quantitative Insights Into Microbial Ecology, Version 1.9.0) software.

[0045] The results of α-diversity analysis of rhizospheric bacterial communities in tomato plants with or without exogenous addition of dipropyl disulfide using the above method are as follows: Figure 3 The principal coordinate analysis results of the β-diversity of tomato rhizospheric bacterial communities based on the Bray-Curtis distance difference between tomato plants with and without exogenous addition of dipropyl disulfide are shown in Figure 2. Figure 4 The results of the effect of dipropyl disulfide on the abundance of bacteria and fungi in tomato rhizosphere soil are shown in Figure 5 shown. Figure 3 and Figure 5 In the figure, the horizontal axis "control" represents the test results of tomato plants without dipropyl disulfide addition, and the "volatiles" represents the test results of tomato plants with exogenous addition of dipropyl disulfide (+ORSV). As can be seen from Figure 3, dipropyl disulfide treatment significantly increased the number of OUT bacterial communities in the tomato rhizosphere. Figure 4 It can be seen that the principal coordinate analysis (PCoA) based on Bray-Curtis differences showed that the bacterial community treated with dipropyl disulfide was significantly different from that of the untreated control. Figure 5 It can be seen that the abundance of bacteria and fungi in the rhizosphere of tomatoes treated with dipropyl disulfide was significantly higher than that in the untreated treatment.

[0046] Example 2

[0047] Effects of dipropyl disulfide on phenylalanine and proline in tomato root exudates

[0048] The above-mentioned experimental device was used to study the effect of dipropyl disulfide on amino acid secretion from tomato roots. Tomato seedlings with 4 true leaves were planted in 38 μm nylon membrane bags containing 1 kg of sterilized soil. Two treatments were set up in the experiment: (1) exogenous addition of dipropyl disulfide (volatile); (2) no dipropyl disulfide addition (control). 20 days after planting, tomato root exudates were collected for analysis of amino acid composition of tomato root secretion. The collection method was as follows: the underground part of the tomato carefully collected from the plastic pot was rinsed with running water until the root surface was clean. Five plants from the same treatment were placed in a 250 ml beaker containing 200 ml of sterile deionized water. The water level was recorded. Each beaker contained 0.5 mM calcium chloride to maintain root osmotic pressure. To prevent light and dust from entering, the plants were surrounded by tin foil. The samples were collected under the light conditions of the plant incubator and water was added to the original water level in a timely manner. After 6 hours, the plants were removed and dried, and the underground parts were cut and weighed to obtain the root fresh weight data. Based on this, the root exudate concentration was adjusted to 1g fresh weight per 10mL. -1 . The roots were then dried to determine the root dry weight. Finally, they were filtered through a 0.22μm microporous filter membrane and stored at -20°C for later use. The root secretions of each treatment were standardized by measuring the total carbon content of the root secretions. Then 20mL of root secretions of each treatment were placed in each culture dish, sealed with plastic wrap, pierced with a toothpick, and placed in a freeze dryer after freezing at -20°C. After freeze-drying, 1mL of sterile ultrapure water was added to each dish to obtain a tomato root secretion concentrate. The types and contents of amino acids in the concentrate were detected and analyzed, and it was found that the contents of phenylalanine and proline in the root secretion concentrates obtained from the two treatments were quite different, such as Figure 6 As shown by Figure 6 It can be seen that dipropyl disulfide treatment significantly increased the content of phenylalanine and proline in tomato root exudates.

[0049] Example 3: Effect of dipropyl disulfide on tomato growth in sterilized soil

[0050] The above experimental device was used to study the effect of dipropyl disulfide in sterilized soil on tomato growth. Tomato seedlings with 4 true leaves were planted in 38 μm nylon membrane bags filled with 1 kg of sterilized soil. The soil was irrigated with 10 7CFU / g PGPR suspension, 20 ml was poured into each tomato plant and mixed thoroughly. The exogenously inoculated PGPR strains were Bacillus (B1) and Pseudomonas (P8) that had been isolated and identified. The experiment set up 6 treatments: (1) no inoculation / exogenous addition of dipropyl disulfide (0PT+), (b) no inoculation / no addition of dipropyl disulfide (0PT-), (c) inoculation of B1 strain / exogenous addition of dipropyl disulfide (1PT+), (d) inoculation of B1 strain / no addition of dipropyl disulfide (1PT-), (e) inoculation of P8 strain / exogenous addition of dipropyl disulfide (8PT+), (f) inoculation of P8 strain / no addition of dipropyl disulfide (8PT-). One tomato plant was transplanted in each device. 20 days after the tomato was planted, the tomato plants were taken out and the biomass and other related indicators were measured. The results are as follows. Figure 7 As shown. Figure 7 It can be seen that when inoculated with B1 and P8 respectively, the dipropyl disulfide treatment significantly increased the aboveground, underground and whole-plant biomass of tomatoes.

[0051] The above description is only a preferred specific embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention within the technical scope disclosed in the present invention, and these changes should be covered by the scope of protection of the present invention.

Claims

1. Application of dipropyl disulfide in promoting tomato growth; The tomatoes are potted tomatoes; the specific application method is: dissolving dipropyl disulfide in a surfactant, adding water to obtain a dipropyl disulfide solution, then placing the solution in a container, and allowing the dipropyl disulfide to evaporate outward from the container, and placing the container containing the dipropyl disulfide solution at the roots of the potted tomato plants; The purity of the dipropyl disulfide is 99 wt %, the surfactant is Tween-80, the mass ratio of the dipropyl disulfide to the surfactant is 1:(1.5-2.5), and the concentration of the dipropyl disulfide solution is 8-12 mmol / L; The rate of volatilization of dipropyl disulfide is 0.8-1.3 μmol / h; The method of promoting tomato growth specifically includes: inducing tomatoes to increase the content of phenylalanine and proline secreted by the root system, increasing the abundance of bacteria and fungi in the rhizosphere soil microorganisms of tomatoes, changing the bacterial and fungal community structure, improving the rhizosphere microecological environment of tomatoes, promoting tomato growth, and improving the resistance of tomatoes to diseases.

2. The use according to claim 1, characterized in that The horizontal distance between the container and the tomato plant is 4 to 5 cm.

3. The use according to claim 1, characterized in that After the tomato plants are established and have grown, a container containing the dipropyl disulfide solution is placed at the roots of the tomato plants.

4. The use according to claim 3, characterized in that The time for dipropyl disulfide to volatilize outwards is from the time the seedlings are transplanted to the time the tomatoes are harvested.

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