Application of 2-naphthalenesulfonic acid and its derivatives in preventing and controlling plant diseases caused by plant pathogens

Abstract

The present invention relates to the field of biological control technology, and in particular to the application of 2-naphthalenesulfonic acid and its derivatives in preventing and treating plant diseases caused by plant pathogens. The present invention proposes for the first time the use of 2-naphthalenesulfonic acid or its pharmaceutically acceptable derivatives as a method for preventing and treating plant diseases caused by plant pathogens, and determines the concentrations used in antibacterial and inducing resistance, respectively. The present invention has a significant effect on preventing and treating plant diseases caused by plant pathogens, can induce and enhance the resistance of the plant itself, and also has good antibacterial properties; in particular, it has a significant effect on inhibiting the growth of Fusarium solani and reducing the lesion area of ​​Alternaria ginseng, can effectively prevent and treat root rot and black spot, and provides a new control agent for preventing and controlling plant diseases, and meets the requirements of sustainable development, and has broad research and market application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of biological control, in particular to application of 2-naphthalenesulfonic acid and its derivatives in preventing and controlling plant diseases caused by plant pathogens. Background Art

[0002] Root rot is a complex soil-borne disease. It is one of the most serious diseases of Panax notoginseng and an important factor affecting the industrial development of Panax notoginseng. The pathogens of Panax notoginseng root rot include fungi, bacteria, nematodes and other microorganisms, and Fusarium solani is the main and more typical root rot pathogen.

[0003] At present, the research on Panax notoginseng root rot mainly focuses on soil fumigation, adding organic matter, biological control and non-host crop rotation. Among them, soil fumigation and other treatment methods seriously affect the quality of medicinal materials, destroy soil structure and inhibit the reproduction of microorganisms in the soil; the application of chemical pesticides is only one of the means to temporarily alleviate plant diseases and insect pests, and the control effect is not stable, and it will also cause serious pollution of the ecological environment, unable to solve the problem from the root, and even lead to soil acidification and salinization; and the rotation period is generally required to be more than 30 years, which is difficult to implement on a large scale in actual production.

[0004] Black spot disease is a common disease in the cultivation and production of Panax notoginseng, with an annual incidence rate of 10% to 20%, and even up to 80% in severe cases. It has become one of the devastating diseases of Panax notoginseng, causing serious impact on the development of Panax notoginseng cultivation. Panax notoginseng black spot disease is caused by Alternaria ginseng, which mainly infects the leaves of Panax notoginseng and causes the disease. The stems, flower axis and young and tender parts of the fruit stalks can be affected, which seriously restricts the yield and quality of Panax notoginseng.

[0005] At present, chemical control is the main method for Panax notoginseng black spot disease, but excessive reliance on fungicides and the increasing use of fungicides can easily lead to excessive pesticide residues in Panax notoginseng and a decline in quality.

[0006] Due to the complexity of root rot and black spot and the limitations of current prevention and control measures, especially the reduced sensitivity of Panax notoginseng pathogens to many commonly used preparations, there is an urgent need to find a new substance that has a significant effect on preventing and controlling Panax notoginseng root rot and black spot. Summary of the invention

[0007] The present invention unexpectedly discovered during the research that, compared with other growth environments, some tree species, when planted together with Panax notoginseng, can effectively prevent and control diseases caused by plant pathogens that occur during the growth process of Panax notoginseng; and after detecting and analyzing a large number of metabolites secreted from the roots of relevant tree species, the present invention found that 2-naphthalenesulfonic acid or its pharmaceutically acceptable derivatives have a significant effect on preventing and controlling diseases of Panax notoginseng trees caused by plant pathogens.

[0008] Based on this, the present invention first provides the use of 2-naphthalenesulfonic acid or its pharmaceutically acceptable derivatives in preventing and treating plant diseases caused by plant pathogens.

[0009] The present invention further provides the use of 2-naphthalenesulfonic acid or its pharmaceutically acceptable derivatives as a preparation for preventing and treating plant diseases caused by plant pathogens.

[0010] Preferably, the preparation comprises a bacteriostatic agent or an inducer.

[0011] Preferably, the plant diseases include root rot or black spot.

[0012] Preferably, the plant pathogenic bacteria include Fusarium solani or Alternaria ginseng.

[0013] The present invention finds that 2-naphthalenesulfonic acid or its pharmaceutically acceptable derivatives are more effective in preventing and treating root rot caused by Fusarium solani or black spot caused by Alternaria ginseng.

[0014] Preferably, the plant belongs to the genus Panax; preferably, the plant is Panax notoginseng.

[0015] In the art, the pathogens of Panax notoginseng are similar to those of Panax notoginseng. Based on 2-naphthalenesulfonic acid or its pharmaceutically acceptable derivatives, the art can prevent and control diseases caused by plant pathogens in Panax notoginseng, and can apply it to other Panax notoginseng plants except Panax notoginseng according to actual needs.

[0016] The invention also provides a bacteriostatic agent for plant pathogenic bacteria, which contains 2-naphthalenesulfonic acid or a pharmaceutically acceptable derivative thereof.

[0017] The present invention finds that 2-naphthalenesulfonic acid or its pharmaceutically acceptable derivatives have inhibitory activity against plant pathogens and can be used to prepare antibacterial agents for plant pathogens.

[0018] Preferably, in the antibacterial agent, the concentration of 2-naphthalenesulfonic acid or a pharmaceutically acceptable derivative thereof is above 50 ppm.

[0019] The present invention finds that when the concentration of 2-naphthalenesulfonic acid or its pharmaceutically acceptable derivatives is within the above range, the antibacterial effect on plant pathogens such as Fusarium solani is better; and when the concentration is above 50 ppm, the antibacterial effect is more obvious as the concentration increases to a certain extent.

[0020] In the present invention, when studying the inhibitory concentration for other pathogens, it is also easy to set up a gradient test in the art to determine the appropriate inhibitory concentration.

[0021] The invention also provides a plant resistance inducer, which contains 2-naphthalenesulfonic acid or a pharmaceutically acceptable derivative thereof.

[0022] The present invention finds that 2-naphthalenesulfonic acid or its pharmaceutically acceptable derivatives can induce and enhance the resistance of plants themselves, and can be used to prepare plant resistance inducers to slow down the occurrence of diseases.

[0023] Preferably, the concentration of 2-naphthalenesulfonic acid or its pharmaceutically acceptable derivatives inducing disease resistance is above 0.1 ppm.

[0024] The present invention finds that 2-naphthalenesulfonic acid or its pharmaceutically acceptable derivatives can significantly improve plant resistance when the concentration is above 0.1 ppm.

[0025] In the present invention, when 2-naphthalenesulfonic acid or its pharmaceutically acceptable derivatives are used to prepare a preparation for preventing and treating plant diseases caused by plant pathogens, those skilled in the art can administer the drug by root irrigation, spraying or other conventional methods in the art according to actual conditions.

[0026] Based on the above technical solution, the beneficial effects of the present invention are:

[0027] (1) The present invention proposes for the first time the use of 2-naphthalenesulfonic acid or its pharmaceutically acceptable derivatives as a method for preventing and treating plant diseases caused by plant pathogens, and determines the concentrations used for inhibition and resistance induction, respectively.

[0028] (2) The present invention has a significant effect on preventing and controlling plant diseases caused by plant pathogens, can induce and enhance the resistance of the plant itself, and also has good antibacterial properties; in particular, it has a significant effect on inhibiting the growth of Fusarium solani and reducing the lesion area of ​​Alternaria ginseng, and can effectively prevent and control root rot and black spot disease, providing a new control agent for the prevention and control of plant diseases, and meeting the requirements of sustainable development. It has broad research and market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 The inhibitory effect of 2-naphthalenesulfonic acid provided by the present invention on Fusarium solani;

[0031] Figure 2 It is a plate photo of the 2-naphthalenesulfonic acid provided by the present invention inhibiting Fusarium solani; wherein, from left to right, the first plate is a control group, and the remaining plates are plate photos of the effects of 2-naphthalenesulfonic acid on Fusarium solani from low to high concentrations from left to right;

[0032] Figure 3This is the result of the induced resistance of 2-naphthalenesulfonic acid to Panax notoginseng black spot provided by the present invention;

[0033] Figure 4 is the number of Panax notoginseng seedlings remaining after root irrigation with 2-naphthalenesulfonic acid provided by the present invention;

[0034] Figure 5 is the dry weight of a single plant of Panax notoginseng roots after root irrigation with 2-naphthalenesulfonic acid provided by the present invention;

[0035] Figure 6 This is the inhibitory effect of spermidine in comparative example 1 on Fusarium solani;

[0036] Figure 7 The plate photos of the inhibition of Fusarium solani by spermidine in comparative example 1; wherein, from left to right, the first plate is the control group, and the remaining plates from left to right are plate photos of the effects of spermidine on Fusarium solani from low to high concentrations;

[0037] Figure 8 This is the result of the induction of resistance of stearic acid to Panax notoginseng black spot in comparative example 2;

[0038] In the above figure, CK is the control group. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0041] Since the tests in the following embodiments and comparative examples were completed at different times and in different batches, a separate control group was required each time.

[0042] Example 1 Antibacterial effect of 2-naphthalenesulfonic acid

[0043] This example provides the verified effect of 2-naphthalenesulfonic acid in inhibiting the growth of Fusarium solani. The specific steps include:

[0044] 1. Place 200 ml of PDA culture medium (40 g of potato, 3 g of agar, 4 g of glucose, and 200 ml of water) in a high-pressure steam autoclave for sterilization for 15 min at 121°C. After sterilization, place it in a clean bench for UV sterilization for 20 min.

[0045] 2. Prepare the compound into a solution: Dissolve 1g of 2-naphthalenesulfonic acid in 10ml of methanol to prepare a mother solution①

[0046] Take 7ml① and add 7ml methanol →②

[0047] Take 7ml② and add 7ml methanol →③

[0048] Take 7ml③ and add 7ml methanol→④

[0049] Take 7ml④ and add 7ml methanol→⑤

[0050] 3. After sterilization, use a pipette to take 800 μl of each of ⑤-① and add them to 200 ml of culture medium to obtain culture medium with concentrations of 25ppm, 50ppm, 100ppm, 200ppm and 400ppm respectively. For the control group, take 800 μl of methanol solution and add it to 200 ml of culture medium. Shake well and pour into a plate and cool.

[0051] 4. Collect Panax notoginseng with root rot in the field, clean the surface soil and other impurities with clean water, select the diseased and healthy junction of the root and cut it into 5mm×5mm size, disinfect it with 2% sodium hypochlorite for 5min in a clean bench, rinse it with sterile water 3 times, and then absorb the surface moisture with sterilized absorbent paper. Culture the disinfected diseased and healthy junction in PDA medium in a clean bench, one piece per dish, invert and culture it in the dark at a constant temperature of 25℃, and observe the growth of hyphae for about 7 days. After hyphae grow around the tissue, pick the hyphae and purify them on PDA medium. The strain obtained by purified culture is identified. The identification mainly uses DNA molecular identification method, combined with fungal morphological identification, to determine the taxonomic status of the isolated fungi. (1) Colony morphological identification: Inoculate the purified fungi on PDA medium, invert and culture it in the dark, and observe the color, size, morphology, etc. of the colonies after the hyphae grow into colonies. Pick a small amount of hyphae and place them on a glass slide, add a drop of sterile water, cover with a coverslip, place under a microscope to observe the morphology of the hyphae, the size and shape of the spores, etc., and take photos and record them. Refer to the "Handbook of Fungal Identification" and related literature to identify the fungi morphologically; (2) Molecular biological identification The purified fungi were inverted and cultured on PDA medium at 25°C in the dark. After one week, the grown hyphae were rinsed into a centrifuge tube with sterile water, and the genomic DNA was extracted using a fungal genomic DNA extraction kit (D2300, Solarbio). The steps were performed according to the instructions. The extracted genomic DNA was sent to Qingke Company for unidirectional sequencing using ITS and primers, and the complete ITS sequence was spliced ​​using the sequencing results. The obtained fungal sequences were compared and analyzed using the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / ) database to preliminarily determine the fungal species name. The fungus with the determined genus name was re-inoculated into the roots of Panax notoginseng, and isolated after the disease occurred. After the above identification, it was determined that the root rot fungus of Panax notoginseng was Fusarium solani.

[0052] 5. Use a 5 mm diameter puncher to punch the purified Fusarium solani into a 5 mm cake. Use an inoculation needle to inoculate the cake into the culture medium containing the compound solution. Place it in an incubator at 26-28°C for 7 days. After 7 days, observe the colony morphology and measure the colony diameter.

[0053] The results are shown in Figure 1 and Figure 2 .

[0054] like Figure 1 , Figure 2As shown, the results showed that when the concentration of 2-naphthalenesulfonic acid was 50 ppm, the mycelial growth of Fusarium solani was significantly lower than that of the control group, and the mycelium could not grow outward. As the concentration increased, the inhibitory effect became more obvious, indicating that 2-naphthalenesulfonic acid has a significant inhibitory effect on Fusarium solani.

[0055] Example 2 The induction effect of 2-naphthalenesulfonic acid

[0056] This example provides the resistance effect of 2-naphthalenesulfonic acid to black spot disease through potted plant experiments. The specific experimental steps are:

[0057] 1. Prepare the compound into a solution: Dissolve 1g of 2-naphthalenesulfonic acid in 100ml of methanol solution to prepare a mother solution①

[0058] Take 1ml① and add 9ml methanol→②

[0059] Take 1ml② and add 1ml methanol →③

[0060] Take 1ml③ and add 9.9ml methanol→④

[0061] 2. Take 1 ml of each of ②-④ and add 1L of water. Mix well and add 100 ml to each tissue culture bottle and place them in a white transparent storage box.

[0062] 3. Sterilize the potting soil with dry heat, then pot it and plant Panax notoginseng, 5 plants per pot. After 7 days, when the growth of Panax notoginseng is stable, add 50 ml of compound solution to each pot. After 24 hours, inoculate the Panax notoginseng black spot disease cake (5mm in diameter) on the Panax notoginseng leaves away from the main veins, and inoculate two leaves per Panax notoginseng plant. After the inoculation, spray sterilized water to keep the Panax notoginseng moist, cover the box and culture at 25-26℃ for 5 days. After 5 days, the Panax notoginseng leaves will be diseased. Remove the cake, take down the leaves, and use a scanner to scan the size of the leaf spots. After the scan, use Adobe Photoshop software to calculate the size of the spots. See the results. Figure 3 .

[0063] like Figure 3 As shown, the results show that 2-naphthalenesulfonic acid can significantly improve the resistance of Panax notoginseng plants at 0.1ppm and reduce the lesion area of ​​leaf black spot disease. When the concentration is greater than 0.5ppm, the lesion area can be significantly reduced. This shows that 2-naphthalenesulfonic acid can reduce the occurrence of black spot disease by enhancing the overall resistance of the plant.

[0064] Example 3 Effect of 2-naphthalenesulfonic acid on the survival rate of Panax notoginseng seedlings and the dry weight of Panax notoginseng single plant

[0065] This example verifies the effect of 2-naphthalenesulfonic acid on increasing the dry weight of Panax notoginseng plants. The specific experimental steps are:

[0066] 1. Prepare the compound into a solution: Dissolve 0.1 g of 2-naphthalenesulfonic acid in 10 ml of methanol to prepare a mother solution①

[0067] Take 1ml① and add 1ml methanol→②

[0068] Take 1ml① and add 2ml methanol→③

[0069] Take 1ml① and add 9ml methanol→④

[0070] 2. Take 1 ml of each of ②-④ and add them to 1L of water. After mixing, add 50 ml of the compound to each pot according to the concentration. For the control, take 1 ml of methanol and add it to 1L of water. Then continue the root irrigation experiment for 6 weeks (once a week). After Panax notoginseng continues to grow for two months, the survival rate of Panax notoginseng is calculated (the results are as follows Figure 4 ), then collect the samples of the potted Panax notoginseng, wash them, put them into envelopes, kill them at 115℃ for 30 minutes, dry them at 60℃ for 3 days, weigh them, and calculate their dry weight (the results are as follows Figure 5 ).

[0071] like Figure 4 As shown, the results showed that there was no significant difference in the survival of Panax notoginseng seedlings after treatment with 2-naphthalenesulfonic acid compared with the control, and there was a trend of increasing the survival of seedlings.

[0072] like Figure 5 As shown, the experimental results showed that the application of 2-naphthalenesulfonic acid to the roots can increase the dry weight of a single Panax notoginseng root at 1 ppm, and can significantly increase the dry weight of a single Panax notoginseng root at 3 ppm. Therefore, the application of 2-naphthalenesulfonic acid can significantly increase the dry weight of Panax notoginseng roots.

[0073] Comparative Example 1

[0074] This comparative example provides another substance detected in soil metabolic secretions - spermidine, and tests its inhibitory effect on Fusarium solani. The specific experimental steps are the same as those in Example 1, except that 2-naphthalenesulfonic acid is replaced by spermidine.

[0075] The results are as follows Figure 6 , Figure 7 As shown, after adding spermidine, at concentrations above 25 ppm, the diameter of the bacterial cake on the plate was significantly higher than that of the control. The results showed that spermidine promoted the growth of Fusarium solani at concentrations of 25 ppm and above.

[0076] Comparative Example 2

[0077] This comparative example verifies the resistance effect of stearic acid, another substance detected in soil metabolic secretions, on black spot disease through a bottle culture experiment. The specific experimental steps are the same as those of Example 2, except that 2-naphthalenesulfonic acid is replaced by stearic acid.

[0078] The results are as follows Figure 8As shown, the results showed that stearic acid could not induce resistance to Panax notoginseng leaf black spot disease.

[0079] In addition to the compounds listed in the above comparative examples, the present invention also verified other numerous compounds obtained from soil metabolic secretions, and found that many other compounds were unable to simultaneously inhibit Fusarium solani and induce resistance to black spot disease, and they will not be listed one by one here.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of 2-naphthalenesulfonic acid in preventing and treating plant root rot caused by Fusarium solani; the plant is Panax notoginseng; the concentration of 2-naphthalenesulfonic acid is above 50 ppm.

2. Application of 2-naphthalenesulfonic acid in preventing and treating plant black spot disease caused by Alternaria ginseng; the plant is Panax notoginseng; the concentration of 2-naphthalenesulfonic acid is above 0.1 ppm. 3.2-naphthalenesulfonic acid is used as a preparation for preventing and treating plant root rot caused by Fusarium solani; the plant is Panax notoginseng; the preparation is an antibacterial agent; in the antibacterial agent, the concentration of 2-naphthalenesulfonic acid is above 50 ppm. 4.2-naphthalenesulfonic acid is used as a preparation for the prevention and treatment of plant black spot disease caused by Alternaria ginseng; the plant is Panax notoginseng; the preparation is an inducer; in the inducer, the concentration of 2-naphthalenesulfonic acid is above 0.1 ppm.

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