Application of small molecule compounds combined with hot scalding treatment in the control of sugarcane smut

Abstract

This invention discloses the application of a small molecule compound combined with hot scalding treatment in the control of sugarcane smut. The small molecule compound is benzyl{4-[1-bromonaphthalen-2-yl)oxybutan-1-amine (N-benzyl-4-(1-bromonaphthalen-2-yl)oxybutan-1-amine, C 21 H 22 BrNO, combined with low-temperature hot water treatment at 42℃~48℃, can effectively inhibit the growth, sexual mating, and germination of sugarcane smut fungus, thereby inhibiting the occurrence of sugarcane smut. At the same time, the temperature of hot water soaking is significantly reduced, the soaking time is significantly shortened, and the germination of sugarcane seed buds is not affected. It has high application value in the prevention and control of sugarcane smut and the improvement of sugarcane yield.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology. More specifically, it relates to the application of a small molecule compound in the control of sugarcane smut and the application of a small molecule compound combined with hot water treatment in the control of sugarcane smut. Background Technology

[0002] Sugarcane smut is a common disease caused by *Sporisorium scitamineum*, primarily occurring in tropical and subtropical regions. The characteristic feature of sugarcane smut is a black, inward-curling whip-like growth at the top of the sugarcane, surrounded by a layer of dark brown teliospores. This disease typically occurs under humid, warm conditions and, in severe cases, can cause sugarcane rot, impacting its growth and yield.

[0003] Currently, the main methods for preventing and controlling sugarcane smut are: (1) Selecting disease-resistant varieties: Selecting sugarcane varieties with strong disease resistance for planting; (2) Removing pathogens: Timely removal of diseased plant debris and weeds in sugarcane fields to reduce the spread of pathogens; (3) Spraying fungicides: Spraying fungicides regularly during the peak period of disease; (4) Crop rotation and intercropping: Adopting crop rotation and intercropping systems to reduce the long-term continuous planting of the same plant species and reduce the probability of disease occurrence; (5) Soaking seeds in warm water: Soaking seed buds in water at 52-54℃ for 2-4 hours. Among them, hot water soaking is the main prevention and control measure. Hot water soaking can kill pathogens or inhibit their growth by using high temperature, thereby effectively reducing the occurrence and spread of diseases. However, long-term high temperature treatment has high requirements for the size and plumpness of seed buds. That is, for heat-resistant seed buds with smaller and plump buds (such as Yuetang 93-159, Yuetang 00-236, etc.), their germination rate is less affected by hot water soaking. However, for seed buds with larger and less plump buds (such as Xintaitang 22, etc.), the germination rate is significantly reduced after hot water soaking, and the growth rate is slow.

[0004] Therefore, it is necessary to develop a method that can reduce the temperature of hot water soaking and shorten the soaking time, while ensuring that the germination of sugarcane seed buds is not affected, so as to more effectively prevent and control sugarcane smut and increase sugarcane yield. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the defects and shortcomings of the existing sugarcane smut control technology, and to provide a method and application of small molecule compound combined with hot water treatment for the control of sugarcane smut, thereby solving the problem of insufficient existing sugarcane smut control technology.

[0006] The first objective of this invention is to provide an application of a small molecule compound combined with hot water treatment in inhibiting sugarcane smut, preventing sugarcane smut disease, or treating sugarcane seed buds.

[0007] The second objective of this invention is to provide the application of a small molecule compound in inhibiting sugarcane smut, preventing sugarcane smut disease, or treating sugarcane seed buds.

[0008] The third objective of this invention is to provide the application of a small molecule compound in the preparation of products that inhibit sugarcane smut, prevent sugarcane smut disease, or treat sugarcane seed buds.

[0009] The fourth objective of this invention is to provide an agent for inhibiting sugarcane smut or preventing sugarcane smut, and to provide a method for preventing sugarcane smut by combining a small molecule compound with hot scalding treatment.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention discovers, through research, the small molecule compound benzyl{4-[1-bromonaphth-2-yl)oxy]butyl}amine (molecular formula C 21 H 22 The combined hot water treatment with BrNO (PubChem CID 2182721) effectively inhibited the mycelial growth of *Ustilago maydis*, the pathogen of sugarcane smut, suppressed sexual maturation, and reduced the germination rate of teliospores. Simultaneously, the hot water treatment significantly reduced the soaking temperature and shortened the soaking time, while ensuring that sugarcane seed germination was not affected. This treatment can be applied to the prevention and control of sugarcane smut and to increase sugarcane yield. The structural formula of this small molecule compound is shown below:

[0012]

[0013] Therefore, the following applications are all within the scope of protection of this invention:

[0014] The application of the above-mentioned small molecule compounds or their derivatives, or pharmaceutically acceptable salts, combined with hot scalding treatment, in inhibiting sugarcane smut, controlling sugarcane smut, or treating sugarcane seed buds. The hot scalding treatment is a soaking treatment at 42℃~48℃.

[0015] The use of the above-mentioned small molecule compounds or their derivatives or pharmaceutically acceptable salts in inhibiting sugarcane smut or in the preparation of products that inhibit sugarcane smut.

[0016] The use of the above-mentioned small molecule compounds or their derivatives or pharmaceutically acceptable salts in the prevention and control of sugarcane smut or in the preparation of products for the prevention and control of sugarcane smut.

[0017] The use of the above-mentioned small molecule compounds or their derivatives or pharmaceutically acceptable salts in the treatment of sugarcane buds or in the preparation of products for the treatment of sugarcane buds.

[0018] The inhibition of sugarcane smut includes inhibiting the growth, sexual mating, or germination of sugarcane smut.

[0019] Based on this, the present invention provides an agent for inhibiting sugarcane smut or preventing sugarcane smut disease, containing benzyl{4-[1-bromonaphth-2-yl)oxy]butyl}amine or its derivatives or pharmaceutically acceptable salts thereof.

[0020] As one of the alternative implementation schemes, the above-mentioned pharmaceutical preparation also contains a pharmaceutically acceptable carrier.

[0021] Furthermore, the present invention also provides a method for preventing and controlling sugarcane smut, specifically, the method is as follows: add the above-mentioned agent to warm water at 42℃~48℃, and then put sugarcane seedlings in and soak them thoroughly.

[0022] The research data of this invention show that at a temperature of 45°C, when C 21 H 22 At a BrNO concentration of 2 μM, treatment for 10 min effectively inhibited sexual mating and hyphal formation of *Ustilago maydis*. When C... 21 H 22 When the BrNO concentration was 20 μM, the growth of *Ustilago maydis* on the culture medium was effectively inhibited after 10 min of treatment; when C 21 H 22 At a BrNO concentration of 25 μM, the growth of *Ustilago maydis* on the culture medium was completely inhibited after 10 min of treatment. Furthermore, when C... 21 H 22 When the BrNO concentration was 20 μM, the germination rate of sugarcane smut spores was significantly reduced after 2 min of treatment, and the incidence of sugarcane smut disease was significantly reduced after 15 min of treatment on sugarcane seed buds, while the germination and growth rate of sugarcane seed buds were not affected.

[0023] Therefore, in the method for controlling sugarcane smut, the concentration of benzyl{4-[1-bromonaphth-2-yl)oxy]butyl}amine or its derivatives or pharmaceutically acceptable salts in the agent is not less than 2 μM.

[0024] Preferably, the concentration of benzyl{4-[1-bromonaphth-2-yl)oxy]butyl}amine or its derivatives or pharmaceutically acceptable salts in the pharmaceutical preparation is not less than 20 μM.

[0025] More preferably, the concentration of benzyl{4-[1-bromonaphth-2-yl)oxy]butyl}amine or its derivatives or pharmaceutically acceptable salts in the pharmaceutical preparation is not less than 25 μM.

[0026] In practical applications, taking into account factors such as cost and efficacy, the concentration of benzyl{4-[1-bromonaphth-2-yl)oxy]butyl}amine or its derivatives or pharmaceutically acceptable salts in the agent is 20 μM to 25 μM; the most preferred concentration is 25 μM.

[0027] In addition, in the method for preventing and controlling sugarcane smut, the soaking time is not less than 2 minutes.

[0028] Preferably, the soaking time is not less than 10 minutes.

[0029] More preferably, the soaking time is not less than 15 minutes.

[0030] In practical applications, considering factors such as efficiency, it is recommended that the soaking time not exceed 2 hours.

[0031] Preferably, the soaking time is 10 min to 60 min.

[0032] More preferably, the soaking time is 15 min to 60 min.

[0033] The present invention has the following beneficial effects:

[0034] 1. This invention is the first to discover small molecule compound C. 21 H 22 The combination of BrNO and low-temperature scalding at 42℃~48℃ can effectively inhibit the growth, sexual mating, and teliospore germination of sugarcane smut, thereby inhibiting the occurrence of sugarcane smut disease.

[0035] 2. The method for preventing and controlling sugarcane smut provided by this invention not only significantly reduces the temperature of hot water soaking and shortens the soaking time, but also ensures that the germination rate of sugarcane seed buds is not affected. It has high application value in preventing and controlling sugarcane smut and increasing sugarcane yield. Attached Figure Description

[0036] Figure 1 It is a small molecule compound C 21 H 22 Effects of BrNO combined with hot scalding on the growth of sugarcane smut. 5–25 μM of small molecule compound C... 21 H 22 The concentration of BrNO, 10 6 10 5 10 4 10 3 The concentration of the sugarcane smut fungus in the solution was 1×10. 6 cells / mL, 1×10 5 cells / mL, 1×10 4 cells / mL, 1×10 3 per mL.

[0037] Figure 2 It is a small molecule compound C 21 H 22Effects of BrNO combined with hot scalding on sexual mating and mycelial growth of *Ustilago maydis*.

[0038] Figure 3 It is a small molecule compound C 21 H 22 Effects of BrNO combined with hot water treatment on the germination of teliospores of *Ustilago maydis*.

[0039] Figure 4 Small molecule compound C 21 H 22 The effects of BrNO combined with hot water treatment on sugarcane seed bud treatment and control of sugarcane smut. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0041] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0042] The small molecule compound C used in the following examples 21 H 22 BrNO was purchased from ChemBridge Chemical Store (https: / / www.hit2lead.com / search.asp?db=SC&SearchPage=id), catalog number 7574494. The small molecule compound was dissolved in DMSO to prepare a 10 mM stock solution for later use.

[0043] The sugarcane smut basidiospores MAT-1 and MAT-2 used in the following examples were isolated, identified and preserved by the mycology laboratory of South China Agricultural University and disclosed in the literature "Transcriptome analysis of Sporisorium scitamineum reveals critical environmental signals for fungal sexual mating and filamentous growth".

[0044] The preparation methods of the culture media used in the following examples are as follows:

[0045] (1) YePS liquid culture medium: 10g yeast extract, 20g peptone, 20g sucrose, add deionized water to make up to 1L, sterilize at 121℃ for 20min.

[0046] (2) YePS solid medium: Add 2% agar powder to YePS liquid medium and sterilize at 121℃ for 20 min.

[0047] (3) Minimal media (MM): Add 2.05g K2HPO4, 1.45g KH2PO4, 0.5g NH4NO3, 0.3g (NH4)2SO4, 0.01g FeSO4, 0.01g CaCl2, and 2g glucose to 5mL Z-Buffer (Z-Buffer contains 0.01% Na2MoO4·2H2O, 0.01% ZnSO4·7H2O, 0.01% H3BO3, 0.01% CuSO4·5H2O, and 0.01% MnSO4·H2O), then add 20g agar powder, and then add deionized water to make up to 1L. Sterilize at 107℃ for 20min.

[0048] Example 1 Small molecule compound C 21 H 22 Effects of BrNO combined with hot scalding on the growth of sugarcane smut

[0049] 1. Use an inoculation loop to pick up sugarcane smut basidiospores MAT-1 and put them into 5 mL of YePS liquid medium. Place the medium in a shaker at 28°C and incubate at 200 rpm for 1-2 days. Centrifuge the cultured solution at 5000 rpm and collect the bacterial cells.

[0050] 2. Resuspend the bacterial cells in sterile water and adjust the bacterial solution to OD. 600 =1.0, 1 μL of small molecule compound C at different concentrations (2 mM, 4 mM, 8 mM, 10 mM) was added to 2 mL centrifuge tubes containing 400 μL of bacterial culture. 21 H 22 BrNO stock solution was prepared to final concentrations of 5 μM, 10 μM, 20 μM, and 25 μM. DMSO was added to 400 μL of bacterial culture as a control. The bacterial cultures treated above were then placed in water baths at 28 °C and 45 °C for 10 min, respectively.

[0051] 3. Dilute the treated bacterial solution with sterile water in a serial gradient to OD values. 600 The values ​​were 1.0, 0.1, 0.01, and 0.001 (i.e., bacterial concentrations of 1×10⁻⁶). 6 cells / mL, 1×10 5 cells / mL, 1×10 4 cells / mL, 1×10 3 (Cells / mL), use a pipette tip to draw 1.5 μL of bacterial suspension and drop it onto YePS solid medium. After drying, seal the plate and place it in a 28℃ incubator for 3 days.

[0052] Experimental results show (see) Figure 1 Small molecule compound C at 45℃ 21 H 22 The effective concentration of BrNO to inhibit the growth of sugarcane smut is 20 μM; when C 21 H 22 The growth of sugarcane smut was completely inhibited at a BrNO concentration of 25 μM.

[0053] Example 2 Small molecule compound C 21 H 22 Effects of BrNO combined with hot scalding on sexual mating and mycelial growth of *Ustilago maydis*.

[0054] 1. Using an inoculation loop, pick up basidiospores MAT-1 and MAT-2 of *Ustilago maydis* and place them in 5 mL of YePS liquid medium. Incubate at 28°C and 200 rpm for 1-2 days. Centrifuge the cultured solution at 5000 rpm and collect the bacterial cells.

[0055] 2. Resuspend the bacterial cells in sterile water and adjust the bacterial solution to OD. 600 =1.0, 1 μL of small molecule compound C at different concentrations (0.4 mM and 0.8 mM) was added to 2 mL centrifuge tubes containing 400 μL of MAT-1 and MAT-2 bacterial cultures, respectively. 21 H 22 Prepare BrNO stock solution to final concentrations of 1 μM and 2 μM, respectively; using DMSO as a control, add 1 μL of DMSO to 400 μL of MAT-1 and MAT-2 bacterial cultures; then place the above treatments in water baths at 28℃ and 45℃ for 10 min, respectively.

[0056] 3. Mix equal volumes of the treated MAT-1 and MAT-2 bacterial solutions, drop 1.5 μL of the bacterial solution onto MM solid medium, incubate at 28℃ for 12 h, and observe the mycelial growth of *Ustilago maydis* using a stereomicroscope.

[0057] Experimental results show (see) Figure 2 Untreated haploid spores of *Ustilago maydis* will sexually mate to produce diploid white mycelia; however, after treatment at 45℃, 1 μM of the small molecule compound C... 21 H 22 BrNO initially inhibits sexual mating and hyphal formation of *Ustilago maydis*, and at 2 μM, it can effectively inhibit sexual mating and hyphal formation of *Ustilago maydis*.

[0058] Example 3 Small molecule compound C 21 H 22 Effects of BrNO combined with hot scalding treatment on teliospore germination of *Ustilago maydis*

[0059] 1. Scrape teliospores from sugarcane smut-infected plants and prepare 1×10⁻⁶ spores using YEPS liquid medium. 6 A suspension of teliospores per mL.

[0060] 2. Add 1 μL of small molecule compound C at different concentrations (2 mM and 8 mM) to 2 mL centrifuge tubes containing 400 μL of teliospore suspension. 21 H 22 BrNO stock solution was prepared to final concentrations of 5 μM and 20 μM, respectively; DMSO was added to 400 μL of teliospore suspension as a control; and the teliospore suspensions treated above were then placed in water baths at 28 °C and 45 °C for 2 min, respectively.

[0061] 3. After treatment, place the spores in a shaker at 28℃ and incubate for 6-8 hours. Observe the germination of teliospores at different concentrations using a stereomicroscope and calculate the germination rate (germination rate = number of germinated spores / total number of spores × 100%).

[0062] Experimental results show (see) Figure 3 After being heated at 45℃, the 20μM small molecule compound C 21 H 22 BrNO significantly reduced the germination rate of teliospores of *Ustilago maydis*.

[0063] Example 4 Small molecule compound C 21 H 22 The effect of BrNO combined with hot scalding treatment on the control of sugarcane smut

[0064] The sugarcane buds used in this embodiment are large but not fully developed buds, and the variety is New Taiwan Sugar No. 22.

[0065] The specific experimental method is as follows:

[0066] 1. Scrape teliospores from sugarcane smut-infected plants and prepare a solution with a concentration of 1×10⁻⁶ ppm using sterile water. 4 Soak sugarcane seedlings in a suspension of teliospores of Ustilago maydis per mL for 10 min.

[0067] 2. Wrap the treated sugarcane sprouts in a damp paper towel and divide them into three groups of 12 sprouts each. One group is soaked in a 52℃ water bath for 2 hours; another group is soaked in a 45℃ water bath containing DMSO (1:1000, v / v) for 15 minutes; and the last group is soaked in a solution containing 20μM C. 21 H 22 Sugarcane seedlings were soaked in a 45°C warm water bath with BrNO (1:1000, v / v) for 15 minutes, and then photographed for record.

[0068] 3. After keeping the treated sugarcane buds moist with a damp paper towel, place them in a 28℃ light incubator. After 4 days, take photos to record the germination of the treated sugarcane buds and plant them in the soil. After 120 days, take photos to record the symptoms of "black whip" and count the incidence rate.

[0069] Experimental results show (see) Figure 4 The incidence rate was very high in the DMSO group. Figure 4 (Figure B) Although the disease incidence rate was significantly reduced in sugarcane treated with 52℃ warm water (Figure B), Figure 4 (Figure B in the middle), but its early germination is slow, and its growth is significantly affected. Figure 4 (Figure A); and after C 21 H 22 BrNO combined with 45℃ hot water treatment not only significantly reduced the incidence of sugarcane smut, but also had minimal impact on the early germination and growth of sugarcane seed buds. These results indicate that 20μM C 21 H 22 The combined BrNO treatment with hot water can effectively prevent the occurrence of sugarcane smut, without affecting the germination and growth rate of sugarcane seedlings.

[0070] In addition, we explored the temperature of the hot ironing process in the above embodiments, and the results showed that the temperature of the hot ironing process is feasible in the range of 42℃ to 48℃.

[0071] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of small molecule compounds or pharmaceutically acceptable salt-combined hot scalding treatments in inhibiting sugarcane smut or controlling sugarcane smut, characterized in that, The structure of the small molecule compound is shown below: ； The hot scalding treatment is a soaking treatment at 42℃~48℃; the soaking time is not less than 2 minutes. The concentration of the small molecule compound or its pharmaceutically acceptable salt is not less than 2 μM.

2. The application according to claim 1, characterized in that, The inhibition of sugarcane whip smut includes inhibiting the growth, sexual mating, or germination of sugarcane whip smut.

3. A method for controlling sugarcane smut, characterized in that, Add the small molecule compound or its pharmaceutically acceptable salt to warm water at 42℃~48℃, and then soak the sugarcane seedlings; the soaking time shall not be less than 2 minutes. The concentration of the small molecule compound or its pharmaceutically acceptable salt is not less than 2 μM; The structure of the small molecule compound is shown below: 。 4. The method according to claim 3, characterized in that, The concentration of the small molecule compound or its pharmaceutically acceptable salt is not less than 20 μM.

5. The method according to claim 4, characterized in that, The concentration of the small molecule compound or its pharmaceutically acceptable salt is 20 μM to 25 μM.

6. The method according to claim 3, characterized in that, The soaking time shall not be less than 10 minutes.

7. The method according to claim 3, characterized in that, The soaking time shall not exceed 2 hours.

8. The method according to claim 3, characterized in that, The soaking time is 10 min to 60 min.

9. The method according to claim 3, characterized in that, The soaking time is 15 min to 60 min.

Images