Application of sakuranetin in prevention and treatment of sugarcane smut

Abstract

The application provides application of prunol in prevention and treatment of sugarcane smut. It is verified through experiments that prunol can inhibit growth of haploid strains JG35 and JG36 of sugarcane smut fungus, and the inhibition on sexual compatibility of the two strains is more prominent, which provides a new way for green prevention and control of sugarcane smut. The method provided by the application has the advantages of green and no residue, simple operation, stable prevention and control effect and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant protection technology, and in particular to the application of a cherry blossom extract in the control of sugarcane smut. Background Technology

[0002] Sugarcane is a perennial grass native to tropical and subtropical regions, widely cultivated globally, and of significant economic value. It has extensive applications in agriculture, food, and industry, and is a major crop for renewable bioenergy. Currently, approximately 160 types of sugarcane diseases have been identified worldwide. Due to differences in the main varieties cultivated in different sugarcane growing areas, varying susceptibility to different pathogens among varieties, and long-term continuous cropping, the types and severity of sugarcane diseases are increasing year by year. Currently, the most serious diseases affecting sugarcane quality and yield include sugarcane smut, top rot, rust, brown spot, white streak, and ratoon dwarf disease, posing serious threats to sugarcane production. Among these, sugarcane smut is the most damaging, causing yield losses of 10%-50% and a decrease in sugar content of 0.5-1 percentage points, severely threatening the stability and development of the sugarcane industry.

[0003] Sugarcane smut is a fungal disease caused by *Ustilago maydis*, a fungus belonging to the genus *Ustilago maydis* of the class Basidiomycetes, and is primarily transmitted through airborne transmission. The complete growth and development of *Ustilago maydis* involves three stages: diploid teliospores, haploid spores, and dikaryotic mycelium. The chlamydospores of *Ustilago maydis* germinate into basidiospores under moist conditions. Each basidiospore contains four haploid basidiospores in haploid cell form. These basidiospores are classified into two mating haploid types: "+" and "-". Different mating haploid types sexually combine to form dikaryotic mycelium, causing disease in sugarcane.

[0004] Sugarcane smut fungus infects sugarcane through both direct and indirect infection. When teliospores of sugarcane smut fungus infect young buds, they germinate basidia. The basidia produce hyphae that spread via plasmodesmata, directly parasitizing the meristem and intercellular spaces of the young buds. These hyphae absorb nutrients from healthy cells and stimulate the apical meristem, accelerating cell differentiation. Infected sugarcane shows initial symptoms in the 2nd to 4th month. Compared to healthy plants, infected plants have smaller stem circumferences and are generally slender. After 6-7 months, a long, unbranched, downward-curling, black whip-like structure emerges from the top, composed of sugarcane parenchyma cells, vascular bundle cells, and sugarcane smut teliospores. Once the whip breaks through the leaf sheath, spores are released from the membrane and spread widely on the ground. The chlamydospores of sugarcane smut fungus are highly adaptable to the environment, making the disease difficult to control once it occurs.

[0005] Breeding and promoting new disease-resistant sugarcane varieties is currently the most widely used method for controlling sugarcane smut, such as Guitang 28 and Guitang 02-761. However, the pathogenicity of sugarcane smut fungus differentiates rapidly, and new races may become dominant, causing newly bred resistant varieties to gradually lose their resistance and become susceptible. Furthermore, the breeding cycle is long. Chemical agents such as propiconazole and triadimefon can effectively inhibit the germination of chlamydospores of sugarcane smut fungus, but due to the thick skin of sugarcane stalks, ordinary agents are difficult to penetrate, and the economic cost is high, while also posing certain environmental hazards. With increasing public concern about food safety and the ecological environment, exploring new strategies for green and sustainable crop production and pathogen control is a new requirement to ensure the stable and healthy development of the agricultural industry. Developing new green and pollution-free antibacterial agents aligns with the concepts of healthy and high-quality sustainable development. Plant protection agents, possessing both environmental protection and stress resistance functions, can be applied to biological control and show great promise. Summary of the Invention

[0006] This invention provides the application of cherry blossom extract in the control of sugarcane smut.

[0007] In a first aspect, the present invention provides the application of sakura extract in the prevention and control of sugarcane smut.

[0008] Secondly, this invention provides the application of cherry blossom extract in the preparation of products for the prevention and control of sugarcane smut.

[0009] As described above, the CAS No. of the sakuranetin is 2957-21-3, and its English name is Sakuranetin.

[0010] As described above, the sugarcane smut is caused by *Ustilago maydis*.

[0011] As described above, the sugarcane smut fungus is formed by crossbreeding sugarcane smut haploid JG35 and sugarcane smut haploid JG36.

[0012] As described above, the prevention and control measures include at least one of the following: prevention, mitigation, control, and treatment of sugarcane smut. The core objective is to effectively reduce the adverse effects of sugarcane smut on sugarcane growth and ensure plant health.

[0013] Thirdly, the present invention provides the application of cherry blossom extract in inhibiting the growth of sugarcane smut fungus.

[0014] Fourthly, the present invention provides the application of cherry blossom extract in the preparation of products that inhibit the growth of sugarcane smut.

[0015] As described above, the sugarcane smut fungus is selected from at least one of sugarcane smut haploid JG35, sugarcane smut haploid JG36, and mycelium formed by crossbreeding sugarcane smut haploid JG35 and sugarcane smut haploid JG36.

[0016] Fifthly, the present invention provides a method for preventing and controlling sugarcane smut, comprising contacting cherry blossom extract with sugarcane to prevent and control sugarcane smut.

[0017] In a sixth aspect, the present invention provides a method for inhibiting the growth of sugarcane smut, comprising contacting safflower extract with sugarcane smut to inhibit the growth of sugarcane smut.

[0018] As described above, the sugarcane smut fungus is selected from at least one of sugarcane smut haploid JG35, sugarcane smut haploid JG36, and mycelium formed by crossbreeding sugarcane smut haploid JG35 and sugarcane smut haploid JG36.

[0019] This invention provides the application of safflower extract in the control of sugarcane smut. Experimental verification shows that safflower extract can inhibit the growth of sugarcane smut fungus, providing a new approach for the green control of sugarcane smut. The method provided by this invention has advantages such as being green and residue-free, simple to operate, and having stable control effects, and has broad application prospects. Attached Figure Description

[0020] Figure 1 The effect of different concentrations of cherry blossom extract on the vegetative growth of haploids of sugarcane smut JG35 or JG36.

[0021] Figure 2 The effect of different concentrations of cherry blossom extract on the concentration of haploid spores of *Ustilago maydis* JG35 or JG36. Figure 3 The effect of different concentrations of cherry blossom extract on sexual mating of *Smuts spp.* in sugarcane; Figure 4 To observe under a microscope the effects of different concentrations of cherry blossom extract on the morphology of dikaryotic hyphae formed after sexual mating of *Ustilago maydis*. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0024] The sugarcane smut fungi JG35 and JG36 involved in the following examples are described in the following literature: Host-induced gene silencing of Sporisorium scitamineum enhances resistance to smut insugarcane.

[0025] The CAS number of the sakuranetin used in the following examples is 2957-21-3, the structural formula is shown in Formula 1, the English name is Sakuranetin, and it was purchased from MCE Company, with the product number HY-N3006.

[0026] .

[0027] The YEPS culture medium involved in the following examples includes: 10 g / L yeast extract, 20 g / L peptone, 20 g / L sucrose, pH adjusted to 6.3 using NaOH / HCl, and sterilized by high temperature and high pressure.

[0028] The YEPS plates involved in the following examples include: 10 g / L yeast extract, 20 g / L peptone, 20 g / L sucrose, and 20 g / L agar powder. The pH was adjusted to 6.3 using NaOH / HCl, and the plates were sterilized by high temperature and high pressure before solidification.

[0029] The 100mM Sakuranetin stock solution involved in the following examples: Sakuranetin powder was dissolved in DMSO to prepare a stock solution with a concentration of 28.629 g / L, filtered through a 0.22 μM filter membrane, and stored at -20°C.

[0030] The following examples involve 0.05 mM / 0.1 mM / 0.3 mM cherry blossom extract YEPS plates: yeast extract 10 g / L, peptone 20 g / L, sucrose 20 g / L, agar powder 20 g / L, pH adjusted to 6.3 using NaOH / HCl, autoclaved, 100 mM cherry blossom extract stock solution added before pouring, and plates poured before solidification.

[0031] The following examples involve 0.05% / 0.1% / 0.3% DMSO-YEPS plates: yeast extract 10g / L, peptone 20g / L, sucrose 20g / L, agar powder 20g / L, pH adjusted to 6.3 using NaOH / HCl, autoclaved, DMSO added before pouring, and plates poured before solidification.

[0032] Example 1: Effects of different concentrations of cherry blossom extract on the vegetative growth of haploids of sugarcane smut JG35 or JG36 Sugarcane smut fungi JG35 and JG36, stored at -80℃, were activated using YEPS plates and cultured at 28℃ for 2-3 days. A portion of the fungal cells was scraped and placed in YEPS culture medium and cultured at 28℃ and 220 rpm for 12 hours. A portion of the bacterial culture was then aspirated, and the OD values ​​of the sugarcane smut fungi JG35 and JG36 bacterial cultures were adjusted to approximately 0.7 using YEPS culture medium. The sugarcane smut fungi JG35 and JG36 bacterial cultures were then diluted 5 times, 25 times, and 125 times, respectively, using YEPS culture medium to obtain four different concentrations of bacterial cultures.

[0033] YEPS plates with concentrations of 0.05 mM, 0.10 mM, and 0.30 mM of safflower extract were prepared, along with corresponding proportions of YEPS plates containing DMSO as negative controls. 5 μL of JG35 and JG36 bacterial suspensions, from highest to lowest concentration, were dropped onto the plates, dried, and incubated upside down at 28°C. After 3 days of incubation, the growth of *Ustilago maydis* JG35 and JG36 was observed.

[0034] The results are as follows Figure 1 As shown in the figure, cherry blossom extract has an inhibitory effect on the haploid growth of sugarcane smut.

[0035] Example 2: Effect of different concentrations of cherry blossom extract on the concentration of haploid spores of *Ustilago maydis* JG35 or JG36. Sugarcane smut fungi JG35 and JG36, stored at -80℃, were activated on YEPS plates and cultured at 28℃ for 2-3 days. A portion of the fungal cells was scraped and placed in YEPS culture medium, and cultured at 28℃ and 220 rpm for 12 hours. YEPS culture medium with concentrations of 0.00 mM, 0.05 mM, 0.10 mM, and 0.30 mM of safflower extract was prepared. 5 mL of each of the sugarcane smut fungi JG35 and JG36 was transferred to 45 mL of YEPS culture medium containing safflower extract and cultured at 28℃ and 220 rpm for 12 hours. The spore concentration of each culture was counted using a hemocytometer, with each group repeated three times.

[0036] The results are as follows Figure 2As shown, cherry blossom extract can effectively inhibit the formation of haploid spores of *Ustilago maydis*.

[0037] Example 3: Effects of different concentrations of safflower extract on sexual mating of *Ustilago maydis*. Sugarcane smut fungi JG35 and JG36, stored at -80℃, were activated on YEPS plates and cultured at 28℃ for 2-3 days. A portion of the fungal cells was scraped and placed in YEPS culture medium and cultured at 28℃ and 220 rpm for 12 hours. A portion of the fungal culture was then aspirated, and the OD values ​​of the sugarcane smut fungi JG35 and JG36 were adjusted to approximately 0.7 using YEPS culture medium. The two fungal cultures were then mixed in equal proportions. The mixed fungal culture was diluted 5 times, 25 times, and 125 times using YEPS culture medium to obtain four different concentrations of fungal culture.

[0038] YEPS plates with concentrations of 0.05 mM, 0.10 mM, and 0.30 mM of safflower extract were prepared, and YEPS plates with DMSO added in the corresponding proportions were prepared as negative controls. 5 μL of the mixed bacterial solution was taken from high to low concentration and dropped onto the plates, dried, and incubated upside down at 28°C. After 3 days of incubation, the sexual mating of *Ustilago maydis* was observed.

[0039] The results are as follows Figure 3 As shown, it can be seen that cherry blossom has a strong inhibitory effect on the sexual mating of sugarcane smut.

[0040] Example 4: Microscopic observation of the effects of different concentrations of cherry blossom extract on the morphology of dikaryotic hyphae formed after sexual mating of *Ustilago maydis*. Sugarcane smut fungi JG35 and JG36, stored at -80℃, were activated on YEPS plates and cultured at 28℃ for 2-3 days. A portion of the fungal cells was scraped and placed in YEPS culture medium and cultured at 28℃ and 220 rpm for 12 hours. A portion of the fungal culture was then aspirated, and the OD values ​​of the sugarcane smut fungi JG35 and JG36 were adjusted to approximately 0.7 using YEPS culture medium. The two fungal cultures were then mixed in equal proportions to obtain a mixed fungal culture.

[0041] YEPS plates with concentrations of 0.00mM, 0.05mM, 0.10mM, and 0.30mM were prepared. 10μL of the mixed bacterial suspension was dropped onto each plate, dried, and covered with a coverslip. The plates were then incubated upright at 28℃. After 3 days of incubation, the coverslip was removed and the plates were placed on a slide to form a compact. Using a Zeiss Observer Z1 bright-field fluorescence microscope, the compact was placed upright under the lens. The objective lens was set to 1×, and the eyepiece lens to 40×. The light intensity and background sharpness were adjusted appropriately before photographing.

[0042] The results are as follows Figure 4 As shown in the figure, it can be seen that cherry blossom extract can effectively inhibit the growth of dikaryotic hyphae formed by sexual mating of *Ustilago maydis*.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of cherry blossom extract in the control of sugarcane smut.

2. Application of cherry blossom extract in the preparation of products for the prevention and control of sugarcane smut.

3. Use according to claim 1 or 2, characterized in that, The sugarcane smut disease is caused by *Smutus canis*.

4. Use according to claim 3, characterized in that, The sugarcane whip smut fungus is formed by the crossbreeding of sugarcane whip smut haploid JG35 and sugarcane whip smut haploid JG36.

5. Application of cherry blossom extract in inhibiting the growth of sugarcane smut.

6. Application of cherry blossom extract in the preparation of products that inhibit the growth of sugarcane smut.

7. Use according to claim 5 or 6, characterized in that, The sugarcane whip smut fungus is selected from at least one of the following: sugarcane whip smut haploid JG35, sugarcane whip smut haploid JG36, and mycelium formed by the mating of sugarcane whip smut haploid JG35 and sugarcane whip smut haploid JG36.

8. A method for controlling sugarcane smut, characterized by, This includes contacting cherry blossom extract with sugarcane to prevent sugarcane smut.

9. A method of inhibiting the growth of Smilax grandis by applying to the plant, soil or water in which the plant is growing a composition comprising a compound of formula (I) as defined in claim 1. This includes contacting cherry blossom extract with *Ustilago maydis* to inhibit its growth.

10. The method according to claim 9, characterized in that, The sugarcane whip smut fungus is selected from at least one of the following: sugarcane whip smut haploid JG35, sugarcane whip smut haploid JG36, and mycelium formed by the mating of sugarcane whip smut haploid JG35 and sugarcane whip smut haploid JG36.

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