Application of methyl zelamin in inhibiting plant pathogenic fungi

Abstract

This invention discloses the application of norzelamin in inhibiting plant pathogenic fungi, belonging to the field of plant protection. This invention confirms that norzelamin has an inhibitory effect on crop anthracnose fungi such as *Anthracnose siamensis* and *Fusarium*. Therefore, the compound norzelamin provided by this invention can be used to prepare small-molecule inhibitors targeting the growth, development, and pathogenicity of crop anthracnose fungi or *Fusarium*, and is expected to become a potential drug against crop anthracnose fungi and *Fusarium*, possessing good development and application value.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology, specifically relating to the application of methyl zelamin in inhibiting plant pathogenic fungi. Background Technology

[0002] Fungal diseases affecting crops, such as anthracnose, wilt, leaf blight, Fusarium head blight, and root rot, cause global agricultural losses exceeding $30 billion annually. Anthracnose comprises 15 species complexes and can infect over 100 crop species, with hosts including fruits, vegetables, cash crops, and forest trees. Fusarium species include more than 44 species, with *Fusarium oxysporum* being the most prominent. F. oxysporum ) and Fusarium graminearum ( F. graminearum Anthracnose and Fusarium are the main representatives, infecting grasses, solanaceae, and tuber crops. Due to their rapid infection rate, long infection duration, high genetic diversity, and dual saprophytic and parasitic abilities, they are extremely difficult to control.

[0003] Anthracnose survives as mycelium, conidiophores, or conidia in diseased plant debris, seed surfaces, and soil. Under suitable conditions, it can directly penetrate the host's plast layer and cell wall, or invade through natural openings such as stomata and lenticels. Numerous conidia on the surface of lesions are spread by wind, rain, and insects, leading to reinfection and exacerbating the disease. Fusarium overwinters as mycelium, chlamydospores, and conidia in diseased plant debris, soil, infected green manure, and seeds. It invades through root wounds or natural openings, multiplies within the vascular bundles, and secretes toxins, damaging vascular tissue and causing wilting, death, and even plant death. It spreads via irrigation water, soil, and agricultural implements, and can also travel long distances through atmospheric deposition and air currents. Especially in tropical regions, the pathogen's generation cycle is greatly shortened, posing a greater risk to control. Currently, the control of fungal diseases in crops mainly relies on chemical pesticides, such as chlorothalonil, carbendazim, thiophanate-methyl, and prochloraz. However, due to long-term and frequent use, many pathogens have developed resistance to these agents in recent years, posing a significant risk of pesticide residue exceeding standards. Therefore, there is a need to develop more efficient, environmentally friendly, and safer pesticides against fungi.

[0004] Norzemarin is an effective monomeric triterpenoid compound extracted from the traditional Chinese medicine Tripterygium wilfordii. Current research only discloses its therapeutic effects on human diseases. However, it offers no technological implications for plant fungal diseases. To date, there are no reports on the use of norzemarin for the control of plant pathogenic fungi. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a new application of nor-zelamin in inhibiting anthracnose and Fusarium. This invention has found that nor-zelamin has a significant inhibitory effect on the radial growth of plant pathogenic fungi such as *Anthracnose siamensis*, *Anthracnose graminearum*, *Anthracnose glomeratus*, *Anthracnose scutellarioides*, *Anthracnose hygroscopicus*, *Fusarium oxysporum*, *Fusarium graminearum*, *Fusarium moniliforme*, *Fusarium equisetifolium*, *Fusarium oxysporum*, and *Fusarium solani*. It also significantly inhibits sporangium release, zoospore germination, and pathogenicity of *Anthracnose*, and significantly inhibits the pathogenicity of *Fusarium*.

[0006] The technical solution of this invention mainly includes the following:

[0007] The use of norzelamin in inhibiting plant pathogenic fungi or in the preparation of plant pathogenic fungi inhibitors, said plant pathogenic fungi including Fusarium and / or Anthracnose.

[0008] The structural formula of nor-zelamin is:

[0009] Furthermore, the Fusarium includes Fusarium graminearum. Fusarium graminearum Fusarium oxysporum Fusarium oxysporum Fusarium moniliforme Fusarium proliferatum Horsetail (Fusarium oxysporum) Fusarium equiseti Fusarium oxysporum Fusarium culmorum Fusarium solani Fusarium solani One or more of the above, wherein the anthrax bacteria include Siamese anthrax bacteria. Colletotrichum siamense、 Anthracnose of Gramineae Colletotrichum graminicola Anthrax bacillus Colletotrichum gloeosporioides Anthrax bacillus flathead Colletotrichum truncatum Watermelon anthracnose Colletotrichum orbiculare One or more of them.

[0010] Furthermore, the application includes at least one of the following (1)-(4):

[0011] (1) Inhibits radial growth of Fusarium and / or Anthrax hyphae;

[0012] (2) Inhibits the production of anthrax conidia;

[0013] (3) Inhibits the germination of anthrax conidia;

[0014] (4) Inhibits the pathogenicity of Fusarium and / or Anthrax.

[0015] Furthermore, the plant includes mango, chili pepper, watermelon, or tomato.

[0016] A method for controlling plant fungal diseases includes the following steps: applying methyl zelamaldehyde or an agent containing methyl zelamaldehyde to the plants to control fungal diseases caused by Fusarium and / or Anthracnose.

[0017] Furthermore, the application to plants includes application to plant stems, leaves, roots, or fruits.

[0018] Furthermore, this invention does not specifically limit the dosage of norzelamin. Those skilled in the art can flexibly adjust the dosage of norzelamin based on the actual pathogen content or the incidence of plant diseases. Norzelamin can also be used in combination with other antibacterial agents to enhance the antibacterial effect or reduce side effects, thereby preventing and controlling anthracnose and wilt diseases and preserving fruits and vegetables.

[0019] Under the experimental conditions of this invention, when the final concentration of methyl zelaminol reaches at least 100 ng / mL, it exhibits good antibacterial or disease-resistant effects, and the effect increases with the increase of the methyl zelaminol concentration. More specifically, when the final concentration of methyl zelaminol reaches 3 μg / mL to 35 μg / mL, it has a significant antibacterial effect against anthracnose and Fusarium; when the final concentration of methyl zelaminol reaches 100 ng / mL to 3000 ng / mL, it has a significant antibacterial effect on the production and germination of conidia of Anthracnose in Siam; and when the final concentration of methyl zelaminol reaches 2 μg / mL to 60 μg / mL, it has a good control effect on mango anthracnose.

[0020] The beneficial effects of this invention are:

[0021] This invention is the first to discover that methyl zelamin has the effect of controlling anthracnose and Fusarium in crops. Studies have shown that methyl zelamin exhibits significant inhibitory activity against *Anthracnose siamensis*, *Anthracnose graminearum*, *Anthracnose glomerulosa*, *Anthracnose scutellarioides*, *Anthracnose hygroscopicus*, *Fusarium oxysporum*, *Fusarium graminearum*, *Fusarium moniliforme*, *Fusarium equisetifolium*, *Fusarium oxysporum*, and *Fusarium solani*. methyl zelamin significantly reduces the lesion area on mango fruits inoculated with *Anthracnose siamensis*. Experiments have confirmed the efficacy of methyl zelamin in controlling mango anthracnose, thus achieving the objective of this invention and having significant implications for the control of crop anthracnose.

[0022] Nor-Zeramol is a natural plant extract used in the food and pharmaceutical fields. It is safe, environmentally friendly, and sustainable, and is not prone to developing resistance. It has great development and application value in the control of crop wilt during the growing season and post-harvest anthracnose in fruits. Attached Figure Description

[0023] Figure 1This is a graph showing the inhibition of radial growth of *Anthracis sinensis* hyphae by methyl zelamin of the present invention; wherein, a: graph showing the inhibitory effect of methyl zelamin on radial growth of *Anthracis sinensis*; b: statistical analysis graph showing the growth diameter of *Anthracis sinensis* after treatment with methyl zelamin; c: statistical analysis graph showing the inhibition rate of growth of *Anthracis sinensis* by methyl zelamin.

[0024] Figure 2 This is a graph showing the inhibition of conidia of *Anthracis sinensis* by norzelamin in this invention; where a: the inhibitory effect of norzelamin on sporulation of *Anthracis sinensis*; b: a statistical analysis graph of the inhibition rate of sporulation of *Anthracis sinensis* by norzelamin.

[0025] Figure 3 This is a graph showing the inhibition of conidia germination by nor-zelamin of the present invention; wherein, a: graph showing the inhibitory effect of nor-zelamin on conidia germination of *Anthracis sinensis*; b: statistical analysis graph showing the inhibition rate of nor-zelamin on conidia germination of *Anthracis sinensis*.

[0026] Figure 4 This invention relates to the effect of methyl zelamin on the pathogenicity of *Anthracis sinensis*. Specifically, a: an image showing the infection effect of *Anthracis sinensis* on mangoes after methyl zelamin treatment; b: a statistical analysis of the area of ​​*Anthracis sinensis* lesions on mangoes after methyl zelamin treatment.

[0027] Figure 5 This is a diagram showing the inhibition of radial hyphal growth of different anthracnose fungi by norzelaminar in this invention; where a: norzelaminar inhibits the growth of anthracnose fungi (…). C. graminicola Anthracnose (capsicum anthracnose) C. gloeosporioides ), flathead anthrax ( C. truncatum ) and watermelon anthracnose ( C. orbiculare a: Radial growth inhibition effect diagram; b: Statistical analysis diagram of the radial growth inhibition rate of nor-zelamin against different anthrax bacteria.

[0028] Figure 6 This is a diagram showing the inhibition of radial hyphal growth of different Fusarium fungi by nor-zelaminar aldehyde according to the present invention; wherein, a: the inhibition of radial hyphal growth of Fusarium graminearum by nor-zelaminar aldehyde (… F. graminearum Fusarium oxysporum ( F. oxysporum Fusarium moniliforme () F. proliferatum ), Fusarium oxysporum ( F. equiseti ), Fusarium oxysporum ( F. culmorum ) and Fusarium solani ( F. solani a) Inhibition effect on radial growth; b) Statistical analysis of the radial growth inhibition rate of nor-zelamin against different Fusarium species. Detailed Implementation

[0029] To facilitate a better understanding of the technical content of this invention by those skilled in the art, the invention will be further described in detail below through specific embodiments.

[0030] Unless otherwise specified, all reagents and agents used in the following examples are commercially available products, and all experimental methods are conventional methods. All pathogens tested were isolated, purified, identified, and preserved by the inventor's laboratory using conventional methods.

[0031] Example 1: Effect of nor-zelamin on the antibacterial activity of Bacillus anthracis in Siamese bacteria.

[0032] Test pathogen: *Anthrax sicca* C. siamense

[0033] Main reagent: Norzemarin was purchased from Sichuan Jingcui Tiancheng Pharmaceutical Technology Co., Ltd., with a purity of 95-99%.

[0034] PDA agar medium is used to culture Siamese anthrax bacteria.

[0035] The inhibitory effect of different concentrations of nor-zelaminaraldehyde on the mycelial growth of pathogenic fungi was determined using the mycelial growth rate method. Specific procedures included:

[0036] 1) Dissolve demethylzelamide aldehyde in DMSO to prepare a mother liquor;

[0037] 2) Melt the V8 culture medium and cool it to 50°C. At approximately C, des-Zeramol stock solution was added to the culture medium and mixed well to achieve final concentrations of des-Zeramol in the culture medium of 3 μg / mL, 10 μg / mL, 18 μg / mL, 27 μg / mL, and 35 μg / mL, respectively. Four replicates were set up for each concentration treatment. A control was prepared by adding the same volume of solvent with half-inhibitory concentration. 20 mL of culture medium was poured into a 9 cm diameter culture dish.

[0038] 3) Using a sterile pipette tip, collect bacterial cakes (d=5 mm) of uniform age from the edge of the activated and cultured anthrax bacteria colony on PDA. Inoculate the bacterial cakes into the center of PDA medium plates containing different concentrations of norzelamin, place them in a 25 ℃ constant temperature incubator for dark incubation, and observe, measure and record their diameters regularly and take photos.

[0039] See results Figure 1 Norzevalerate has a significant antibacterial effect against Siamese anthrax bacteria. When the concentration of norzevalerate reaches 12.94 μg / mL, the antibacterial rate against Siamese anthrax bacteria reaches 50%.

[0040] Example 2: Effect of nor-Zeramol on the antibacterial activity of *Anthrax sicca* conidia

[0041] Test pathogen: *Anthrax sicca* C. siamense

[0042] Preparation of pathogenic conidial suspension: Using an inoculation loop, scrape an appropriate amount of *Anthracis sicca* mycelium into 100 mL of PDB liquid medium and incubate for 28 hours. C. Incubate on a shaker at 180 rpm for 36 h, filter through four layers of gauze, and adjust the spore concentration to 1×10⁻⁶. 5 The spores were prepared as a suspension at a density of 1 spore per mL.

[0043] Prepare a stock solution containing norzelaminaraldehyde using the same method as in Example 1. Accurately add the prepared norzelaminaraldehyde stock solution to PDB medium to achieve final concentrations of norzelaminaraldehyde of 100 mL of PDB medium of 100 ng / mL, 300 ng / mL, 500 ng / mL, 700 ng / mL, and 1000 ng / mL, respectively. Each concentration treatment is performed in triplicate, with a control containing the same volume of solvent as the half-inhibitory concentration.

[0044] Take 20 μL of *Bacillus anthracis* conidia suspension and add them to PDB medium containing different concentrations of norzelaminaraldehyde. Incubate at 28°C. The culture was carried out at a constant temperature of 180 rpm in a shaker. The samples were taken out at 12 h and 24 h and observed under a microscope to observe the sporulation. Each bottle was counted 5 times.

[0045] Preparation of WA medium containing norzelaminaraldehyde: Melt the WA medium and then cool it to 50°C. At approximately 100°C, the stock solution of des-zelamin was accurately added to the culture medium and mixed thoroughly to achieve final concentrations of 100 ng / mL, 300 ng / mL, 700 ng / mL, 1500 ng / mL, and 3000 ng / mL, respectively. Each concentration treatment was performed in triplicate, with a control containing the same volume of solvent at half the inhibitory concentration. 15 mL of culture medium was poured into 9 cm diameter culture dishes.

[0046] Take 100 μL of anthrax bacillus conidia suspension and spread them on WA plates containing different concentrations of norzelaminaraldehyde. Incubate at 25 ℃. When the conidia of the control plate have germinated to more than 80%, take them out and observe their germination under a microscope. Count 5 times for each plate.

[0047] See results Figure 2 and Figure 3Norgelamin significantly inhibited both conidial production and germination of *Anthracis sinensis*. Treatment with norgelamin at concentrations above 100 ng / mL for more than 12 h significantly inhibited both conidial production and germination, while DMSO treatment showed no inhibitory effect. These results indicate that norgelamin significantly inhibits conidial production and germination of *Anthracis sinensis*.

[0048] Example 3: Effects of nor-zelamin on the pathogenicity and virulence of Bacillus anthracis

[0049] The tested bacterial strain was *Anthrax sicca*, and the tested mango variety was *Guifei*, purchased from the market.

[0050] After disinfecting the surface of mango fruits with 75% alcohol for 5 minutes, wash them three times with sterile water and air-dry them in a fume hood. The dried mango fruits were then immersed in DZL solutions of different concentrations (2 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL) for 8 minutes, air-dried, and inoculated with 0.5 cm diameter *Anthracis sinensis* mycelial blocks, with 6 blocks inoculated per fruit. A commercially available prochloraz drug with an active ingredient of 450 g / L, purchased from Shandong Xinbang Biochemical Co., Ltd., was used as a positive control. The inoculated fruits were kept moist and placed at 28 °C. After 6 days, photographs were taken, and the lesion area was statistically analyzed using ImageJ. One-way ANOVA was used for significance analysis. Each experimental group was repeated 5 times.

[0051] See results Figure 4 Nor-Zeramol has a good preventive effect against mango anthracnose, and the preventive effect increases with the increase of Nor-Zeramol concentration.

[0052] Example 4: Inhibitory effect of nor-zelamin on different anthrax bacteria

[0053] Tested pathogens: C. graminicola, C. gloeosporioides, C. truncatum, C. orbiculare

[0054] PDA agar medium was used to culture *Bacillus anthracis*. The inhibitory effect of different concentrations of nor-zelaminaraldehyde on the mycelial growth of the pathogen was determined using the mycelial growth rate method. Specific procedures included:

[0055] 1) Dissolve demethylzelamide aldehyde in DMSO to prepare a mother liquor;

[0056] 2) After melting the V8 medium, reduce the temperature to 50°C. At approximately C, the des-Zeramol stock solution was accurately added to the culture medium and mixed well to achieve final concentrations of 3 μg / mL, 10 μg / mL, 18 μg / mL, 27 μg / mL, and 35 μg / mL, respectively. Four replicates were set up for each concentration treatment. A control was prepared by adding the same volume of solvent with half-inhibitory concentration. 20 mL of culture medium was poured into 9 cm diameter culture dishes.

[0057] 3) Using a sterile pipette tip, create a mycelial cake (d=5 mm) of uniform age at the edge of the activated anthrax bacteria colony on V8 medium. Inoculate the mycelial cake into the center of a V8 medium plate containing different concentrations of norzelaminaraldehyde. Place the plate in a 25 ℃ constant temperature incubator for dark incubation. Observe, measure and record the diameter of the mycelial cake regularly and take photos.

[0058] Table 1

[0059]

[0060] See results Figure 5 As shown in Table 1, norzelamin has a significant antibacterial effect against Bacillus anthracis. C. graminicola , C. gloeosporioides , C. truncatum , C. EC of orbiculare 50 The concentrations of 0.7091, 16.39, 18.59 and 34.76 μg / mL showed varying degrees of antibacterial effect against different anthrax bacteria.

[0061] Example 5: Inhibitory effect of nor-zelamin on different Fusarium species

[0062] Tested pathogens: F. graminearum, F. oxysporum, F. proliferatum, F. equiseti, F. culmorum, F. solani .

[0063] PDA agar medium was used to culture Fusarium. The specific method is the same as in Example 4.

[0064] Table 2

[0065]

[0066] See results Figure 6 As shown in Table 2, norzelamin has a significant antibacterial effect against Fusarium. F. graminearum , F. oxysporum , F. proliferatum , F. equiseti , F. culmorum , F. solani EC 50The concentrations of 15.16 μg / mL, 5.635 μg / mL, 14.89 μg / mL, 27.14 μg / mL, 21.62 μg / mL and 24.16 μg / mL, respectively, all showed varying degrees of antibacterial effect against different Fusarium species.

[0067] The embodiments described above are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. Use of norzelandin aldehyde for inhibiting Fusarium or Colletotrichum, characterized in that, The Fusarium is one of Fusarium graminearum Fusarium graminearum , Fusarium oxysporum Fusarium oxysporum , Fusarium verticillioides Fusarium proliferatum , Fusarium equiseti Fusarium equiseti , Fusarium chlamydosporum Fusarium culmorum or Fusarium solani Fusarium solani ; and the Colletotrichum is one of Colletotrichum siamense Colletotrichum siamense、 , Colletotrichum graminicola Colletotrichum graminicola , Colletotrichum capsici Colletotrichum gloeosporioides , Colletotrichum truncatum Colletotrichum truncatum or Colletotrichum lagenarium Colletotrichum orbiculare .

2. Use according to claim 1, characterized in that, The applications include at least one of the following (1)-(4): (1) inhibiting radial growth of the Fusarium or Colletotrichum mycelium; (2) inhibiting conidial production of the Colletotrichum; (3) inhibiting conidial germination of the Colletotrichum; (4) inhibiting pathogenicity of the Fusarium or Colletotrichum.

3. A method of controlling fungal diseases of mango, characterized in that, Administering norezaramindial or a formulation containing norezaramindial to mangoes to control fungal diseases caused by fusarium oxysporum Fusarium oxysporum or glomerella cingulata Colletotrichum siamense ​ 4. The method of claim 3, wherein, The applying to the mango includes applying to a mango stem, a leaf, a root system, or a fruit.

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