Use of sanguinarine and analogs thereof

Abstract

The present application relates to the technical field of plant disease prevention and treatment, and particularly relates to the application of sanguinarine and its analogues. The specific technical scheme is as follows: the present application provides a new use of sanguinarine and its analogues, which can be used in the agricultural field for the prevention and treatment of plant bacterial diseases and plant fungal diseases including rice bacterial leaf blight, gong capsule anthracnose, tea tree anthracnose and sorghum anthracnose, and provides a new approach for the research and development of new pesticides.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, specifically to the application of sanguisorbin and its analogues. Background Technology

[0002] As essential crops for human survival, rice is susceptible to damage from various diseases during cultivation. Bacterial and fungal infections can lead to severe symptoms such as wilting, canker, soft rot, and anthracnose, directly resulting in reduced crop yields and loss of economic value. Taking rice as an example, chemical control has become an important technology for controlling plant diseases. However, due to its low efficiency, high toxicity, high residue, single mode of action, and frequent use, bacterial resistance has increased. For instance, the commercial fungicide tebuconazole, used to control bacterial leaf blight in rice, only achieved a control rate of 25.49% at a high dosage of 200 μg / mL. Therefore, there is an urgent need to develop novel fungicides with novel structures, unique mechanisms of action, and high efficiency and low toxicity to replace traditional fungicides. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides an application of sanguinarine and its analogues.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] This invention discloses the application of sanguisorbin and its analogues in the prevention and control of plant diseases.

[0006] Preferably, the structural formula of sanguinarine and its analogues is:

[0007]

[0008] Preferably, the plant diseases include fungal diseases and bacterial diseases, wherein the bacterial diseases are caused by bacteria that cause rice bacterial blight, and the fungal diseases are caused by Colletotrichum candida, Colletotrichum teaii, and Colletotrichum sorghumii.

[0009] Preferably, the plant diseases include rice bacterial leaf blight, rice bacterial leaf streak, citrus canker, cabbage black rot, tomato bacterial spot, kiwifruit canker, tobacco bacterial wilt, cucumber bacterial angular leaf spot, potato bacterial wilt, soybean bacterial spot, tomato canker, *Colletotrichum gloeosporioides*, *Colletotrichum gloeosporioides*, sorghum *Colletotrichum gloeosporioides*, cotton verticillium wilt, tomato root rot, watermelon vine blight, rapeseed damping-off, osmanthus leaf blight, rose black spot, corn large leaf spot, and small leaf spot. Wheat stripe rust, wheat powdery mildew, cucumber gray mold, Chinese cabbage downy mildew, loquat sooty mold, cucumber black spot, tobacco black shank, cucumber sclerotinia stem rot, apple white mold, Chinese cabbage damping-off, rice sheath blight, rice blast, rice false smut, rice bakanae disease, pepper damping-off, tomato early and late blight, cucumber target spot, water chestnut stem blight, tomato downy mildew, wheat take-all, tomato leaf mold, grape canker, pepper wilt, rapeseed sclerotinia stem rot, eggplant verticillium wilt, wheat scab.

[0010] The present invention has the following beneficial effects:

[0011] This invention discloses that sanguinarine and its analogues have an inhibitory effect on FtsZ of *Bacillus oryzae*, effectively inhibiting bacterial binary fission, leading to bacterial filamentation, lysis, and death, thereby inhibiting the bacterial infection process and achieving disease resistance. Simultaneously, it also exhibits certain inhibitory effects on plant fungal pathogens, including *Colletotrichum gloeosporioides*, *Colletotrichum teaensis*, and *Colletotrichum sorghum*. This invention provides a new application for sanguinarine and its analogues in the agricultural field for the control of various plant bacterial and fungal diseases such as rice bacterial blight, *Colletotrichum gloeosporioides*, tea anthracnose, and sorghum anthracnose, and provides a new avenue for the research and development of new pesticides. Attached Figure Description

[0012] Figure 1 Image of rice blight fungus treated with sanguisorbin and its analogues at a concentration of 50 mg / L. Detailed Implementation

[0013] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0014] Unless otherwise specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.

[0015] This invention discloses the application of sanguisorbin and its analogues in the prevention and control of plant diseases.

[0016] Preferably, the structural formula of sanguinarine and its analogues is:

[0017]

[0018]

[0019] Furthermore, the plant diseases include fungal diseases and bacterial diseases. The bacterial diseases are caused by bacteria that cause rice bacterial blight, and the fungal diseases are caused by Colletotrichum candida, Colletotrichum teaii, and Colletotrichum sorghumii.

[0020] Furthermore, the plant diseases mentioned include rice bacterial leaf blight, rice bacterial leaf streak, citrus canker, cabbage black rot, tomato bacterial spot, kiwifruit canker, tobacco bacterial wilt, cucumber bacterial angular leaf spot, potato bacterial wilt, soybean bacterial spot, tomato canker, *Colletotrichum gloeosporioides*, *Colletotrichum gloeosporioides*, sorghum *Colletotrichum gloeosporioides*, cotton verticillium wilt, tomato root rot, watermelon vine blight, rapeseed damping-off, osmanthus leaf blight, rose black spot, corn large leaf spot, and small leaf spot. Wheat stripe rust, wheat powdery mildew, cucumber gray mold, Chinese cabbage downy mildew, loquat sooty mold, cucumber black spot, tobacco black shank, cucumber sclerotinia stem rot, apple white mold, Chinese cabbage damping-off, rice sheath blight, rice blast, rice false smut, rice bakanae disease, pepper damping-off, tomato early and late blight, cucumber target spot, water chestnut stem blight, tomato downy mildew, wheat take-all, tomato leaf mold, grape canker, pepper wilt, rapeseed sclerotinia stem rot, eggplant verticillium wilt, wheat scab.

[0021] The present invention will be further described below with reference to specific embodiments.

[0022] Example 1

[0023] The inhibition rate and effective median concentration (EC50) of sanguinarine and sanguinarine analogues against rice bacterial blight pathogens were tested using the turbidimetric method. 50

[0024] The experimental subject was rice bacterial blight pathogen (Xoo). Thiazole (20% wettable powder) was used as a positive control, and DMSO dissolved in NB medium was used as a blank control. Each experiment was repeated three times.

[0025] 1. Inoculate the rice bacterial blight pathogen into NB medium (1.5 g beef extract, 2.5 g peptone, 0.5 g yeast powder, 5.0 g glucose and 500 ml distilled water; pH 7.0-7.2) and culture it in a constant temperature shaker at 28℃ and 180 rpm until it reaches the logarithmic growth phase for later use.

[0026] 2. Prepare NB liquid culture media containing different concentrations of sanguinarine, sanguinarine analogues, and thiabendazole (20% wettable powder) at concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.7813 mg / L using the two-fold dilution method. Take 5 mL of each medium and add it to a test tube.

[0027] 3. Add 40 μL of NB liquid culture medium containing rice bacterial blight pathogen to the test tubes containing different concentrations of reagents in step 2, and culture them in a constant temperature shaker at 28℃ and 180 rpm for 36 hours.

[0028] 4. Measure the OD of the bacterial solutions at various concentrations using a spectrophotometer. 595 The value was also measured, and the OD of the corresponding concentration of sterile NB liquid culture medium containing the toxin was also determined. 595 value.

[0029] Corrected OD value = OD value of culture medium containing bacteria - OD value of sterile culture medium.

[0030] Inhibition rate % = [(OD value of bacterial suspension in calibration control medium - OD value of toxic culture medium in calibration) / OD value of bacterial suspension in calibration control medium] × 100%.

[0031] EC 50 Median effective concentration (MEC) is an important indicator for evaluating the sensitivity of plant pathogens to compounds, and it is also a crucial parameter for setting the compound concentration when studying the mechanism of action of target compounds. In concentration gradient experiments, eight suitable concentrations (100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.7813 mg / L) were established using the two-fold dilution method. Finally, the inhibition rate of the agent against the plant pathogen and the agent concentration were converted to logarithmic values, and the toxicity curve was obtained through regression analysis using SPSS software. The EC50 was then calculated. 50 .

[0032] The results are shown in Table 1.

[0033] Table 1. Anti-bacterial activity of sanguinarine and sanguinarine analogues against rice bacterial blight.

[0034]

[0035]

[0036] As shown in Table 1, sanguinarine exhibits excellent in vitro antibacterial activity against *Bacillus thuringiensis*, the causal agent of rice bacterial blight, with its EC50... 50 =0.96 mg / L, its in vitro antibacterial activity was significantly better than that of the commercial control drug thiabendazole (EC). 50=23.9 mg / L); Chelidonium hydrochloride also showed excellent in vitro antibacterial activity against rice bacterial blight pathogen, with its EC50 concentration being 23.9 mg / L. 50 =0.93 mg / L, its in vitro antibacterial activity was significantly better than that of the commercial control drug, tebuconazole (EC). 50 =23.9 mg / L). At a concentration of 50 mg / L, the inhibition rates of sanguisorbin and chelidonine hydrochloride were 99.7% and 99.8%, respectively, comparable to the commercial control drug thiabendazole (98.9%); at a concentration of 25 mg / L, the inhibition rates of sanguisorbin and chelidonine hydrochloride were 98.8% and 98.0%, respectively, significantly higher than the commercial control drug thiabendazole (61.6%).

[0037] Example 2

[0038] Filamenting temperature-sensitive pertain Z (FtsZ) is a prokaryotic cell division protein with GTPase activity. It is a homolog of tubulin and plays a crucial role in bacterial division. Observing whether bacteria exhibit filamentation after drug treatment can provide a preliminary indication of whether the drug potentially affects FtsZ.

[0039] Will grow to OD 595 Xanthomonas oryzae at a concentration of 0.1% and a compound at a concentration of 50 mg / L were cultured at 28 °C and 180 rpm for 24 h. Then, 10 μL of the bacterial culture was dropped onto a copper grid containing a pre-hydrophilized carbon-supported membrane for approximately 30 s. The solution was then removed with filter paper, and 1% phosphotungstic acid was added for 15 s. After the liquid was absorbed by the filter paper and the cells were air-dried, they were ready for transmission electron microscopy (TEM). The effects of sanguinarine and its analogues on the morphology of Xoo cells were investigated using TEM.

[0040] like Figure 1 As shown, after incubation with five sanguisorbin analogues at a concentration of 50 mg / L, some elongated filamentous bacterial cells were observed under the presence of sanguisorbin and celandine hydrochloride compounds, which have high antibacterial activity. These cells exhibited significant bacterial filamentation compared to the other three analogues, indicating that these two sanguisorbin analogues can block the binary fission and normal reproduction of Xoo, thereby leading to bacterial death.

[0041] Example 3

[0042] The in vitro antibacterial efficacy inhibition rates of sanguisorbin, corydaline, chelidonine hydrochloride, and dihydrosanguisorbin against plant pathogenic fungi were tested using the mycelial growth rate method.

[0043] The experimental subjects, *Colletotrichum candida*, *Colletotrichum teaii*, and *Colletotrichum sorghumii*, used DMSO in sterile distilled water as a negative control and cyazofamid as a positive control.

[0044] 1. Dissolve sanguisorbin, corydaline, chelidonine hydrochloride and dihydrosanguisorbin separately in DMSO (1.0 mL), then add to 9.0 mL of sterile water containing Tween 20 (1%), and then mix with potato dextrose agar (PDA, 90.0 mL). The concentration of sanguisorbin, corydaline, chelidonine hydrochloride and dihydrosanguisorbin is 25 mg / L.

[0045] 2. Transfer the stock solution evenly into three 9cm diameter petri dishes.

[0046] 3. Then, cut a mycelial culture dish with a diameter of about 4 mm from the culture medium and aseptically inoculate it in the center of a PDA plate.

[0047] 4. After inoculation, the plates were incubated at 27±1℃ for 3-5 days, with 3 replicates for each treatment condition.

[0048] 5. The radial growth of fungal colonies was measured, and the data were statistically analyzed. The antibacterial effect was calculated using the formula I = [(CT) / (C-0.4)] × 100%, where C is the diameter of fungal growth on the untreated PDA, T is the diameter of fungal growth on the treated PDA, and I is the inhibition rate.

[0049] The results are shown in Table 2.

[0050] Table 2. Inhibition rates (%) of the in vitro antibacterial efficacy of sanguisorbin, corydaline, celandine hydrochloride, and dihydrosanguisorbin against plant pathogenic fungi *Colletotrichum gloeosporioides*, *Colletotrichum teaii*, and *Colletotrichum sorghum*.

[0051]

[0052] As shown in Table 2, sanguinarine exhibits excellent in vitro antibacterial activity against plant pathogenic fungi *Colletotrichum gloeosporioides*, *Colletotrichum teaensis*, and *Colletotrichum sacchariformis*, with inhibition rates of 71.48%, 60.61%, and 100%, respectively, at a concentration of 25 mg / L. Its in vitro antibacterial activity against *Colletotrichum sacchariformis* is significantly higher than that of the commercial control drug thiabendazole (inhibition rate of 92.9%).

[0053] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of sanguisorbin in the prevention and control of plant diseases, characterized by: The plant disease in question is sorghum anthracnose caused by Colletotrichum sorghumus.

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