Application of florfenicol in preventing and treating plant bacterial diseases and inhibitors

Abstract

The application discloses application of florfenicol in prevention and treatment of plant bacterial diseases and an inhibitor. The application provides a new application of florfenicol, which can be used for preventing and treating Xanthomonas oryzae pv. oryzae, Xanthomonas campestris pv. oryzae, Xanthomonas axonopodis pv. citri, Pseudomonas syringae pv. tabaci, Pseudomonas syringae pv. lachrymans, Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. lactucae, Pseudomonas syringae pv. actinidiae, Pseudomonas syringae pv. angulata and the like, and provides a new idea for preventing and treating the above-mentioned plant bacterial diseases. Experiments prove that florfenicol has high bactericidal activity on the above-mentioned plant bacterial diseases.

Description

Technical Field

[0001] This invention relates to a new application of florfenicol, particularly its application in the prevention and control of plant bacterial diseases. Background Technology

[0002] Rice is highly susceptible to infection by numerous pathogenic microorganisms, leading to severe yield reductions. Among these, rice bacterial blight and rice bacterial leaf streak, caused by the corresponding Xanthomonas oryzae, are the two most widespread and damaging bacterial diseases affecting rice. While bacterial diseases of rice occur worldwide, they are particularly severe in my country, prevalent in all major rice-growing regions, with the most severe outbreaks in East, South, and Central China. Bacterial diseases typically reduce rice yields by 10%-20%, and in severe cases, can cause difficulty in heading, resulting in yield losses exceeding 50%, causing enormous economic losses to my country's rice production annually.

[0003] Currently, commercially available antibacterial agents used to control rice bacterial blight and rice bacterial leaf streak are mainly tebuconazole, thiamethoxam zinc, thiabendazole copper, and agricultural streptomycin. However, prolonged use has led to varying degrees of drug resistance in pathogens, resulting in decreased efficacy. Therefore, finding and developing novel, highly effective, and low-risk green pesticides for controlling rice bacterial blight and rice bacterial leaf streak has become an important direction in the current development of green pesticides.

[0004] Meanwhile, the annual affected area of ​​bacterial diseases in my country is approximately 150 million mu (10 million hectares), resulting in a large demand for pesticides for their control, with a conservative market size estimated at 3.5 billion yuan. Citrus canker, in particular, affects over 3 million mu (200,000 hectares), causing annual losses exceeding 500 million yuan to the citrus industry. However, due to regional distribution and market factors, there is a lack of suitable pesticides for controlling citrus canker, with copper-based formulations being the primary method, supplemented by agricultural antibiotics and antagonistic microbial agents. Inorganic copper, represented by Bordeaux mixture and copper hydroxide, remains the mainstream in the market, accounting for more than half. Therefore, the high-dose, high-frequency use of copper-based formulations not only directly harms crops but also causes permanent soil pollution and ecological damage. To avoid the harm caused by the overuse of copper-based formulations, the development of safe and efficient non-copper bactericides is urgently needed.

[0005] In addition to the aforementioned plant bacterial diseases, other common plant bacterial diseases in my country include tobacco bacterial wilt, melon leaf spot, tomato bacterial wilt, watermelon fruit rot, cabbage soft rot, peach tree shot-hole disease, and cabbage black rot, which cause serious losses to the corresponding plants every year.

[0006] Florfenicol, also known as florfenicol, is a fluorine derivative at the 3-position of thiamphenicol, a broad-spectrum antibacterial drug. It is a new generation of chloramphenicol antibiotics that can replace chloramphenicol and thiamphenicol. It has broad-spectrum antibacterial activity, strongly interfering with bacterial protein synthesis, and inhibits both Gram-negative and Gram-positive bacteria.

[0007] The modification and alteration of the florfenicol structure gives it a significant advantage over chloramphenicol and thiamphenicol in terms of safety and efficacy. Structurally, the hydroxyl group at the 3-carbon position of the propane chain in chloramphenicol and thiamphenicol is replaced by a fluorine atom, which prevents bacterial acetylation and thus prevents inactivation by the enzyme. Therefore, it is less likely to develop plasmid-mediated drug resistance.

[0008] Currently, florfenicol is mainly used as a broad-spectrum antibiotic in the veterinary chloramphenicol class and is widely used for the prevention and control of infectious diseases in animals. However, during the synthesis and activity screening of antimicrobial drugs, we discovered that florfenicol exhibits excellent antibacterial activity against bacterial diseases of plants such as rice bacterial blight, rice bacterial leaf streak, citrus canker, tobacco bacterial wilt, melon fine angular rot, tomato bacterial wilt, watermelon fruit rot, cabbage soft rot, peach leaf spot, and cabbage black rot. Research on florfenicol against plant bacterial diseases is scarce; therefore, developing florfenicol for the control of plant pathogenic bacteria is of great significance. Summary of the Invention

[0009] The purpose of this invention is to provide an application and inhibitor of florfenicol in the control of plant bacterial diseases. This invention provides a new application of florfenicol for the control of plant bacterial diseases such as rice bacterial leaf blight, rice bacterial leaf streak, citrus canker, tobacco bacterial wilt, melon leaf blight, tomato bacterial wilt, watermelon fruit rot, cabbage soft rot, peach leaf spot, and cabbage black rot, providing a new approach to the control of these plant bacterial diseases.

[0010] The technical solution of this invention: the application of florfenicol in the prevention and control of plant bacterial diseases.

[0011] Preferably, the aforementioned application of florfenicol in the prevention and control of plant bacterial diseases refers to the following plant bacterial diseases: rice bacterial blight, rice bacterial leaf streak, citrus canker, tobacco bacterial wilt, melon fine angular rot, tomato bacterial wilt, watermelon fruit rot, cabbage soft rot, peach tree shot-hole disease, or cabbage black rot.

[0012] Preferably, in the aforementioned application of florfenicol in the control of plant bacterial diseases, the chemical structural formula of the florfenicol is shown below, CAS: 73231-34-2, purity > 98%:

[0013]

[0014] An inhibitor of plant bacterial diseases containing florfenicol.

[0015] Preferably, the aforementioned inhibitor of plant bacterial diseases is an aqueous solution or a powder.

[0016] A method for controlling plant bacterial diseases involves preparing a solution of florfenicol with water and spraying it on the affected areas.

[0017] Preferably, in the aforementioned method for controlling plant bacterial diseases, the concentration of florfenicol in the pesticide solution is 10-200 μg / ml.

[0018] Preferably, in the aforementioned method for controlling plant bacterial diseases, the concentration of florfenicol in the pesticide solution is 200 μg / ml.

[0019] Preferably, in the aforementioned method for controlling plant bacterial diseases, the plant bacteria are rice bacterial blight pathogens, rice bacterial leaf streak pathogens, citrus canker pathogens, tobacco bacterial wilt pathogens, melon fine angular pathogens, tomato bacterial wilt pathogens, watermelon fruit rot pathogens, cabbage soft rot pathogens, peach tree shot-hole pathogens, or cabbage black rot pathogens.

[0020] Beneficial effects of the present invention

[0021] This invention provides a new application for florfenicol, which can be used to control bacterial diseases of plants such as rice bacterial leaf blight, rice bacterial leaf streak, citrus canker, tobacco bacterial wilt, melon fine angular rot, tomato bacterial wilt, watermelon fruit rot, cabbage soft rot, peach leaf spot, and cabbage black rot, providing a new approach to the control of these bacterial diseases.

[0022] Experiments have shown that florfenicol has high bactericidal activity against the aforementioned plant bacterial diseases. Attached Figure Description

[0023] Appendix Figure 1 The chemical structural formula of florfenicol;

[0024] Appendix Figure 2 To compare the protective activities of different agents against rice bacterial blight pathogen;

[0025] Appendix Figure 3 To compare the therapeutic activities of different agents against rice bacterial blight pathogen;

[0026] Appendix Figure 4 To compare the protective activities of different agents against rice leaf streak pathogen. Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0028] Embodiments of the present invention

[0029] Example 1:

[0030] In vitro antibacterial activity assay of florfenicol compounds

[0031] The strains used in this experiment were strains that were frozen in the laboratory at -80℃ with 50% glycerol.

[0032] Step 1): Take out the frozen strains (rice bacterial blight pathogen, rice bacterial leaf streak pathogen, citrus canker pathogen, tobacco bacterial wilt pathogen, melon fine angular pathogen, tomato bacterial wilt pathogen, watermelon fruit rot pathogen, cabbage soft rot pathogen, peach tree shot-hole pathogen, and cabbage black rot pathogen), and streak them on NA solid medium (preparation method: weigh 3g beef extract, 5g peptone, 1g yeast extract, 10g glucose and 18g agar into 1L beakers, add 1L deionized water, adjust pH to about 7 with 5M NaOH solution, sterilize at 120℃ for 20min and store at room temperature for later use). Incubate at 28℃ until single colonies grow.

[0033] Step 2): Pick single colonies from the solid culture medium and transfer them to NB liquid culture medium (preparation method: weigh 3g beef extract, 5g peptone, 1g yeast extract and 10g glucose into a 1L beaker, add 1L deionized water and adjust the pH to about 7 with 5M NaOH solution, sterilize at 121℃ for 20min and store at room temperature for later use). Incubate on a constant temperature shaker at 28℃ and 180rpm until the logarithmic growth phase is reached for later use.

[0034] Step 3): Florfenicol, thiabendazole copper, and thiamethoxam zinc were prepared into NB liquid culture media with concentrations of 100, 10, and 1 μg / mL, respectively. 40 μL of the aforementioned NB liquid culture media containing pathogens causing rice bacterial blight, rice bacterial streak, citrus canker, tobacco bacterial wilt, melon pod rot, tomato bacterial wilt, watermelon fruit rot, cabbage soft rot, peach tree perforation, and cabbage black rot were added accordingly. Blank controls were NB liquid culture media containing only equal volumes of DMSO, 0.1% Tween 20, and 40 μL of bacterial suspension. The cultures were incubated at 28℃ and 180 rpm in a shaker for 24–48 h until the bacterial suspension in the blank control group reached the logarithmic growth phase. The OD values ​​(OD values) of each concentration of bacterial suspension were measured using a microplate reader. 595 Furthermore, the OD values ​​(OD values) of sterile NB liquid culture media containing the compound, thiabendazole copper, and thiamethoxam zinc at concentrations of 100, 10, and 1 μg / mL, as well as a blank control, were determined. 595 The OD value caused by the culture medium itself is corrected. The formulas for calculating the corrected OD value and inhibition rate are as follows:

[0035] Correction: OD value = OD value of culture medium containing bacteria - OD value of culture medium without bacteria.

[0036] Inhibition rate (%) = (OD value of bacterial suspension in control culture medium after correction - OD value of bacterial suspension in drug-containing culture medium after correction) / OD value of bacterial suspension in control culture medium after correction × 100.

[0037] Step 4): Five corresponding concentration gradients of compounds florfenicol, thiabendazole copper, and thiamethoxam zinc were established using a stepwise dilution method. The in vitro EC50 values ​​of these compounds against *Bacillus oryzae* (rice bacterial leaf blight), *Bacillus streak* (rice bacterial leaf streak), *Citrus canker* (citrus canker), *Rhizoctonia solani* (tobacco bacterial wilt), *Fragaria lobata* (melon leaf blight), *Rhizoctonia solani* (tomato bacterial wilt), *Fragaria sclerotium* (watermelon fruit rot), *Fragaria caudatum* (cabbage soft rot), *Fragaria lobata* (peach leaf spot), and *Fragaria caudatum* (cabbage black rot) were then determined. 50 Values. All experiments were conducted in triplicate. The inhibition rates of compounds florfenicol, thiabendazole copper, and thiamethoxam zinc are shown in Tables 1 and 2. EC50 50 The values ​​are shown in Tables 3 and 4.

[0038] Table 1

[0039]

[0040]

[0041] Note: "-" indicates inactivity.

[0042] Table 2

[0043]

[0044] Note: "-" indicates inactivity.

[0045] As shown in Tables 1 and 2, the florfenicol compounds involved in this invention exhibit excellent antibacterial activity against plant pathogenic bacteria. At a concentration of 10 μg / mL, the inhibitory activities against *Rhizoctonia solani*, *Rhizoctonia solani*, *Citrus canker*, *Rhizoctonia solani*, *Rhizoctonia solani*, *Rhizoctonia solani*, *Rhizoctonia solani*, *Rhizoctonia solani*, *Rhizoctonia solani*, *Rhizoctonia solani*, *Rhizoctonia solani*, *Rhizoctonia solani*, *Rhizoctonia solani*, *Rhizoctonia solani*, *Rhizoctonia solani*, *Rhizoctonia solani*, and *Rhizoctonia solani* were 100%, 92.1%, 84.1%, 94.1%, 56.1%, 91.4%, 55.1%, 85.4%, 43.3%, and 93.8%, respectively, all of which were superior to the inhibitory activities of the control agents thiamethoxam zinc and thiamethoxam.

[0046] Table 3

[0047]

[0048] Table 4

[0049]

[0050] As shown in Tables 3 and 4, the florfenicol compounds involved in this invention exhibit EC50 efficacy against *Rhizoctonia solani*, *Rhizoctonia solani*, *Citrus canker*, *Rhizoctonia solani ... and *Rhizoctonia solani*. 50 The values ​​were 0.3 μg / mL, 0.5 μg / mL, 1.1 μg / mL, 1.3 μg / mL, 8.1 μg / mL, 2.3 μg / mL, 9.3 μg / mL, 0.4 μg / mL, 18.2 μg / mL and 0.7 μg / mL, respectively, all of which were superior to the control agents thiabendazole copper and thiamethoxam zinc.

[0051] Example 2:

[0052] Determination of the protective activity of florfenicol compounds against rice bacterial blight pathogens in live pots

[0053] At a concentration of 200 μg / mL, the protective activities of florfenicol compounds, thiabendazole copper, and thiamethoxam zinc against rice bacterial blight in live pots were determined using the leaf-cutting method.

[0054] Florfenicol compounds and control agents thiabendazole copper and thiamethoxam zinc were prepared into 200 μg / mL solutions using 0.1% Tween 80 solution. These solutions were sprayed onto the surface of rice leaves until droplets fell. After 24 hours, the leaf tips were cut off 1–2 cm from the leaf tip using scissors dipped in rice bacterial blight fungus. A blank control was established, consisting of rice seedlings inoculated only with rice bacterial blight fungus solution without any treatment. Each treatment consisted of 20 rice seedlings, with three replicates. Disease incidence was checked 14 days after application, and the length of lesions on the rice leaves was recorded. The disease index and control efficacy were calculated, as shown in Table 5.

[0055] The calculation method for the protective effect is as follows:

[0056] Efficacy (%) = (Disease index of blank control group - disease index of drug group) / disease index of blank group × 100.

[0057] Table 5

[0058]

[0059] All results are expressed as mean ± SD; statistical analysis was performed by analysis of variance (ANOVA) in SPSS 17.0 software, with different lowercase letters indicating differences in activity between different drug groups, and P < 0.05.

[0060] Example 3:

[0061] Determination of the therapeutic activity of florfenicol compounds against live potted plants of *Bacterium tumefaciens*, the causal agent of rice bacterial blight.

[0062] The therapeutic activities of florfenicol compounds, thiabendazole copper, and thiamethoxam zinc against rice bacterial blight in live pots were determined using the leaf-cutting method at a concentration of 200 μg / mL.

[0063] Cut off the tips of rice leaves 1-2 cm from the leaf tip using scissors dipped in rice bacterial blight fungus, and soak the wounds in the fungal solution for about 10 seconds. 24 hours later, prepare florfenicol compound and control agents thiabendazole copper and thiamethoxam zinc solutions at a concentration of 200 μg / mL using 0.1% Tween 80 solution, and spray the solutions onto the rice leaf surface until droplets fall. Simultaneously, establish a blank control group of rice seedlings inoculated only with rice bacterial blight fungal solution without any treatment. Each treatment consisted of 20 rice seedlings, with 3 replicates. Disease incidence was checked 14 days after application, and the length of lesions on the rice leaves was recorded. The disease index and control efficacy were calculated, as shown in Table 6.

[0064] The method for calculating the preventive effect is the same as in Example 2.

[0065] Table 6

[0066]

[0067] All results are expressed as mean ± SD; statistical analysis was performed by analysis of variance (ANOVA) in SPSS 17.0 software, with different lowercase letters indicating differences in activity between different drug groups, and P < 0.05.

[0068] As shown in Tables 5 and 6, the florfenicol compounds involved in this invention exhibited protective and curative activities of 60.6% and 55.3% against live potted plants of rice bacterial blight pathogen, respectively, both of which were superior to the protective and curative activities of the control agents thiabendazole copper (39.2% and 35.1%) and thiamethoxam zinc (46.4% and 43.9%).

[0069] Example 4:

[0070] Determination of the protective activity of florfenicol compounds against rice bacterial leaf streak pathogen in live pots

[0071] At a concentration of 200 μg / mL, the protective activities of florfenicol compounds, thiabendazole copper, and thiamethoxam zinc against rice bacterial leaf streak pathogens in live pots were determined using the pressure osmosis method.

[0072] Florfenicol compounds and control agents thiabendazole copper and thiamethoxam zinc were prepared into 200 μg / mL solutions using 0.1% Tween 80 solution. These solutions were sprayed onto the surface of rice leaves until droplets fell. After 24 hours, a mixed bacterial solution of *Bacillus streakus*, which had reached the logarithmic growth stage, was poured into an integrated syringe. Using this syringe, 100 μL of bacterial solution was injected into the leaf tips of rice seedlings via pressure osmosis inoculation. One well was used per leaf, and 20 seedlings were treated with each agent. A blank control group of seedlings inoculated only with *Bacillus streakus* was established. The treatment was repeated three times. Fourteen days after application, the disease incidence was checked, and the length of lesions on the rice leaves was recorded. The average lesion length and control efficacy were calculated, as shown in Table 7.

[0073] The calculation method for the protective effect is as follows:

[0074] Efficacy (%) = (Average lesion length in the blank control group - Average lesion length in the drug group) / Average lesion length in the blank group × 100.

[0075] Table 7

[0076]

[0077] All results are expressed as mean ± SD; statistical analysis was performed by analysis of variance (ANOVA) in SPSS 17.0 software, with different lowercase letters indicating differences in activity between different drug groups, and P < 0.05.

[0078] As shown in Table 7, the florfenicol compound involved in this invention provides 67.6% protection against live potted plants of rice bacterial leaf streak, which is superior to the control agent thiabendazole copper (47.6%).

[0079] In summary, the florfenicol compound described in this invention exhibits excellent antibacterial activity against plant pathogenic bacteria, possesses broad-spectrum antibacterial activity, and demonstrates excellent in vivo activity against rice bacterial blight pathogen, thus possessing significant research value and importance.

[0080] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An application of florfenicol in the control of plant bacterial diseases, wherein the plant bacterial diseases are rice bacterial blight pathogen, rice bacterial leaf streak pathogen, citrus canker pathogen, tobacco bacterial wilt pathogen, and tomato bacterial wilt pathogen; the concentration of florfenicol is 1-200 μg / mL.

2. The application of florfenicol in the control of plant bacterial diseases according to claim 1, characterized in that: The concentration of florfenicol was 200 μg / mL.

Images