Application of Isoquinoline-3-carboxylic Acid in Preventing and Controlling Plant Bacterial Diseases

Abstract

The present invention discloses the application of isoquinoline-3-carboxylic acid in preventing and controlling plant bacterial diseases. The isoquinoline-3-carboxylic acid provided by the present invention has an obvious inhibitory effect on plant bacterial diseases, and the plant bacterial diseases are Ralstonia solanacearum, Acidovorax avenae subsp. Citrulli, Pseudomonas syringae pv. actinidiae, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, Alternaria brassicae, Xanthomonas citri subsp. citri, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas fragariae, Pseudomonas syringae pv. lachrymans, Dickeya dadantii, Xanthomonas axonopodis pv. mangiferaeindicae. The isoquinoline-3-carboxylic acid of the present invention has protective and therapeutic effects on Acidovorax avenae subsp. Citrulli.

Description

Technical Field

[0001] The present invention relates to the field of prevention and control of plant bacterial diseases, and particularly to the application of isoquinoline-3-carboxylic acid in the prevention and control of plant bacterial diseases. Background Art

[0002] Plant bacterial diseases cause a significant reduction in crop yields, resulting in huge economic losses. For example, rice bacterial blight and bacterial leaf streak caused by Xanthomonas are common diseases of rice, resulting in about 10% reduction in rice yields every year, with a spread area of 330,000 - 660,000 hectares; bacterial fruit blotch of cucurbits caused by Acidovorax, and infected seeds are the main means of transmission, causing serious economic losses to the industries of crops such as watermelon and melon; bacterial wilt caused by the Gram-negative bacterium Ralstonia solanacearum can infect tobacco and solanaceous vegetables, resulting in losses of more than 20%. At present, China mainly responds to plant bacterial diseases by using disease-resistant varieties, physical control, chemical control, biological control, etc. Among them, the most important and effective control method is still chemical control. However, the existing types of pesticides far cannot meet the market demand. There are about 280 registered bactericides in China, accounting for only 2.6% of the total registered fungicides. The control agents are mainly copper preparations, thiazole-based and antibiotics. Such as thiodiazole copper, thiazole zinc, quinoline copper, kasugamycin, etc. However, copper preparations are prone to phytotoxicity and cause the rampant of harmful mites; thiazole-based agents have a great impact on non-target organisms; long-term use of antibiotics results in serious resistance, and with the ban of streptomycin in agriculture in 2019, it has even made the bacterial diseases suffer from a shortage of drugs. Therefore, it is urgent to develop novel-structured, highly efficient and low-toxic green fungicides to replace traditional agents.

[0003] Searching for lead compounds from natural products and using them as probes for the design of natural bionic pesticides is one of the effective ways to create new pesticides. Isoquinoline-3-carboxylic acid is an isoquinoline compound with the molecular formula C 10 H7NO2 and a relative molecular mass of 173.17. Since the isolation of the tetrahydroisoquinoline natural product naphthriydinomycin with antitumor activity in 1974, the application of isoquinoline alkaloids in antitumor has received extensive attention. Since isoquinoline can directly act on the DNA of cancer cells, isoquinoline-3-carboxylic acid and its derivatives have the potential for antitumor. By modifying its amino acid benzyl ester, the in vitro anti-proliferation activity and in vivo antitumor activity can be improved. However, there is no relevant report at home and abroad on the application of isoquinoline compounds in the prevention and control of agricultural diseases, nor is there a report on the inhibitory effect of isoquinoline-3-carboxylic acid on plant pathogenic bacteria. Summary of the Invention

[0004] In view of this, the present invention provides the use of isoquinoline-3-carboxylic acid in preventing and controlling plant bacterial diseases to solve the above problems.

[0005] The technical solution of the present invention is achieved as follows:

[0006] Use of isoquinoline-3-carboxylic acid in preventing and controlling plant bacterial diseases.

[0007] Furthermore, the isoquinoline-3-carboxylic acid is the active ingredient.

[0008] Furthermore, the concentration of the isoquinoline-3-carboxylic acid is above 200 μg / mL.

[0009] Furthermore, the isoquinoline-3-carboxylic acid can be formulated into a suspension concentrate, emulsion in water, microemulsion, emulsifiable concentrate, wettable powder, water dispersible granule or ultra-low volume liquid.

[0010] Furthermore, the plant bacterial diseases are Ralstonia solanacearum causing tomato bacterial wilt, Acidovorax citrulli causing bacterial fruit blotch of melon, Pseudomonas syringae pv. actinidae causing kiwifruit canker, Pectobacterium carotovorum subsp. brasiliense causing potato soft rot, Xanthomonas oryzae pv. oryzae causing rice bacterial blight, X. oryzae pv. oryzicola causing rice bacterial leaf streak, X. campestris pv. campestris causing black spot of cabbage, X. axonopodis pv. citri causing citrus canker, P. carotovorum subsp. carotovorum causing soft rot of Chinese cabbage, X. fragariae causing bacterial angular leaf spot of strawberry, P. syringae pv. lachrymans causing bacterial angular leaf spot of cucumber, Dickeya zeae causing bacterial soft rot of banana, X. campestris pv. mangiferaeindicae causing bacterial angular leaf spot of mango.

[0011] Furthermore, the isoquinoline-3-carboxylic acid has an antibacterial effect on plant bacterial diseases.

[0012] Furthermore, the isoquinoline-3-carboxylic acid has protective and therapeutic effects on bacterial fruit blotch of melon.

[0013] Furthermore, the concentration of the isoquinoline-3-carboxylic acid is above 50 μg / mL.

[0014] Further, isoquinoline-3-carboxylic acid can be chemically synthesized. The compound is a white powder with a CAS number of 6624-49-3, and its molecular formula is C 10 H7NO2, and its molecular weight is 173.17.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] When the tested concentration of the isoquinoline-3-carboxylic acid provided by the present invention is 50 μg / mL, it has excellent antibacterial activity against Ralstonia solanacearum, Acidovorax avenae subsp. citrulli, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas oryzae pv. oryzicola, Alternaria brassicae, Xanthomonas citri subsp. citri, Pectobacterium carotovorum subsp. odoriferum, and Xanthomonas fragariae, and the inhibition rate of all of them is above 93%.

[0017] The results of the pot experiment show that isoquinoline-3-carboxylic acid has a strong protective effect on Acidovorax avenae subsp. citrulli. When the tested concentration is 200 μg / mL, its protective effect is 68.56%, which has no significant difference from the control agent kasugamycin; it also has a strong therapeutic effect on Acidovorax avenae subsp. citrulli. When the tested concentration is 200 μg / mL, its therapeutic effect is 62.11%, which is significantly better than the control agent thiodiazole copper.

[0018] In summary, the compound provided by the present invention has a significant inhibitory effect on phytopathogenic bacteria, providing a natural antibacterial active substance for agricultural disease control; this compound can be used to control various important agricultural diseases such as tomato bacterial wilt, rice bacterial leaf streak, mango bacterial black spot, Alternaria brassicae, Pectobacterium carotovorum subsp. odoriferum, and Acidovorax avenae subsp. citrulli; as a fungicide, its characteristics of high efficiency, low toxicity, and safety meet the requirements of current new pesticide development. Specific Embodiments

[0019] To better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0020] The isoquinoline-3-carboxylic acid used in the present invention is a white powder with a CAS number of 6624-49-3, and its molecular formula is C 10 H7NO2, and its molecular weight is 173.17. It is purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.

[0021] Determination of the inhibitory activity of isoquinoline-3-carboxylic acid against phytopathogenic bacteria

[0022] 1. Tested strains

[0023] Plant pathogenic bacteria including Ralstonia solanacearum, Acidovorax citrulli, Pseudomonas syringae pv. actinidae, Pectobacterium carotovorum subsp. brasiliense, Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, X. campestris pv. campestris, X. axonopodis pv. citri, P. carotovorum subsp. carotovorum, X. fragariae, P. syringae pv. lachrymans, Dickeya zeae, and X. campestris pv. mangiferaeindicae. All of the above plant pathogenic bacteria were provided by the Green Pesticide Innovation and Application Research Group of the Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences.

[0024] 2. Experimental methods

[0025] 2.1 In vitro activity detection

[0026] The present invention uses the 96-well plate method to evaluate the antibacterial activity of isoquinoline-3-carboxylic acid against plant pathogenic bacteria (Li, G.H.; Qiao, M.Y.; Guo, Y.; Wang, X.; Xu, Y.F.; Xia, X.D. Effect of subinhibitory concentrations of chlorogenic acid on reducing the virulence factor production by Staphylococcus aureus. Foodborne Pathog. Dis. 2014, 11(9), 677-683). The prepared bacterial suspension was added to a 96-well microplate, 10 μL per well. The sample control group was added with 10 μL of the bacterial suspension and 200 μL of the liquid medium, the blank control group was added with 200 μL of the liquid medium, and a control of the compound solution was set, 200 μL per well. The medicament solution was mixed with the bacterial suspension, and the samples were placed in an incubator and cultured with vibration (500 r / min). The culture temperature was set at 28-30 °C, and the culture time was 18-24 h. The absorbance of the bacterial solution was measured with a microplate reader. The inhibitory effect of the compound on the pathogen was calculated using the following formula.

[0027] Inhibitory rate (%) = { (OD of the control group 600 — OD of the blank control group 600 ) — (OD600 of the experimental group — OD600 of the compound solution control)} / (OD600 of the control group — OD600 of the blank control group) × 100%

[0028] 2.2 Data analysis

[0029] The antibacterial activity of isoquinoline-3-carboxylic acid against 13 plant pathogenic bacteria was tested indoors, and the results are shown in Table 1. When the concentration of isoquinoline-3-carboxylic acid was 50 μg / mL, it had excellent antibacterial activity against Ralstonia solanacearum, Acidovorax citrulli, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas oryzae pv. oryzicola, Alternaria brassicae, Xanthomonas citri subsp. citri, Pectobacterium carotovorum subsp. odoriferum, and Xanthomonas fragariae, and its inhibition rate ranged from 93.10% to 98.56%.

[0030] Table 1 Antibacterial activity of isoquinoline-3-carboxylic acid against 13 plant pathogenic bacteria

[0031]

[0032] 2.2 Pot experiment method

[0033] The in vivo pot experiment of isoquinoline-3-carboxylic acid against Acidovorax citrulli on melons was slightly modified based on the reference method (Li, B.; Shi, Y.; Shan, C. L.; Zhou, Q.; Ibrahim, M.; Wang, Y. L.; Wu, G. X.; Li, H. Y.; Xie, G. L.; Sun, G. C. Effect of chitosan solution on the inhibition of Acidovorax citrulli causing bacterial fruit blotch of watermelon. J. Sci. Food Agric. 2013, 93(5), 1010 - 1015): Five treatments of agent concentration 1, concentration 2, concentration 3, 2% kasugamycin aqueous solution at 100 μg / mL, and water were evenly sprayed on the seedlings of melons (cultivar: Yangjiaomi), and then the suspension of Acidovorax citrulli (concentration: 1×10 9 CFU / mL) was sprayed on each pot for inoculation. Each treatment was repeated 3 times, with 6 pots in each repetition and 1 plant in each pot. The plants were cultured under moisturized light conditions, and the disease incidence was observed 7 - 10 days after cultivation, and the disease index (Formula 1) and control effect (Formula 2) were calculated. The grading of leaf disease levels is as follows:

[0034] Grade 0: No disease spots;

[0035] Level 1: The lesion area accounts for less than 5% of the entire leaf area;

[0036] Level 3: The lesion area accounts for 6 - 10% of the entire leaf area;

[0037] Level 5: The lesion area accounts for 11 - 20% of the entire leaf area;

[0038] Level 7: The lesion area accounts for 21 - 50% of the entire leaf area;

[0039] Level 9: The lesion area accounts for more than 51% of the entire leaf area;

[0040] Pharmacodynamic calculation formula:

[0041] Disease index = ∑(number of diseased leaves at each level × relative level value) / (total number of leaves surveyed × highest disease level value) × 100 (1)

[0042] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100 (2)

[0043] Pot experiments were conducted to test the protective and therapeutic effects of isoquinoline - 3 - carboxylic acid against Acidovorax avenae subsp. citrulli. The results showed that isoquinoline - 3 - carboxylic acid had excellent protective effects against Acidovorax avenae subsp. citrulli. The protective effect at a tested concentration of 200 μg / mL was 68.56%, showing no significant difference from the control agent 2% kasugamycin aqueous solution (72.48%), and was superior to the control agent 20% thiodiazole copper suspension (64.62%); in addition, isoquinoline - 3 - carboxylic acid also had strong therapeutic effects against Acidovorax avenae subsp. citrulli. The therapeutic effect at a treatment concentration of 200 μg / mL against Acidovorax avenae subsp. citrulli was 62.11%, which was superior to the control agent 20% thiodiazole copper suspension (56.84%).

[0044] Table 2 Protective and therapeutic effects of isoquinoline - 3 - carboxylic acid against Acidovorax avenae subsp. citrulli

[0045]

[0046] The results of in - vivo pot experiments showed that the compound had good protective and therapeutic effects against Acidovorax avenae subsp. citrulli. The above results indicate that isoquinoline - 3 - carboxylic acid has broad - spectrum antibacterial effects against phytopathogenic bacteria and the potential to develop new green bactericides with it as a lead compound.

[0047] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of isoquinoline-3-carboxylic acid in preventing and controlling plant bacterial diseases, characterized in that, The plant bacterial disease is a disease caused by Acidovorax avenae subsp. Citrulli ( Acidovorax citrulli ), The application concentration of the isoquinoline-3-carboxylic acid is 200 μg / mL or more.

2. The application according to claim 1, wherein Using isoquinoline-3-carboxylic acid as the active ingredient, a suspending agent, an emulsion in water, a microemulsion, an emulsifiable concentrate, a wettable powder, a water dispersible granule or an ultra-low volume liquid is prepared.

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