CN116806828B - Application of hinokitiol in preventing and/or controlling bacterial diseases - Google Patents
Application of hinokitiol in preventing and/or controlling bacterial diseasesInfo
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- CN116806828B CN116806828B CN202310046454.1A CN202310046454A CN116806828B CN 116806828 B CN116806828 B CN 116806828B CN 202310046454 A CN202310046454 A CN 202310046454A CN 116806828 B CN116806828 B CN 116806828B
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Abstract
本发明属于植物病害防治技术领域，具体涉及一种预防和/或防治植物细菌性病害中应用。本发明通过筛选，发现桧木醇对青枯病菌LS2020、丁香假单胞菌Pto DC3000、黄单胞杆菌Xcv 85‑10有抑制作用，从而对植物青枯病、斑点病进行有效防治。本发明公开了桧木醇抑制青枯病菌、番茄斑点病病原菌的致死浓度，为植物细菌性病害防治提供了一种新的途径。The present invention belongs to the technical field of plant disease prevention and control, and specifically relates to an application in preventing and/or controlling plant bacterial diseases. Through screening, the present invention finds that hinokitiol has an inhibitory effect on bacterial wilt LS2020, Pseudomonas syringae Pto DC3000, and Xanthomonas Xcv 85-10, thereby effectively preventing and controlling plant bacterial wilt and spot disease. The present invention discloses the lethal concentration of hinokitiol for inhibiting bacterial wilt and tomato spot disease pathogens, and provides a new way for plant bacterial disease prevention and control.
Description
Technical Field
The invention belongs to the technical field of plant disease control, and particularly relates to application of hinokitiol in preventing and/or controlling plant bacterial diseases.
Background
Bacterial wilt bacteria (Ralstonia solanacearum) are worldwide destructive plant pathogenic bacteria, have a wide host range, can infect more than 400 plants of more than 50 families and cause bacterial wilt. The disease is particularly serious in occurrence and hazard of important solanaceous crops such as tomatoes, peppers, eggplants, potatoes, tobacco and the like in China, and has serious influence on agricultural production. The bacterial wilt bacteria survive in soil throughout the year, invade through the root of the plant, colonize in the vascular bundle of the plant and cause wilting of the whole plant. The field control of bacterial wilt is very challenging due to the complexity of root infestation and vascular bundle colonization. Based on its academic and economic importance, bacterial wilt is considered as a second major class of phytopathogenic bacteria in the world, and has become an important factor restricting sustainable development of related industries.
Bacterial wilt is caused by the fact that pathogenic bacteria are propagated in plant vascular bundles, so that the vascular bundles are blocked, and water transport of plants is blocked, so that the plants show wilting symptoms. The main symptom of bacterial wilt is that plants wilt and die quickly, and stems and leaves remain green. The brown stain part of the disease stem is extruded by hands, and the milky white fungus liquid is discharged. Under high temperature and high humidity conditions, the disease is very easy to occur. The bacterial wilt is fast in onset, serious and difficult to prevent and treat, and brings great attention to people. Therefore, effective control of bacterial wilt is necessary.
At present, three methods of physical control, chemical control and biological control are mainly used for controlling bacterial wilt.
The existing method for physically preventing and controlling bacterial wilt comprises the steps of adding an improver into a tobacco planting field in patent CN103190222A in 2013 Ding Wei and the like, balancing soil nutrients through soil improvement, controlling tobacco bacterial wilt, sterilizing contaminated soil in time and replacing bed soil in common physical prevention and control in agricultural production, making a reasonable rotation plan, and preventing and controlling by utilizing grafting cultivation, burning infected plants and the like. However, the control method is single, the control effect is general, the symptoms and the root causes are not treated, the environment is easy to pollute, and the cooperative control is usually carried out by other methods.
The method for preventing and treating bacterial wilt by using chemical agents comprises the following steps of spraying 50% of the wettable powder of the bacterial wilt in fields of dry-land eggplants for 6 times in 2000, wherein the residual effect period is as long as 5 months, and the like in 2004, kong Yufan, wherein the 20% of the wettable powder of the bacterial wilt can reach 62.1% -81.5% and the effect is obviously better than that of 72% of the wettable powder of agricultural streptomycin. Although the chemical control effect is good, the chemical pollution is caused to cause great damage to the environment.
In recent years, methods for biologically preventing and treating bacterial wilt are provided, such as 2020, yang Liang and the like, that hydroxycoumarin compounds can destroy bacterial wilt cell membrane structures and inhibit bacterial wilt growth by regulating and controlling bacterial wilt lipopolysaccharide synthesis, 2016, li and the like, research discovers that protocatechuic aldehyde can reduce bacterial wilt mobility and inhibit bacterial wilt biofilm formation and destroy cell membrane structures, the prevention and control effect of root irrigation treatment on tobacco bacterial wilt is 92.01%, 2013, kiirika and the like, that comprehensive use of chitosan and silicon is utilized for preventing and treating tomato bacterial wilt, 2011, li and the like discover that polymyxa and bacillus macerans are antagonistic bacteria of tomato bacterial wilt and inhibit bacterial wilt biofilm formation, and 2008, jiang Ning and the like in plant protection indicate that magnolia bark extract of purple oil can effectively inhibit the bacterial wilt. Although the biological agent can effectively inhibit bacterial wilt, the biological agent has larger dosage and higher control cost. In conclusion, the main means for green prevention and control of the bacterial wilt is economic and effective biological prevention and control.
Bacterial spot disease, tomato scab, is commonly referred to as spot disease (Bacterial spot) in tomato production, and can generally result in a 20% -43% yield reduction. Pseudomonas syringae tomato pathogenic variety (Pseudomonas syringaepathovar tomato, pst) Pto DC3000 is a saprophyte in soil that can invade through wounds or water holes of plants, causing bacterial leaf spot on tomato leaves. Pseudomonas syringae Pto DC3000 infects mainly leaves, petioles, flowers and fruits of plants, with the leaf margin and immature fruits being most significantly affected. After the pathogenic bacteria are infected, plant growth is delayed, black disease spots appear on stems and leaf vascular bundles, and the yield and quality of tomatoes are seriously affected. Xanthomonas campestris scab pathogen (Xanthomonas campestris pv. Vesitaria) Xcv 85-10 is a gram-negative bacterium capable of overwintering on seeds and weeds in soil, surviving for more than 9 months and becoming a source of infection for the next year. The pathogenic bacteria can be transmitted by means of rainwater, wind, soil and other mediums, and can cause tomato and pepper scab again. Xanthomonas Xcv 85-10 is a major hazard to pepper and tomato leaves, stems and fruits, especially on leaves, causing irregular or circular yellow lesions, i.e., bacterial blotches, on plant leaves. The CN201210105218.4 patent discloses the use of 2-bromo-2-nitro-1, 3-propanediol to control tomato scab and the CN202111524823.0 patent discloses the use of boron-resistant lysine bacillus bacteriocin analogues to control tomato bacterial blotch.
The green control is an extension of integrated control, and is to select a pest control material with minimum risk by using pest control and green pest control, and the pest control has higher requirements on the pest control material than the integrated control, and requires the use of organic materials (plants) or materials of natural sources.
Sabina chinensis wood alcohol, CAS No.499-44-5, also known as 2-hydroxy-4-isopropyl-2, 4, 6-cycloheptatrien-1-one, is a compound extracted from the trunk of Taiwan cypress in China in 1948 by Japanese scientist AndersonThe monoterpene natural compound of the phenolic ketone skeleton belongs to a tolphenolic ketone compound, has good antibacterial property, moisture retention and pest repellent effect, is a high-safety plant component, has wider biological activity, and has aromatic smell and good effect. Generally, hinokitiol is applied to the control of fungal diseases, and it is not disclosed that hinokitiol can be used to control plant bacterial diseases.
Disclosure of Invention
Aiming at the problems, the invention discloses a method for preventing and treating bacterial diseases by using hinokitiol and inhibiting the lethal concentration of bacterial wilt by using hinokitiol, which can effectively inhibit bacterial wilt by using low-concentration hinokitiol, is an effective method for preventing and treating bacterial wilt and has no pollution to the environment. In addition, the cypress alcohol is used for inhibiting or killing pseudomonas syringae pathogenic bacteria and xanthomonas.
The technical scheme of the invention is as follows:
The application of hinokitiol in preventing and/or controlling plant bacterial diseases.
The application of hinokitiol in preparing medicines for preventing and/or treating plant bacterial diseases.
Preferably, the plant bacterial pathogen is one or more of bacterial wilt pathogen, pseudomonas syringae pathogen and xanthomonas.
Preferably, the plant is tomato or tobacco.
Preferably, when the plant bacterial pathogen is bacterial wilt pathogen LS2020, the concentration of the sabinol is 30-700 mu M.
Preferably, when the plant bacterial pathogen is pseudomonas syringae pathogen Pto DC3000, the concentration of the sabinol is 200 mu M-300 mu M.
Preferably, when the plant bacterial pathogen is xanthomonas Xcv 85-10, the concentration of the sabinol is 200 mu M-300 mu M.
The invention has the beneficial effects that:
Through screening, the invention discovers that the hinokitiol has an inhibiting effect on bacterial wilt LS2020, pseudomonas syringae Pto DC3000 and xanthomonas Xcv 85-10, thereby effectively preventing and treating plant bacterial wilt and alternaria leaf spot. The invention discloses a method for inhibiting the lethal concentration of bacterial wilt and tomato spotted disease pathogenic bacteria by using hinokitiol, and provides a new way for preventing and controlling plant bacterial diseases.
Drawings
FIG. 1 is a graph showing the results of the lowest concentration range of hinokitiol having an inhibitory effect on bacterial wilt LS 2020.
Fig. 2 is a graph showing the results of the lowest concentration range of hinokitiol having a bactericidal effect against ralstonia solanacearum LS 2020.
FIG. 3 is a graph showing the results of the lowest concentration range of hinokitiol having bactericidal effect against Pseudomonas syringae Pto DC 3000.
FIG. 4 is a graph showing the results of the lowest concentration range of hinokitiol having bactericidal effect against Xanthomonas Xcv 85-10.
Fig. 5 is a graph showing the result of inhibiting bacterial wilt in soil by hinokitiol.
FIG. 6 is a graph showing the result of a bacterial wilt proliferation test of hinokitiol on the leaf of Nicotiana benthamiana.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The preparation method of the hinokitiol solution comprises the following steps:
weighing a small amount of hinokitiol, dissolving in dimethyl sulfoxide (DMSO) solution, and preparing into mother solution, wherein the dosage and concentration can be diluted with water as required.
Spectrophotometry is adopted for determining antibacterial effect of hinokitiol.
Spectrophotometry, namely measuring the absorbance of bacterial solutions added with hinokitiol with different concentrations by using an enzyme-labeled instrument, and accurately measuring the inhibition effect of the bacterial wilt. The calculation formula of the bacteriostasis rate is shown as formula (I):
Inhibition = ((OD negative blank control -OD sabina chinensis wood alcohol )/OD negative blank control ) x 100% (I)
Example 1
In the embodiment, spectrophotometry is adopted to detect the inhibition effect of the juniper alcohol with the lowest concentration range on the ralstonia solanacearum LS 2020.
Bacterial wilt LS2020 is subjected to shake culture in a liquid B culture medium (tryptone 10g, yeast extract 1.0g, casein hydrolysate 1.0g, ddH 2 O is added to 1000mL of the culture medium, sterilization is performed at 121 ℃ for 20 min) at 150r/min and 28 ℃ for 24h to obtain bacterial suspension. Sabina alcohol was dissolved in dimethyl sulfoxide to prepare a 10mM solution, and the solution was filtered through a 0.22 μm microporous membrane to remove the effect of bacteria. 3mL of B liquid medium was added to each tube, and hinokitiol (30. Mu.M, 40. Mu.M, 50. Mu.M) and dimethyl sulfoxide at different concentrations were set as negative controls. Inoculating bacterial wilt LS2020 in the test tube, repeating for 3 times, and shake culturing at 150r/min and 28deg.C overnight. Finally 200 mu L of bacterial suspension in a test tube is added into a 96-well plate, and is read at 600nm by a spectrophotometer, and the bacteriostasis rate is calculated according to the formula (I).
The result of the graph 1 shows that the minimum concentration range of the cypress fungus LS2020 with the inhibition effect on the cypress fungus LS2020 is 30 mu M-50 mu M (0.000005 g/mL-0.00008 g/mL), and the inhibition rate is 85% -100%.
Example 2
The sterilization effect of the hinokitiol in the lowest concentration range on the bacterial wilt bacteria LS2020 is detected in the embodiment.
LS2020 is shake cultured in a B liquid culture medium at 150r/min and 28 ℃ for 24 hours to obtain bacterial suspension. Sabina alcohol was dissolved in dimethyl sulfoxide to prepare a 100mM solution, and the solution was filtered through a 0.22 μm microporous membrane to remove the effect of bacteria. 3mL of bacterial wilt LS2020 with an OD600 of 0.1 was added to each tube, and hinokitiol (250. Mu.M, 300. Mu.M) and dimethyl sulfoxide at different concentrations were set as negative controls. 3 repeats are carried out, bacterial solutions are respectively diluted to 10 -1、10-2、10-3、10-4、10-5 when the bacterial solutions are subjected to shake culture for 1h, 2h, 6h and 12h at the temperature of 150r/min and 28 ℃, and 5 mu L of bacterial colony statistics is carried out.
The results of the graph 2 show that the minimum concentration range of the cypress alcohol with the sterilization effect on LS2020 is 200 mu M-300 mu M (0.000032 g/mL-0.00048 g/mL), and the antibacterial rate of the cypress alcohol after 12 hours of drug treatment is 60% -90%.
Example 3
This example examined the bactericidal effect of hinokitiol in the lowest concentration range on pseudomonas syringae Pto DC 3000.
Pseudomonas syringae Pto DC3000 is shake-cultured in LB liquid medium (tryptone 10g, yeast extract 5g, naCl 10g, ddH 2 O is added to volume to 1000m L, sterilization is performed at 121 ℃ for 20 min) at 150r/min and 28 ℃ for 24h to obtain bacterial suspension. Sabina alcohol was dissolved in dimethyl sulfoxide to prepare a 100mM solution, and the solution was filtered through a 0.22 μm microporous membrane to remove the effect of bacteria. 3mL of Pseudomonas syringae Pto DC3000 with an OD600 of 0.1 was added to each tube, and various concentrations of hinokitiol (200. Mu.M, 300. Mu.M) and dimethyl sulfoxide were set as negative controls. 3 repeats are carried out, bacterial solutions are respectively diluted to 10 -1、10-2、10-3、10-4、10-5 when the bacterial solutions are subjected to shake culture for 1h, 4h, 10h and 20h at the temperature of 150r/min and 28 ℃, and 5 mu L of bacterial colony statistics is carried out.
The result of the graph 3 shows that the minimum concentration range of the cypress alcohol with the sterilization effect on pseudomonas syringae PtoDC 3000 is 200 mu M-300 mu M (0.000032 g/mL-0.00048 g/mL), and the antibacterial rate of the medicine treatment for 10 hours is 75% -85%.
Example 4
This example examined the bactericidal effect of hinokitiol on Xanthomonas Xcv 85-10 in the lowest concentration range.
The xanthomonas Xcv 85-10 is shake cultured for 24 hours in LB liquid medium at 150r/min and 28 ℃ to obtain bacterial suspension. Sabina alcohol was dissolved in dimethyl sulfoxide to prepare a 100mM solution, and the solution was filtered through a 0.22 μm microporous membrane to remove the effect of bacteria. Each tube was charged with 3mL of Xanthomonas Xcv 85-10 having an OD600 of 0.1, and various concentrations of hinokitiol (200. Mu.M, 300. Mu.M) and dimethyl sulfoxide were set as negative controls. 3 repeats are carried out, bacterial solutions are respectively diluted to 10 -1、10-2、10-3、10-4、10-5 when the bacterial solutions are subjected to shake culture for 1h, 6h, 10h and 20h at the temperature of 150r/min and 28 ℃, and 5 mu L of bacterial colony statistics is carried out.
The results of FIG. 4 show that the minimum concentration range of the hinokitiol with bactericidal effect on xanthomonas Xcv 85-10 is 200 mu M-300 mu M (0.000032 g/mL-0.00048 g/mL), and the antibacterial rate of the medicine treatment for 20 hours is 50% -85%.
Example 5
This example illustrates the efficacy test of hinokitiol inoculated in greenhouse bacterial wilt LS 2020.
Bacterial wilt LS2020 is subjected to shaking culture in a B liquid culture medium (tryptone 10g, yeast extract 1.0g, casein hydrolysate 1.0g, ddH 2 O is added to a volume of 1000m L, and sterilization is performed at 121 ℃ for 20 min) at 150r/min and at 28 ℃ for overnight to obtain bacterial liquid. For tomato irrigation of around 3 weeks, sabinol (700. Mu.M, 5 mL) and ralstonia solanacearum LS2020 (5 mL) with OD600 of 0.2, dimethyl sulfoxide was set as control group, and 10 replicates were set.
After 4 days of irrigation, tomato plants started to develop disease, a first disease investigation was performed on day 4, and a second disease investigation was performed on day 6, with the results shown in fig. 5 below. The result shows that after 700 mu M (0.000112 g/mL) of hinokitiol with effective concentration is added into tomato soil with bacterial wilt, the disease condition of the tomato is reduced, which indicates that the hinokitiol has a prevention and treatment effect on bacterial wilt in the soil.
Example 6
This example illustrates the proliferation assay of sabinol against ralstonia solanacearum LS2020 on leaf blades of Nicotiana benthamiana.
And (3) culturing the bacterial wilt LS2020 in the B liquid culture medium at 150r/min and 28 ℃ under shaking overnight to obtain bacterial liquid. The leaf of Benshi tobacco with good growth condition about 3 weeks is selected, a disposable sterile injector is used for injecting hinokitiol (700 mu M) and ralstonia solanacearum LS2020 with OD600 of 0.0001 on the leaf, dimethyl sulfoxide is used as a control group, and 3 repetitions are set.
The results in FIG. 6 show that hinokitiol at an effective concentration of 700. Mu.M (0.000112 g/mL) is capable of significantly inhibiting the proliferation of ralstonia solanacearum LS2020 on Nicotiana benthamiana leaves.
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