Method for inducing peach bacterial shot hole resistance to coumaric acid

Abstract

The invention discloses a method for inducing peach bacterial shot hole resistance by coumaric acid. According to the method, p-coumaric acid is used for carrying out root irrigation treatment on potted high-susceptibility peach germplasm resources, living body inoculation is carried out on leaves after root irrigation treatment, and the induced resistance effect is evaluated by counting disease indexes. The test result shows that the disease index of the p-coumaric acid aqueous solution with the concentration of 1.0 mmol / L after root irrigation treatment is 35.55, and the disease index of clear water control is 65.05, which indicates that the p-coumaric acid can effectively induce the resistance of peach germplasm resources to the peach bacterial shot hole disease.

Description

Technical Field

[0001] The invention belongs to the technical field of development of peach bacterial punch hole disease inducers, and particularly relates to a method for inducing peach bacterial punch hole disease resistance with p-coumaric acid. Background Art

[0002] Peach bacterial punchhole is a common global disease on peach trees and one of the important diseases in peach producing areas. The pathogen is Xanthomonas arboricola pv.pruni (Xap), which mainly infects leaves, branches and fruits, and invades the tissues through the stomata, bud scars and lenticels of the leaves, showing water-soaked lesions on the leaves. In the later stage, the lesions dry up and fall off in a perforated shape, causing a large number of leaves to fall early. The infected fruits lead to a decline in fruit quality, and the serious infection of fruits leads to a significant reduction in production or even a total crop failure, causing huge economic losses. In addition to harming peach trees, the pathogen also harms stone fruit trees such as plum, apricot, cherry, and almond. With the prevalence of peach bacterial punchhole in peach producing areas, the demand for effective prevention and control agents is increasing. There are few types of chemical agents available for the prevention and control of peach bacterial shothole in production. Among them, copper preparations and antibiotics are widely used effective agents for the prevention and control of peach bacterial shothole. However, their dependence and long-term use have led to increasingly prominent problems such as drug resistance, tree toxicity, environmental pollution and pesticide residues.

[0003] Induced resistance is currently an effective plant disease resistance strategy. Compared with the inherent disease resistance of the plant itself, the disease resistance induced in plants has the following characteristics:

[0004] (1) Broad-spectrum resistance, that is, plant-induced resistance, can provide resistance to a variety of diseases caused by a variety of pathogens.

[0005] (2) Persistence: the induced disease resistance can be maintained for a long time, even throughout the entire growth cycle.

[0006] (3) Transmissibility and non-inheritance: Plants can transmit the acquired local disease resistance to other parts, so that the entire plant acquires disease resistance, but this disease resistance cannot be inherited to the next generation through seeds.

[0007] (4) Safety. The induction factor itself does not have a direct antibacterial effect, but rather exerts disease resistance by inducing the plant's own immune system. It does not produce toxic or harmful substances or residues, and therefore does not endanger the safety of humans, animals, and the environment.

[0008] At present, a few inducers have been reported, including chitosan, methyl jasmonate, amino oligosaccharides, nano-NANOZnO and mineral elements (Si). Summary of the invention

[0009] In view of the fact that the existing peach bacterial punch hole prevention and control drugs mainly use copper preparations and antibiotics, which lead to drug resistance, tree toxicity, environmental pollution and pesticide residues, the present invention provides a method for inducing peach bacterial punch hole resistance with p-coumaric acid, and also the application of p-coumaric acid in inducing peach bacterial punch hole resistance. The present invention uses three compounds (methyl ferulate, p-coumaric acid and methyl caffeate) with good inhibitory effects on the pathogens of peach bacterial punch hole to perform root irrigation treatment on highly susceptible peach germplasm resources, and then performs live inoculation after treatment, and evaluates the induction resistance effect by disease index.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A method for inducing resistance to bacterial puncture of peach by p-coumaric acid, comprising the following steps:

[0012] S1. Test materials: The "Fenghua Pantao", a peach variety highly susceptible to bacterial perforation disease, selected in the previous stage, was introduced from the National Peach Germplasm Resource Nursery of Beijing Academy of Agriculture and Forestry Sciences and planted in Guilin, Guangxi. It is an annual potted seedling;

[0013] S2. Root irrigation treatment: Use purified water to prepare a 0.25-1.0 mmol / L aqueous solution of p-coumaric acid, and irrigate the roots of the potted test seedlings. Irrigate the roots again every 3 days, with a root irrigation volume of 1000 mL / plant each time, for a total of 2 times;

[0014] S3. Live inoculation:

[0015] S3-1. Preparation of test bacterial suspension: Take the preserved bacteria with an inoculation loop, inoculate them onto the LB solid plate by streak method, and culture at 37℃±1℃ for 24h; Take 20mL of LB liquid culture medium and add it into a sterile conical flask with a capacity of 100mL, take a single colony on the LB solid plate with an inoculation loop and inoculate it into the LB liquid culture medium, and culture at 37℃±1℃ for 12h~18h; Use LB liquid culture medium to adjust the bacterial concentration after culture to an OD value of 0.65, and use this as the test bacterial suspension;

[0016] S3-2, live inoculation: 6 days after the root irrigation treatment, all leaves that have just turned green were selected from different branches of each tree, and the needle injection method was used for inoculation. The specific inoculation operation was as follows: first, a sterile syringe needle was used to puncture holes symmetrically on both sides of the veins in the middle of the leaf, and the front of the leaf was held up by fingers. The syringe nipple was aligned with the puncture holes from the back of the leaf, and the piston handle was lightly pressed to completely penetrate the bacterial suspension into the leaf flesh tissue until a clear bacterial liquid circle was formed. Four holes were inoculated on each leaf;

[0017] S4. Evaluation of induced resistance effect:

[0018] S4-1. Measurement of diseased hole area: 15 days after inoculation, the leaves were collected after the lesion tissues completely fell off, and the diseased holes were covered with a transparent grid film ruler. The number of grids occupied by the diseased holes on the transparent grid film ruler was calculated (the area of ​​each grid was 1 mm 2 ), area greater than 0.5mm 2 Count 1 grid, area less than 0.5mm 2 Count 0;

[0019] S4-2. Disease grade classification standard: The disease grade is classified according to the size of the lesion hole, as follows:

[0020] Level 0: The lesion area is 0 to 1.0 mm 2 ;

[0021] Level 1: The lesion area is 1.1 to 2.0 mm 2 ;

[0022] Level 2: The lesion area is 2.1 to 3.0 mm 2 ;

[0023] Level 3: The lesion area is 3.1-4.0 mm 2 ;

[0024] Level 4: The lesion area is 4.1-5.0 mm 2 ;

[0025] Level 5: The lesion area is ≥5.1mm 2 ;

[0026] S4-3. Evaluation of the effect of induced resistance: Substitute the disease grade into the disease index calculation formula:

[0027]

[0028] The disease index is 34.55-50.24.

[0029] In the present invention:

[0030] Furthermore, in step S1, the peach bacterial punch hole disease refers to a disease caused by peach trees being infected with the tree-borne Xanthomonas arboricola pv. Pruni pathogenic bacteria.

[0031] Preferably, in step S2, a p-coumaric acid aqueous solution with a concentration of 1.0 mmol / L is prepared using purified water.

[0032] The feasibility of the method is evaluated according to the disease index in step S4.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] The present invention discloses a method for inducing resistance to bacterial punchhole disease of peach by using p-coumaric acid, and also discloses the application of p-coumaric acid in inducing resistance to bacterial punchhole disease of peach, thereby providing a compound with good resistance induction effect. p-coumaric acid is a natural product with abundant sources, and there is no report in the prior art on the application of p-coumaric acid in inducing resistance to bacterial punchhole disease of peach. Therefore, p-coumaric acid is expected to become an inducer with good effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a diagram of the induction effect of a method for inducing peach bacterial punch hole resistance using p-coumaric acid as described in an embodiment of the present invention (A, p-coumaric acid; B, methyl ferulate; C, methyl caffeate; D, clean water control). DETAILED DESCRIPTION

[0036] The specific implementation modes of the present invention are further described below in conjunction with examples.

[0037] Example:

[0038] A method for inducing resistance to bacterial puncture of peach by p-coumaric acid, comprising the following steps:

[0039] 1. Materials:

[0040] 1.1 Test materials:

[0041] The "Fenghua Pantao", a peach variety highly susceptible to bacterial perforation disease that was screened out in the early stage, was used as the test material, introduced to the National Peach Germplasm Resource Nursery of Beijing Academy of Agricultural and Forestry Sciences, and planted in Guilin, Guangxi, as an annual potted seedling.

[0042] 1.2 Drugs and reagents:

[0043] p-Coumaric acid, caffeic acid methyl ester, and ferulate methyl ester were purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0044] LB broth powder and nutrient agar (NA) were purchased from Haibo Biotechnology Co., Ltd.;

[0045] Culture medium:

[0046] LB liquid medium was prepared by weighing 33.0 g of LB broth powder, adding it to 1000 mL of distilled water, boiling to dissolve, adjusting the pH to 7.0 ± 0.2, dispensing into conical flasks, and sterilizing at 120 °C for 20 min;

[0047] Solid culture medium was prepared by weighing 33.0 g of LB broth powder, adding it to 1000 mL of distilled water, boiling to dissolve, adjusting the pH to 7.0 ± 0.2, dispensing into 250 mL conical flasks, and sterilizing at 120 °C for 20 min;

[0048] LB solid plate, solid culture medium is sterilized at 120℃, cooled to about 55℃, 15mL is taken into a sterile culture dish, and waited for it to solidify;

[0049] 1.3 Bacterial suspension:

[0050] Use an inoculating loop to take the preserved bacteria, inoculate them onto the LB solid plate by the streak method, and culture them at 28℃±1℃ for 24h; take 20mL of LB liquid culture medium and add it into a sterile conical flask with a capacity of 100mL, use an inoculating loop to take a single colony on the LB solid plate and inoculate it into the LB liquid culture medium, culture it at 28℃±1℃ for 48h, then transfer it to a 50mL sterile centrifuge tube, centrifuge it at 4000r / min, discard the liquid culture medium, and suspend the bacteria in sterile water to obtain a bacterial suspension.

[0051] 2. Experimental methods:

[0052] 2.1 Root irrigation treatment

[0053] A 1.0 mmol / L aqueous solution of p-coumaric acid, methyl ferulate and methyl caffeate was prepared with purified water and used for root irrigation of potted test seedlings. The root irrigation was repeated after 3 days, with purified water as the control. The root irrigation volume was 1000 mL per plant each time, and the root irrigation was performed twice in total.

[0054] 2.2 Live inoculation: 6 days after the root irrigation treatment, select all the leaves that have just turned green on different branches of each tree and inoculate with the fungus using the needle injection method; the specific inoculation operation is: first use a sterile syringe needle to puncture holes symmetrically in the middle of the leaf and on both sides of the veins, hold up the front of the leaf with your fingers, aim the syringe nipple at the puncture holes from the back of the leaf, and lightly press the piston handle to allow the bacterial suspension to completely penetrate the mesophyll tissue until a clear bacterial liquid circle is formed. Inoculate 4 holes on each leaf.

[0055] 2.3 Evaluation of induced resistance effect

[0056] 2.3.1 Measurement of diseased hole area: 15 days after inoculation, the leaves were collected after the lesion tissues completely fell off, and the diseased holes were covered with a transparent grid film ruler. The number of grids occupied by the diseased holes on the transparent grid film ruler was calculated (each grid area was 1 mm 2 ), area greater than 0.5mm 2 Count 1 grid, area less than 0.5mm 2 Count 0.

[0057] 2.3.2 Disease grade classification standard: The disease grade is classified according to the size of the lesion hole, as follows:

[0058] Level 0: The lesion area is 0 to 1.0 mm 2 ;

[0059] Level 1: The lesion area is 1.1 to 2.0 mm 2 ;

[0060] Level 2: The lesion area is 2.1 to 3.0 mm 2 ;

[0061] Level 3: The lesion area is 3.1 to 4.0 mm 2 ;

[0062] Level 4: The lesion area is 4.1 to 5.0 mm 2 ;

[0063] Level 5: The lesion area is ≥5.1mm 2 ;

[0064] 2.3.3 Disease index statistics: Substitute the disease grade into the disease index calculation formula to obtain the disease index:

[0065]

[0066] 2.4 Evaluation of the induction resistance effect of different concentrations of p-coumaric acid according to the methods of 2.1-2.3

[0067] 3 Experimental results

[0068] 3.1 Induced resistance effects of different compounds

[0069] According to Table 1, the best induction resistance effect of the same concentration (1.0mmol / L) treatment was p-coumaric acid, followed by caffeic acid methyl ester. After root irrigation with 1.0mmol / L p-coumaric acid solution, the disease index was 34.55, while the disease index of the clear water control was 65.05.

[0070] Table 1 Disease index after root irrigation with different compounds:

[0071]

[0072] 3.2 Induced resistance effect of different concentrations of p-coumaric acid

[0073] p-Coumaric acid is slightly soluble in cold water and becomes turbid when the concentration is 1.25mmol / L, so 4 concentrations of 0.25, 0.5, 0.75 and 1.0mmol / L were set to investigate the induction effect of p-coumaric acid at different concentrations. According to Table 2, as the concentration of p-coumaric acid increases, the induction resistance effect increases, and the optimal induction concentration is 1.0mmol / L.

[0074] Table 2 Disease index after root irrigation with different concentrations of p-coumaric acid:

[0075]

[0076]

[0077] Figure 1 It is a diagram of the induction effect of p-coumaric acid (A), methyl ferulate (B), methyl caffeate (C) and a clear water control (D) in a method for inducing resistance to bacterial scorch disease of peach with p-coumaric acid (application of p-coumaric acid in inducing resistance to bacterial scorch disease of peach) in an embodiment.

[0078] The above description is only a preferred embodiment of the present invention. It should be pointed out that it is possible for a person skilled in the art to make several improvements and changes without departing from the creative concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for inducing resistance to bacterial puncture of peach by p-coumaric acid, characterized in that: The steps include: S1. Test materials: The "Fenghua Pantao", a peach variety highly susceptible to bacterial perforation disease, selected in the previous stage, was introduced from the National Peach Germplasm Resource Nursery of Beijing Academy of Agriculture and Forestry Sciences and planted in Guilin, Guangxi. It is an annual potted seedling; S2. Root irrigation treatment: Use purified water to prepare a 0.25-1.0 mmol / L aqueous solution of p-coumaric acid, and irrigate the roots of the potted test seedlings. Irrigate the roots again every 3 days, with a root irrigation volume of 1000 mL / plant each time, for a total of 2 times; S3. Live inoculation: S3-1. Preparation of test bacterial suspension: Take the preserved bacteria with an inoculation loop, inoculate them onto the LB solid plate by streak method, and culture at 37℃±1℃ for 24h; Take 20mL of LB liquid culture medium and add it into a sterile conical flask with a capacity of 100mL, take a single colony on the LB solid plate with an inoculation loop and inoculate it into the LB liquid culture medium, and culture at 37℃±1℃ for 12h~18h; Use LB liquid culture medium to adjust the bacterial concentration after culture to an OD value of 0.65, and use this as the test bacterial suspension; S3-2, live inoculation: 6 days after the root irrigation treatment, all leaves that have just turned green were selected from different branches of each tree, and the needle injection method was used for inoculation. The specific inoculation operation was as follows: first, a sterile syringe needle was used to puncture holes symmetrically on both sides of the veins in the middle of the leaf, and the front of the leaf was held up by fingers. The syringe nipple was aligned with the puncture holes from the back of the leaf, and the piston handle was lightly pressed to completely penetrate the bacterial suspension into the leaf flesh tissue until a clear bacterial liquid circle was formed. Four holes were inoculated on each leaf; S4. Evaluation of induced resistance effect: S4-1. Measurement of diseased hole area: 15 days after inoculation, the leaves were collected after the lesion tissues completely fell off, and the diseased holes were covered with a transparent grid film ruler. The number of grids occupied by the diseased holes on the transparent grid film ruler was calculated. The area of ​​each grid was 1 mm 2 , area greater than 0.5mm 2 Count 1 grid, area less than 0.5mm 2 Count 0; S4-2. Disease grade classification standard: The disease grade is classified according to the size of the lesion hole, as follows: Level 0: The lesion area is 0 to 1.0 mm 2 ; Level 1: The lesion area is 1.1 to 2.0 mm 2 ; Level 2: The lesion area is 2.1 to 3.0 mm 2 ; Level 3: The lesion area is 3.1-4.0 mm 2 ; Level 4: The lesion area is 4.1-5.0 mm 2 ; Level 5: The lesion area is ≥5.1mm 2 ; S4-3. Evaluation of the effect of induced resistance: Substitute the disease grade into the disease index calculation formula: The disease index is 34.55-50.

24.

2. The method for inducing resistance to bacterial puncture of peach by p-coumaric acid according to claim 1, characterized in that: In step S1, the peach bacterial punch hole disease refers to a disease caused by peach trees being infected with the tree-borne xanthomonas arborigenic bacteria Xanthomonas arborigenic bacteria pv. Pruni.

3. The method for inducing resistance to bacterial punch hole of peach by p-coumaric acid according to claim 1, characterized in that: In step S2, a p-coumaric acid aqueous solution with a concentration of 1.0 mmol / L is prepared using purified water.

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