Application of pipecolic acid as plant disease-resistant activator in preventing and treating apple leaf diseases
阅读说明：本技术 哌啶酸作为植物抗病激活剂在防治苹果叶部病害中的应用 (Application of pipecolic acid as plant disease-resistant activator in preventing and treating apple leaf diseases ) 是由 王彩霞 孙子豪 韩春雪 周娟 练森 李保华 于 2021-01-12 设计创作，主要内容包括：本发明公开了哌啶酸作为植物抗病激活剂在防治苹果叶部病害中的应用,属于植物诱导抗病性技术领域。浓度为0.2mmol·L～(-1)～0.5mmol·L～(-1)的哌啶酸溶液可用于诱导苹果叶片抗病性,有效防治苹果炭疽叶枯病。本发明的哌啶酸溶液本身无离体的杀菌或抑菌活性,在活体条件下才能诱发植物抗病性,诱导苹果叶片产生的抗病性具有持效期长和抗病谱广特点,且环保安全,不宜导致病原菌产生抗药性；使用方法简单,浓度低,用量少；还能提高植物体内防御酶的活性并提高抗病相关基因的表达量,并且能提高生防菌丁香假单胞菌B-1对苹果炭疽叶枯病的防治效果。(The invention discloses application of piperidine acid as a plant disease-resistant activator in prevention and treatment of apple leaf diseases, and belongs to the technical field of plant induced disease resistance. The concentration is 0.2 mmol.L ‑1 ～0.5mmol·L ‑1 The piperidine acid solution can be used for inducing the disease resistance of apple leaves and effectively preventing and treating anthracnose and leaf blight of apples. The piperidine acid solution disclosed by the invention has no in-vitro bactericidal or bacteriostatic activity, can induce plant disease resistance under the living condition, has the characteristics of long duration and wide disease resistance spectrum for inducing the disease resistance generated by apple leaves, is environment-friendly and safe, and is not suitable for causing pathogenic bacteria to generate drug resistance; the use method is simple, the concentration is low, and the dosage is less; can also improve the activity of defense enzymes in plantsThe sex is improved, the expression quantity of disease-resistant related genes is improved, and the control effect of the biocontrol bacterium pseudomonas syringae B-1 on apple anthracnose leaf blight can be improved.)
1. Use of pipecolic acid for enhancing the activity of a defence enzyme in a plant and/or for increasing the expression level of a defence-related gene in a plant.
2. The use according to claim 1, wherein said defensive enzymes are PAL, PPO, POD and SOD; the defense related genes are PR1, PR5, beta-1, 3-glucanase gene and chitinase gene.
3. The application of the pipecolic acid in improving the control effect of the biocontrol bacterium Pseudomonas syringae (Pseudomonas syringae) B-1 on apple anthracnose leaf blight is provided.
4. Use according to any one of claims 1 to 3, wherein the plant material is apple leaves.
5. A plant disease-resistant activator is characterized in that the effective component is piperidine acid.
6. The use of the plant disease-resistant activator according to claim 5, wherein the pipecolic acid is used for inducing apple leaf disease resistance and controlling apple diseases.
7. The use of the plant disease-resistant activator according to claim 6, wherein the apple disease is apple anthracnose leaf blight, apple brown spot or apple rust caused by pathogenic bacteria of apple leaf disease.
8. A method for inducing disease resistance of apple leaves is characterized in that a piperidine acid solution is used.
9. The method of claim 8, wherein the concentration of the piperidine acid solution is 0.2 mmol-L-1～0.5mmol·L-1。
10. The method of claim 8 or 9, wherein the piperidine acid solution is sprayed on the apple tree leaves by using a sprayer.
Technical Field
The invention belongs to the technical field of plant induced disease resistance, and particularly relates to application of piperidine acid as a plant disease resistance activator in prevention and treatment of apple leaf diseases.
Background
The apple is used as an important economic crop and an export fruit in China, is one of important post industries for increasing income of farmers in northern China, but the disease seriously restricts the healthy development of the apple industry in China. More than 100 kinds of diseases damaging apples are reported, and the diseases are divided into leaf diseases, branch diseases, fruit diseases and root diseases according to different damage parts.
Apple anthracnose leaf blight (GLS) caused by small cluster shells (glomerilla cingulata) is a newly discovered disease in apples in China in recent years, leaves and fruits of apples are mainly damaged, the leaves are scorched and fall off in advance, and the serious orchard leaf falling rate reaches 90%; after the fruits are infected, black disease spots with the diameter of 1-2 mm are formed on the surfaces of the fruits, so that the yield and the quality of the fruits are directly reduced, serious economic loss is caused, the tree vigor can be seriously weakened, the disease resistance of the trees is reduced, and the harm of branch diseases and root diseases is induced. The anthracnose leaf blight of the apples is a new disease on the apples, the disease is firstly found in Brazil in 1988, then serious harm of the disease is also reported in the United states, the harm of the anthracnose leaf blight is firstly reported in China in 2012, and the anthracnose leaf blight of the apples is spread and spread to most of main apple producing areas in China in 2014. Gala, Jinguan, Qinhuan and other varieties are highly susceptible to diseases, and Fuji, Hongxing and other varieties have strong resistance.
At present, the control of the anthracnose leaf blight of the apples mainly depends on chemical agents, and frequent use of chemical bactericides can continuously deteriorate the ecological environment of an orchard and bring great hidden danger to food safety; and the anthracnose and leaf blight of the apples are rapidly attacked, the incubation period is only 2-4 days, and once pathogenic bacteria invade host tissues, the application control time is almost not available. If the anthracnose leaf blight can not be effectively controlled, the good varieties with diseases such as Gala and Qinhuan and the like face the risk of being eliminated in China. The chemical bactericide is frequently used in a large amount, not only brings serious threats to human health and environmental safety, but also causes increasingly prominent drug resistance of pathogenic bacteria. Therefore, the search for safe and effective control measures capable of replacing chemical bactericides for controlling apple diseases becomes a problem to be solved urgently in apple production.
Many researches show that the method controls diseases by exciting the defense system of plants and is an important way for developing efficient, safe and green pesticides. The compound capable of inducing plants to generate disease resistance is called a Plant activator, and various commercial disease-resistant activators are applied to crops such as tomatoes, grapes, tobaccos and the like at present, and achieve remarkable disease prevention effect. Induced resistance in plants begins with the recognition by the plant of signal substances from pathogenic bacteria, including increased expression levels of defense genes, increased activity of defense enzymes, accumulation of defense compounds, and the like. The induction resistance is utilized to control plant diseases, has the advantages of long resistance duration, wide disease resistance spectrum, no environmental pollution and the like, and is considered as a new strategy and a feasible way for preventing and controlling the plant diseases.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the application of the piperidine acid as the plant disease-resistant activator in preventing and treating apple leaf diseases. The invention provides a method for inducing disease resistance of apple leaves aiming at the problems of serious harm and difficult control of apple anthracnose leaf blight, environmental pollution caused by chemical agents, pathogenic bacteria resistance and the like in the existing agricultural production, and the method can be used for effectively controlling the apple anthracnose leaf blight. Therefore, the using times and the using amount of the chemical agent are reduced, the harm to the environment and the influence on the human health are weakened, the appearance of the bacterial strains for resisting and resisting the anthracnose blight of the apples can be delayed, and the apple industry develops towards the safe, efficient and sustainable direction.
In order to achieve the purpose, the technical scheme of the invention is as follows:
use of pipecolic acid for enhancing the activity of a defence enzyme in a plant and/or for increasing the expression level of a defence-related gene in a plant.
On the basis of the scheme, the defensive enzyme is PAL, PPO, POD and SOD; the defense related genes are PR1, PR5, beta-1, 3-glucanase gene and chitinase gene.
Based on the same invention concept, the invention provides the application of the piperidine acid in improving the control effect of the biocontrol bacterium Pseudomonas syringae (Pseudomonas syringae) B-1 on apple anthracnose leaf blight.
On the basis of the scheme, the plant body is an apple leaf.
Based on the same invention concept, the invention provides a plant disease-resistant activator, the effective component of which is piperidine acid.
On the basis of the scheme, the piperidine acid is used as a plant disease-resistant activator and is used for inducing apple leaf disease resistance and preventing and treating apple diseases.
On the basis of the scheme, the apple diseases are apple anthracnose leaf blight, apple brown spot or apple rust diseases caused by apple leaf disease pathogenic bacteria.
Based on the same conception, the invention provides a method for inducing apple leaf disease resistance, which is characterized in that piperidine acid solution is used.
Based on the scheme, the concentration of the piperidine acid solution is 0.2 mmol.L-1～0.5mmol·L-1。
On the basis of the scheme, a sprayer is used for spraying a piperidine acid solution on the apple tree leaves.
The invention has the beneficial effects that:
the compound piperidine acid provided by the invention can induce plant disease resistance, belongs to a plant disease resistance activator, and has the following advantages compared with the traditional pesticide:
(1) the plant has no in-vitro sterilization or bacteriostasis function, and can induce the immune system of the plant to resist the attack of diseases under the condition of living bodies;
(2) the disease resistance generated by the induction of the piperidine acid belongs to the physiological metabolic reaction of plant organisms, is safe to the environment and is not suitable for generating drug resistance;
(3) the use method is simple, the concentration is low, and the dosage is less;
(4) the disease resistance generated by induction has long-lasting effect and broad-spectrum characteristics, the apple leaves treated by the piperidine acid have obvious control effect on apple anthracnose leaf blight, also have obvious control effect on apple brown spot and apple rust, the control effect on the diseases can reach more than 70 percent, and the apple leaves have very high economic benefit;
(5) the use of the pipecolic acid can also improve the activities of defense enzymes PAL, PPO, POD and SOD in the apple leaves, and can induce the expression levels of disease-resistant related genes PR1, PR5, beta-1, 3-glucanase gene and chitinase gene to be obviously improved;
(6) the use of the piperidine acid can improve the control effect of the biocontrol bacterium Pseudomonas syringae (Pseudomonas syringae) B-1 on apple anthracnose leaf blight.
Drawings
FIG. 1 shows the effect of pipecolic acid of different concentrations on spore germination and hypha growth of apple colletotrichum gloeosporioides, wherein A is spore germination rate of apple colletotrichum gloeosporioides, and B is colony diameter of apple colletotrichum gloeosporioides;
FIG. 2 shows the control effect of piperidine acid with different concentrations on apple anthracnose leaf blight, wherein A is the influence of piperidine acid with different concentrations on the disease index of apple anthracnose leaf blight, and B is the influence of piperidine acid with different concentrations on the number of spots per leaf of apple anthracnose leaf blight;
FIG. 3 is the variation of the effect of pipecolic acid on controlling apple anthracnose leaf blight with the interval of use;
FIG. 4 is a graph of the effect of pipecolic acid on defensive enzyme activity in apple leaves;
FIG. 5 is the effect of pipecolic acid on the expression of disease resistance related genes in apple leaves;
FIG. 6 shows that piperidine acid improves the control effect of the biocontrol bacterium Pseudomonas syringae (Pseudomonas syringae) B-1 on apple anthracnose leaf blight.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the contents in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention relates to a method for inducing disease resistance of apple leaves, which comprises the steps of applying piperidine acid aqueous solution with effective concentration to the apple leaves, and exciting the disease resistance of the leaves after absorption, thereby improving the resistance of the apple leaves to diseases.
The piperidine acid of the present invention has the following structure:
example 1
The influence of pipecolic acid on spore germination and hypha growth of apple colletotrichum anthracnose:
preparation of spore suspension of apple Ralstonia anthracis (glomeriella cingulata) with concentration of 105each.mL-1Separately, an aqueous piperidine acid solution was added to each spore suspension to give a final concentration of 0.2 mmol. multidot.L-1And 0.5 mmol. multidot.L-1And (5) observing the spore germination condition for 12 hours under the condition of 25 ℃.
PDA medium was prepared, and an aqueous piperidine acid solution was added thereto to give a final concentration of 0.2 mmol.L-1And 0.5 mmol. multidot.L-1Respectively inoculating activated apple anthracnose leaf blight bacteria in the PDA culture medium, performing dark culture at a constant temperature of 25 ℃ for 3-5 days, and measuring the colony diameter.
The PDA culture medium comprises the following components in percentage by weight: peeling potatoes, weighing 200g, cutting into small pieces, boiling in water for 15-20 min, filtering with four layers of gauze, adding 20g of glucose and 15g of agar powder, fixing the volume to 1000mL, keeping the pH value natural, and sterilizing with high-pressure steam at 121 ℃ for 20 min.
The results show that: 0.2 mmol. L-1And 0.5mmol·L-1The pipecolic acid has no obvious influence on spore germination and colony diameter of the apple anthracnose leaf blight germ, which shows that the compound with the concentration has no inhibition effect on spore germination and hypha growth of pathogenic bacteria.
The effect of pipecolic acid on spore germination and hypha growth of apple colletotrichum anthracnose, the results are shown in FIG. 1.
Example 2
The effect of the piperidine acid on controlling apple anthracnose leaf blight is as follows:
preparing spore suspension of apple colletotrichum gloeosporioides, culturing pathogenic bacteria on PDA at constant temperature in dark, scraping off aerial hyphae with inoculating ring when hyphae grow to 2/3 flat dish, allowing generation of orange conidia after 2-3 days, configuring conidia suspension, and adjusting concentration to 5 × 104each.mL-1。
The solution was disposed at 0.2 mmol. multidot.L-1And 0.5 mmol. multidot.L-1Selecting Gala apple branches with completely unfolded leaves of one to two years of consistent growth, respectively and uniformly spraying the piperidine acid solutions with different concentrations until water drops drop from the leaves, and taking sterile water as a reference. And (3) after the piperidine acid solutions with different concentrations are treated, spraying and inoculating the prepared apple colletotrichum gloeosporioides spore suspension, moisturizing for 24 hours by using a plastic bag after field inoculation, and regularly observing and recording the disease condition of leaves.
Grading according to GB/T17980.124-2004 'test criteria for pesticide field efficacy (II)', and calculating disease index.
Level 0: no disease spots; level 1: the lesion area accounts for less than 10% of the whole leaf area; and 3, level: the lesion area accounts for 11 to 30 percent of the leaf area; and 5, stage: the lesion area accounts for 30 to 50 percent of the leaf area; and 7, stage: the lesion area accounts for 51 percent of the leaf area; and 9, stage: and (4) falling leaves.
Calculating the disease index as 100 x (the number of each disease leaf multiplied by the disease value)/(the total number of investigated leaves multiplied by the highest value); the prevention and treatment effect is (contrast disease index-piperidine acid solution treatment disease index)/contrast disease index multiplied by 100 percent
The control effect of the piperidine acid solutions with different concentrations on apple anthracnose leaf blight is shown in figure 2, and the piperidine acid solutions with different concentrationsAfter the liquid treatment, the number of the anthracnose leaf blight spots of the apple and the disease index are both obviously reduced, and the concentration is 0.2 mmol.L-1And 0.5 mmol. multidot.L-1The effect of the piperidine acid solution on controlling the anthracnose leaf blight is over 75 percent.
Example 3
The effect of the piperidine acid on preventing and controlling the anthracnose leaf blight of the apples is changed along with the use interval time:
apple tree leaves were treated according to the method of example 2 to yield 0.2 mmol.L-1Uniformly spraying the piperidine acid aqueous solution on the leaves until water drops, naturally airing, respectively spraying and inoculating the apple anthracnose leaf blight spore suspension at 1d, 2d, 3d, 5d, 7d, 11d and 14d after treatment, and measuring the change of the control effect of the piperidine acid along with the use interval time.
The results are shown in FIG. 3: the pathogenic bacteria are inoculated at different time intervals, the control effect is obviously different, wherein the time intervals are 3-7 days, the control effect is the best, and the control effect is over 77 percent.
Example 4
The effect of the pipecolic acid on controlling the cercospora leaf spot and the rust disease of the apple is as follows:
collecting leaf of cercospora leaf (apple maculopathy), moistening, picking conidia angle, preparing spore suspension, and regulating conidia concentration to 105each.mL-1(ii) a After rainfall, the apple rust (Gymnosporoangium yamadai) winter spore angle on cypress trees is taken, and after germination to generate basidiospores, a spore suspension is prepared.
The solution was disposed at 0.2 mmol. multidot.L-1Selecting Fuji apple branches with completely unfolded leaves of one to two years with consistent growth vigor, uniformly spraying the piperidine acid solution until water drops on the leaves, and taking sterile water as a reference. And 3d after the piperidine acid solution is treated, spraying and inoculating the prepared apple brown spot pathogen and apple rust pathogen spore suspension, moisturizing for 24 hours by using a plastic bag after field inoculation, and regularly observing and recording the disease condition of the leaves.
Grading according to GB/T17980.124-2004 'test criteria for pesticide field efficacy (II)', and calculating disease index.
Level 0: no disease spots; level 1: the lesion area accounts for less than 10% of the whole leaf area; and 3, level: the lesion area accounts for 11 to 30 percent of the leaf area; and 5, stage: the lesion area accounts for 30 to 50 percent of the leaf area; and 7, stage: the lesion area accounts for 51 percent of the leaf area; and 9, stage: and (4) falling leaves.
Calculating the disease index as 100 x (the number of each disease leaf multiplied by the disease value)/(the total number of investigated leaves multiplied by the highest value); the prevention and treatment effect is (contrast disease index-piperidine acid solution treatment disease index)/contrast disease index multiplied by 100 percent
0.2mmol·L-1After the Fuji apple leaves are treated by the piperidine acid aqueous solution, pathogenic bacteria of the apple brown spot and the apple rust are inoculated, the control effect is remarkable, and the control effect on the apple brown spot and the apple rust is 76.4 percent and 70.5 percent respectively.
Example 5
The piperidine acid induces the apple leaf disease-resistant mechanism:
apple tree leaves are treated according to the method of example 2, and 0.2 mmol.L is evenly sprayed-1And (3) taking the piperidine acid aqueous solution as a control of the leaves treated by the sterile water, respectively sampling 0, 0.5, 1, 2, 3, 5, 7 and 11d after treatment, and determining the change trend of defense-related enzyme activity and disease-resistant related genes in leaf tissues.
The results are shown in FIGS. 4 and 5: 0.2 mmol. L-1After the apple leaves are treated by the piperidinic acid, the activities of Phenylalanine Ammonia Lyase (PAL), polyphenol oxidase (PPO), Peroxidase (POD) and superoxide dismutase (SOD) in the leaf tissues are obviously improved compared with the control, and the gene expression levels of disease course related Protein (PRs) genes PR1, PR5, beta-1, 3-glucanase gene and chitinase are obviously increased, which indicates that the piperidinic acid can induce the resistance of the apple leaves to diseases by improving the activity of defense enzymes and the expression of disease-resistant genes in the leaves.
Example 6
The piperidine acid improves the control effect of Pseudomonas syringae (Pseudomonas syringae) B-1 on apple anthracnose leaf blight:
pseudomonas syringae (Pseudomonas syringae) B-1 used in this example was deposited at 31 months 12 in 2015 at: china center for type culture Collection, accession number: m2015813, the preservation address is as follows: wuhan city, Hubei province, the flood mountain area eight ways.
Prepared at a concentration of 105cfu·L-1Mixing the suspension of biocontrol bacterium Pseudomonas syringae (Pseudomonas syringae) B-1 with a piperidine acid solution to obtain a final piperidine acid concentration of 0.2 mmol.L-1. And selecting the gala apple branches with consistent growth vigor and completely unfolded leaves for one to two years, respectively and uniformly spraying the bacterial suspension of the bacterial strains B-1 with different concentrations until water drops on the leaves, and taking sterile water as a reference. And (3) mixing the pseudomonas syringae B-1 bacterial suspension with a piperidine acid solution to treat apple leaves, inoculating the apple colletotrichum gloeosporioides spore suspension according to the method in the embodiment 2, and calculating the control effect.
The results are shown in FIG. 6: the piperidine acid can obviously improve the control effect of the pseudomonas syringae B-1, and the control effects of the single treatment of the pseudomonas syringae B-1 are respectively 80.5% and 71.2% at 7d and 14d after inoculation; the control effect of the mixed treatment of the pipecolic acid and the pseudomonas syringae B-1 respectively reaches 90.6 percent and 80.3 percent.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.