Rapid screening method for rust-resistant pepper and application of rapid screening method
By inoculating and cultured pepper leaves with spore liquid in the laboratory, rapid screening of anti-rust pepper varieties is achieved, and the problems of low screening efficiency and environmental dependence in the prior art are solved, and an efficient and repeatable screening method is provided.
Patent Information
- Application Number
- CN202510020164.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-07
- Publication Date
- 2025-03-04
AI Technical Summary
The existing technology is difficult to effectively screen rust-resistant pepper varieties, mainly due to the long growth cycle, large area, large field environment influence and long rust onset cycle, which makes the ex vivo screening method difficult to succeed.
A rapid screening method for anti-rust peppercorns is provided, including preserving the leaves of peppercorns with special preservative liquid, and inoculating spore liquid in the laboratory, and observing the disease of the leaves after 13 days. The preservative composition of the method includes pepper leaf extract, citric acid, luteolin, L-proline, methyl jasmonate, ascorbic acid, acetic acid, chitin, yellow collagen, ethanol and sterile water.
This method can quickly and effectively screen rust-resistant pepper varieties in the laboratory. The cycle is short, the screening amount is large, and it can be repeated multiple times, and is not affected by the field environment. The data obtained is consistent with the field test data, providing theoretical basis and technical support for the breeding of rust-resistant pepper varieties.
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Figure CN119547724A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of disease-resistant breeding, and in particular to a rapid screening method for rust-resistant Zanthoxylum bungeanum and an application thereof. Background Art
[0002] Pepper rust is an important disease in the pepper cultivation process. It occurs from August to October every year in the north, and occurs all year round in green pepper in the south. When the plant is infected by the pathogen, the leaves fall off, affecting the nutrition of the tree, and then affecting the yield of pepper in the next year. At present, the prevention and control of pepper rust mainly relies on chemical agents, but it requires continuous spraying of pesticides, which has high manpower and material costs, and the pathogen gradually develops resistance, making it more difficult to prevent. Screening pepper varieties resistant to rust can provide a better strategy for the prevention and control of pepper rust, but there is currently no method for screening pepper rust-resistant varieties. Screening methods for rust varieties have been established on crops such as wheat and soybeans. They are mainly through field inoculation tests to investigate the incidence, disease index and symptoms of different varieties to identify disease-resistant varieties. Crops such as wheat and soybeans are agricultural crops with short growth cycles, and the test is easier to complete. Pepper belongs to forestry crops with long growth cycles and large land area, which limits the screening of a large number of rust-resistant varieties in the field. And based on the existing in vitro screening technology, it was found that due to the long disease cycle of rust, the disease often takes 11 days to occur after inoculation, and the leaves are often easy to rot in the in vitro environment, making the in vitro screening method not easy to succeed. Summary of the invention
[0003] The purpose of the present invention is to provide a method for quickly screening rust-resistant Zanthoxylum bungeanum and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention can screen rust-resistant varieties of Zanthoxylum bungeanum in a laboratory, has the characteristics of short cycle, large screening amount, multiple repetitions, and no influence from field environment, and provides a theoretical basis and technical support for the breeding of rust-resistant Zanthoxylum bungeanum varieties.
[0004] To achieve the above object, the present invention provides the following solutions:
[0005] The present invention provides a method for quickly screening rust-resistant Zanthoxylum bungeanum, comprising the following steps:
[0006] Taking Zanthoxylum bungeanum leaves and preserving them with a preservative solution, inoculating spore solution for cultivation, and observing the disease condition of the leaves after 13 days;
[0007] The fresh-keeping solution comprises the following components in mass fractions: 4% of Zanthoxylum bungeanum leaf extract, 1% of citric acid, 0.5% of luteolin, 2.5% of L-proline, 1.5% of methyl jasmonate, 2% of ascorbic acid, 0.5% of acetic acid, 0.35% of chitosan, 1% of xanthocollagen, 10% of ethanol and 76.65% of sterile water.
[0008] Optionally, the fresh-keeping solution is prepared by the following method:
[0009] The Zanthoxylum bungeanum leaves are dried and crushed into powder, ethanol is added for ultrasonic extraction, zirconium oxide beads are added for continued oscillation extraction, and the Zanthoxylum bungeanum leaf extract is obtained by filtration and reduced pressure distillation;
[0010] The extract of Zanthoxylum bungeanum leaf, luteolin, methyl jasmonate, chitosan and xanthocollagen are dissolved in ethanol, half amount of sterile water is added, stirred in an anaerobic tank, and then allowed to stand, and then the remaining sterile water, acetic acid, ascorbic acid, citric acid and L-proline are added, stirred and ultrasonicated again to obtain the fresh-keeping solution.
[0011] Optionally, the ratio of the powder to ethanol is 1 g: (10-20) mL.
[0012] Optionally, the conditions for the ultrasonic extraction are: power of 40 W, temperature of 60° C., and time of 1 h.
[0013] Optionally, the oscillation condition is: 180r / min oscillation for 24h.
[0014] Optionally, the conditions for the second ultrasound are: power of 40 W and time of 1 h.
[0015] Optionally, the preservation includes the steps of dipping cotton into the diluted preservation solution and wrapping the cotton around the petiole of the pepper leaf in a ring shape; and the dilution includes diluting it ten times.
[0016] Optionally, the culture conditions are: temperature of 25°C, light:darkness of 16h:8h, light intensity of 10000LX, and humidity of 75%.
[0017] The invention also provides application of the screening method in breeding rust-resistant Zanthoxylum bungeanum varieties.
[0018] The present invention also provides a special fresh-keeping solution for isolated Zanthoxylum bungeanum leaves, which comprises the following components in mass fractions: 4% of Zanthoxylum bungeanum leaf extract, 1% of citric acid, 0.5% of luteolin, 2.5% of L-proline, 1.5% of methyl jasmonate, 2% of ascorbic acid, 0.5% of acetic acid, 0.35% of chitosan, 1% of xanthocollagen, 10% of ethanol and 76.65% of sterile water.
[0019] The present invention discloses the following technical effects:
[0020] The present invention provides a method for rapid screening of rust-resistant pepper and a preservative solution for pepper leaves, aiming at the characteristics of long disease-resistant screening cycle of forest varieties, large land area, and large field impact. By using the preservative solution prepared by the present invention, the problem that isolated pepper is often easy to rot is overcome. At the same time, the screening method can be operated in the laboratory, has the characteristics of short cycle, large screening amount, good repeatability, etc., and the data obtained are consistent with the field test data. The present invention provides a theoretical basis and technical support for the breeding of rust-resistant pepper varieties. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0022] Figure 1 The picture shows the collection of pepper rust spores; from left to right, the collection device, the drying process, and the dried spore powder;
[0023] Figure 2 For indoor vaccination pictures;
[0024] Figure 3 The left side shows the field performance of disease-resistant varieties; the right side shows the field performance of disease-susceptible varieties. DETAILED DESCRIPTION
[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.
[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0030] Example 1
[0031] 1. Collection of spore powder and preparation of spore suspension
[0032] Collect the living leaves with bacteria, put them in a sterilized beaker, add sterile water and stir to disperse the spore powder in the water, take out the leaves, use a vacuum filtration device to filter, and separate with a 0.45 micron filter membrane. Put the obtained spore powder into silica gel and dry it naturally for later use ( Figure 1 ).
[0033] Prepare a 0.01% Tween-20 solution with sterile water, put the collected spores into the solution, stir evenly, check the spore concentration under a microscope, and adjust the spore suspension concentration to 1×10 5 Pieces / mL.
[0034] 2. Preparation of pepper leaves
[0035] Healthy 2-year-old green pepper leaves were collected, mainly the new leaves 4-5 leaves from the top for the test. The leaves were rinsed in tap water for 3 minutes, then soaked in 10% sodium hypochlorite solution for 5 minutes, and then soaked in 75% ethanol for 30 seconds. They were immediately washed three times with sterile water and placed in a sterilized culture dish for later use. The culture dish was padded with sterilized moist cotton.
[0036] 3. Preparation of leaf preservation solution
[0037] Select healthy Zanthoxylum bungeanum leaves, clean them and dry them at low temperature, grind them into powder with a grinder, weigh 5g and put them into a conical flask, add 70% ethanol at a ratio of 1g: (10-20)mL, ultrasonically extract at 40W and 60℃ for 1h, then add 6mm zirconia beads and shake at 180r / min for 24h, filter with filter paper, and use a rotary evaporator to perform vacuum distillation on the filtrate to remove the extraction solvent to obtain Zanthoxylum bungeanum leaf extract.
[0038] Prepare 4% Zanthoxylum leaf extract, 1% citric acid, 0.5% luteolin, 2.5% L-proline, 1.5% methyl jasmonate, 2% ascorbic acid, 0.5% acetic acid, 0.35% chitin, 1% xanthocollagen, 10% ethanol and 76.65% sterile water according to mass fraction. Dissolve Zanthoxylum leaf extract, luteolin, methyl jasmonate, chitin and xanthocollagen in ethanol solution, add half amount of sterile water (i.e. 38.325%), stir evenly and mix into anaerobic tank, stir for 24h, pour out solution, stand at constant temperature of 25℃ for 12h, then add remaining sterile water (i.e. 38.325%), acetic acid, ascorbic acid, citric acid and L-proline, stir for 2h, and ultrasonicate at 40W at room temperature for 1h to obtain self-made leaf preservative solution, filter the preservative solution with 0.22 micron microporous filter membrane and seal and store in 4℃ refrigerator for standby use.
[0039] 4. Keep pepper leaves fresh
[0040] Dilute the homemade leaf preservative solution 10 times with sterile water, dip sterilized cotton in the diluted preservative solution, wrap the petiole of the pepper with the cotton in a ring shape, and put it in the prepared sterile culture dish for inoculation.
[0041] 5. Inoculation
[0042] Dip a sterilized brush into the spore suspension and gently apply it on the back of the leaves. Place the leaves coated with spores in a culture dish and culture them in a light incubator. The culture conditions are: 25°C, 16h:8h=light:dark, light intensity 10000LX, humidity 75%. During this period, add sterile water appropriately. Inoculate 30 leaflets of each variety, repeat 3 times, and use only 0.01% Tween-20 solution as a blank control (CK) ( Figure 2 ).
[0043] During the in vitro screening process, a certain amount of fresh-keeping liquid was sucked out with a syringe to moisten the cotton at the base of the petiole every three days to ensure the fresh-keeping effect.
[0044] 6. Data Recording
[0045] After 13 days of cultivation, the leaf incidence and disease index were observed and recorded to evaluate the disease resistance of each variety.
[0046] References for disease index grading standards: Cao Zhimin, Tian Chengming, Liang Yingmei. Preliminary study on pepper rust_Cao Zhimin [J]. Shaanxi Forestry Science and Technology, 1989, (04): 63-65.).
[0047] If the disease index is <10, it is judged as a disease-resistant variety; if the disease index is 10-25, it is judged as a moderately susceptible variety; if the disease index is >30, it is judged as a susceptible variety.
[0048] 7. Results
[0049] Three in vitro tests on green pepper were conducted in May, August and October respectively. The varieties used were the existing green pepper varieties A, B, C, D, E, F and G in Guizhou Province (A, B, C, D, E and F were selected and bred from Dingtan pepper, namely Dingtan No. 1, Dingtan No. 2, Dingtan No. 3, Dingtan No. 4, Dingtan No. 5 and Dingtan No. 6 respectively, and G was the Jiuyeqing variety), all of which were provided by the pepper research team of Guizhou Academy of Forestry Sciences. Jiuyeqing was known to be a susceptible variety. Its incidence rate and disease index through in vitro tests are shown in Table 1.
[0050] Furthermore, it is found that the problem that isolated Zanthoxylum bungeanum is prone to rot is overcome by using the fresh-keeping solution prepared by the present invention.
[0051] Table 1 In vitro test data results
[0052]
[0053]
[0054] After three in vitro tests, it was shown that variety A had the lightest incidence and disease index, and could be identified as a disease-resistant variety, while variety G was the most susceptible to the disease, and was a susceptible variety.
[0055] Example 2 Field test
[0056] The varieties were planted in the field, with 20 trees and 2 rows of each variety. All varieties were sprayed with fungi in September, and the incidence and disease index of each variety were counted in October. The results are shown in Table 2 and Figure 3 .
[0057] Table 2 Field test data results
[0058] variety Incidence Disease index A 15％ 6.7 B 22.4％ 9.8 C 30.5％ 12.5 D 44.5％ 16.8 E 55.8％ 22.5 F 60％ 32.4 G 68.5％ 35.8
[0059] According to the field test data, variety A is a disease-resistant variety, while variety G is susceptible to the disease, and the susceptibility of the seven varieties is consistent with the indoor in vitro leaf test, indicating that the in vitro screening method has a certain degree of reliability and can be used for preliminary resistance screening tests.
[0060] The present invention can quickly, efficiently and conveniently screen out disease-resistant varieties, and provide a theoretical basis and technical support for pollution-free prevention and control of pepper rust and resistance breeding.
[0061] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. A rapid screening method for rust-resistant Zanthoxylum bungeanum, characterized in that: The following steps are involved: Taking Zanthoxylum bungeanum leaves and preserving them with a preservative solution, inoculating spore solution for cultivation, and observing the disease condition of the leaves after 13 days; The fresh-keeping solution comprises the following components in mass fractions: 4% of Zanthoxylum bungeanum leaf extract, 1% of citric acid, 0.5% of luteolin, 2.5% of L-proline, 1.5% of methyl jasmonate, 2% of ascorbic acid, 0.5% of acetic acid, 0.35% of chitosan, 1% of xanthocollagen, 10% of ethanol and 76.65% of sterile water.
2. The screening method according to claim 1, characterized in that The fresh-keeping solution is prepared by the following method: The Zanthoxylum bungeanum leaves are dried and crushed into powder, ethanol is added for ultrasonic extraction, zirconium oxide beads are added for continued oscillation extraction, and the Zanthoxylum bungeanum leaf extract is obtained by filtration and reduced pressure distillation; The extract of Zanthoxylum bungeanum leaf, luteolin, methyl jasmonate, chitosan and xanthocollagen are dissolved in ethanol, half amount of sterile water is added, stirred in an anaerobic tank, and then allowed to stand, and then the remaining sterile water, acetic acid, ascorbic acid, citric acid and L-proline are added, stirred and ultrasonicated again to obtain the fresh-keeping solution.
3. The screening method according to claim 2, characterized in that The ratio of the powder to ethanol is 1 g: (10-20) mL.
4. The screening method according to claim 2, characterized in that The conditions of the ultrasonic extraction are: power of 40W, temperature of 60°C, and time of 1h.
5. The screening method according to claim 2, characterized in that The oscillation condition is: 180 r / min oscillation for 24 hours.
6. The screening method according to claim 2, characterized in that The conditions for the second ultrasound treatment are: power of 40 W and time of 1 h.
7. The screening method according to claim 1, wherein The preservation method comprises the steps of dipping cotton into the diluted preservation liquid and wrapping the cotton in a ring shape around the petiole of the pepper leaf; the dilution multiple is ten times.
8. The screening method according to claim 1, characterized in that The culture conditions are as follows: temperature of 25° C., light:darkness of 16 h:8 h, light intensity of 10000 LX, and humidity of 75%.
9. Use of the screening method according to any one of claims 1 to 8 in breeding rust-resistant Zanthoxylum bungeanum varieties.
10. A special fresh-keeping liquid for isolated Zanthoxylum bungeanum leaves, characterized in that: The fresh-keeping solution comprises the following components in mass fractions: 4% of Zanthoxylum bungeanum leaf extract, 1% of citric acid, 0.5% of luteolin, 2.5% of L-proline, 1.5% of methyl jasmonate, 2% of ascorbic acid, 0.5% of acetic acid, 0.35% of chitosan, 1% of xanthocollagen, 10% of ethanol and 76.65% of sterile water.