Application of luteolin in identification of peanut fruit rot resistant variety

Abstract

The invention belongs to the technical field of agricultural biology, and particularly discloses application of luteolin in identification of peanut fruit rot resistant varieties. The invention discloses application of luteolin in identification of peanut fruit rot resistant varieties and a method for identifying the peanut fruit rot resistant varieties by utilizing the luteolin. The method comprises the following steps: S1, measuring the content of the luteolin in shells of a to-be-detected peanut variety; s2, the resistance level of the peanuts to the fruit rot is determined according to the comparison result of the luteolin content measured in the S1 and a preset threshold value, and the luteolin content is in positive correlation with the fruit rot resistance. The invention discloses application of luteolin in identification of peanut fruit rot resistance, resistance can be judged by directly measuring the content of luteolin in peanut shells, dependence on a field planting period or a pathogenic bacteria culture process is not needed, identification time is remarkably shortened, resistance screening of a large number of germplasm resources can be rapidly completed, and the method is simple and convenient to operate. The requirement of efficient identification in breeding work is met.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and particularly relates to the application of luteolin in identifying peanut varieties resistant to fruit rot. Background Art

[0002] Peanuts are an important cash crop and oilseed crop in my country, and their yield and quality are directly related to food and oil security and the sustainable development of the peanut industry. In recent years, peanut pod rot has shown a trend of sudden, frequent, and recurring outbreaks, particularly in plots planted with continuous crops and in rainy environments. This disease can cause peanut yield losses exceeding 20%, with losses exceeding 50% or even total crop failure in recurrently affected plots, posing a significant threat to the peanut industry. Peanut pod rot primarily affects peanut pods. In the early stages of the disease, brown to black lesions appear on the pods. As the disease progresses, the lesions gradually expand, and the kernels inside become small and hard due to poor development. In severe cases, the entire pod turns black and rots. Because the aboveground parts of diseased peanuts are not significantly different from normal plants, and there is no noticeable leaf drop at harvest time, early warning through appearance is difficult, making disease prevention and control extremely difficult.

[0003] Currently, identifying and utilizing disease-resistant varieties is the most economical, effective, and safe approach to combating peanut fruit rot. However, existing methods for identifying peanut fruit rot resistance have significant limitations: Traditional field identification is affected by factors such as annual climate variations, fluctuations in disease severity, and human error, resulting in poor stability. While indoor manual inoculation identification can control environmental conditions, it is still time-consuming and labor-intensive, making it difficult to meet the needs of large-scale germplasm screening. Therefore, establishing a rapid, stable, and quantifiable resistance identification method is crucial for the selection and promotion of disease-resistant peanut varieties. Summary of the Invention

[0004] The present invention aims to provide an application of luteolin in identifying peanut varieties resistant to fruit rot, solve the problems of low efficiency and poor stability of traditional identification methods, and provide a new method for the rapid screening of disease-resistant peanut varieties.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] Application of luteolin in identifying peanut varieties resistant to fruit rot.

[0007] The present invention also provides a method for identifying peanut varieties resistant to fruit rot using the luteolin, comprising the following steps:

[0008] S1. Determine the content of luteolin in the shell of the peanut variety to be tested;

[0009] S2. Determine the resistance level of peanuts to fruit rot based on a comparison result of the luteolin content determined in S1 with a preset threshold value, wherein the luteolin content is positively correlated with the resistance to fruit rot.

[0010] Preferably, in S1, the method for determining the luteolin content comprises the following steps:

[0011] After defatting, the peanut shell sample was extracted with 60% ethanol solution at 80℃ for 2h. The extracts were combined, concentrated to volume and filtered. The product was separated by reverse phase chromatography using Compass C18 column with 0.1% phosphoric acid aqueous solution and methanol as mobile phases for gradient elution at a flow rate of 1.0mL / min and detection wavelength of 360nm.

[0012] Preferably, in S2, the method for determining the preset threshold comprises the following steps:

[0013] The luteolin content in the fruit shells of known highly resistant and highly susceptible varieties was determined, and the lower limit of the luteolin content in the highly resistant varieties was used as the high-resistance threshold, while the upper limit of the luteolin content in the highly susceptible varieties was used as the high-susceptibility threshold.

[0014] Preferably, the fruit rot is peanut fruit rot caused by Fusarium.

[0015] The present invention also provides the use of luteolin in preparing a reagent or a kit for identifying peanut varieties resistant to fruit rot.

[0016] Preferably, the identification reagent or kit is used to quantitatively detect the luteolin content in peanut shells, and to judge the resistance level of peanuts to fruit rot based on the content.

[0017] The present invention also provides a detection kit for peanut varieties resistant to fruit rot, using the luteolin as a biomarker.

[0018] The present invention also provides the use of luteolin in preparing a preparation for preventing and treating peanut fruit rot.

[0019] The present invention also provides a preparation for preventing and treating peanut fruit rot, wherein the preparation contains the luteolin.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The present invention discloses the application of luteolin in identifying peanut varieties resistant to fruit rot. Resistance can be determined by directly measuring the luteolin content in peanut shells, without relying on field planting cycles or pathogen culture processes. This significantly shortens the identification time and can quickly complete resistance screening of a large number of germplasm resources, meeting the demand for efficient identification in breeding work.

[0022] The present invention uses luteolin content as a molecular marker for resistance identification, which can not only be used for rapid judgment of resistance phenotypes, but also provide a clear targeting indicator for the selection and breeding of disease-resistant varieties, helping to directional cultivate disease-resistant peanut varieties with high luteolin content, and fundamentally improving the resistance level of peanuts to fruit rot. In summary, by establishing a correlation between luteolin content and peanut fruit rot resistance, the present invention provides an efficient, stable, and simple identification method, which solves the technical problems of traditional identification methods such as long cycle, high interference, and high workload, and plays an important role in promoting the discovery and breeding of disease-resistant peanut varieties and the healthy development of the peanut industry.

[0023] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a photo of the disease resistance identification of indoor in vitro peanut pods in Example 1;

[0025] Figure 2 This is a statistical chart of the luteolin content in the shells of highly susceptible and highly resistant peanut varieties in Example 1, wherein: Figure 2 A in the figure is the statistical chart of luteolin content in the shells of peanut varieties Yuhua 22, Jihua 11, Shanyouhong 2, and Nongdahua 108. Figure 2 Figure B is a statistical graph of luteolin content in highly susceptible and highly resistant varieties. Lowercase letters on the columns represent significant differences between different treatment groups.

[0026] Figure 3 This is a statistical graph showing the effect of luteolin soaking on the incidence of peanut fruit rot in Example 1. The lowercase letters on the columns represent significant differences between different treatment groups. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0028] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0029] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in this field and can be purchased through commercial channels.

[0030] Example 1

[0031] Identification of peanut fruit rot resistance in natural field nurseries

[0032] Yuhua 22, Jihua 11, Shanyouhong 2, and Nongdahua 108 were planted on May 29, 2022, and June 13, 2023, respectively, in a plot with long-term continuous peanut cultivation and severe annual fruit rot (Fusarium spp. peanut fruit rot). The soil was sandy loam with medium-to-high fertility, uniform fertility, flat terrain, and convenient irrigation and drainage. Field management was based on average to above-average standards for local field production. After emergence, the peanuts were promptly tilled, weeded, watered, and pest controlled; no fungicides were used. The test varieties were arranged in randomized blocks, with five rows of each variety planted, 40 cm apart and 350 cm long. The planting density was 10,000 peanut holes per mu, with two peanuts per hole. One row was left blank between varieties, and 80 cm was left for walkways to facilitate variety identification. A fruit rot survey was conducted one week before harvest. Fifteen peanut plants from each variety plot were selected for the survey. The disease severity of all pods on each plant was recorded, and the disease index for each test variety was calculated.

[0033] The peanut fruit rot classification standards are as follows:

[0034] Level 0: no lesions on pods; Level 1: pod lesion area <10%; Level 3: pod lesion area 10%-25%; Level 5: pod lesion area 25%-50%; Level 7: pod lesion area 50%-75%; Level 9: pod lesion area >75%.

[0035] Disease index (DI) = ∑ (number of pods at each level × representative value of each level) / (total number of pods × highest representative value) × 100;

[0036] Relative resistance index (RRI) = 1-disease index of a certain variety / disease index of the most susceptible variety;

[0037] Peanut disease resistance was categorized into different levels based on the relative disease resistance index (RRI): RRI = 1.0, considered immune (I); RRI = 0.80-0.99, considered highly resistant (HR); RRI = 0.60-0.79, considered moderately resistant (MR); RRI = 0.40-0.59, considered resistant (R); RRI = 0.20-0.39, considered susceptible (S); and RRI < 0.20, considered highly susceptible (HS). The results are shown in Table 1.

[0038] Table 1 Identification of peanut fruit rot resistance levels in natural field nurseries

[0039]

[0040] As shown in Table 1, Yuhua 22 and Yuhua 11 are highly susceptible varieties, Shanyouhong 2 and Nongdahua 108 are highly resistant varieties, among which Shanyouhong 2 has not been reported to be resistant to fruit rot.

[0041] Identification of peanut fruit rot resistance using fresh fruit in vitro:

[0042] Freshly harvested peanut pods were collected and disinfected with 1% NaCl solution for 30 seconds, rinsed with sterile water and dried, and then immersed in a suspension of Fusarium solani spores (10 6 The peanut pods were inoculated with 100 spores / mL of spores and then placed in 9 cm Petri dishes lined with moistened filter paper. The pods were incubated at 28°C, 75% relative humidity, and darkness for 7 days. The severity of peanut pod disease was calculated according to the disease classification described above, and the disease index was calculated. Each treatment consisted of 25 pods, with 5 pods per replicate, for a total of 5 replicates. Sterile water was used as a blank control.

[0043] According to the disease index, peanut disease resistance is divided into the following levels: High resistance (HR): 0 <DI≤25;抗病(R):25<DI≤45;中抗(MR):45<DI≤60;感病(S):60<DI≤75;高感(HS):DI> 75. The result is Figure 1 and as shown in Table 2.

[0044] Table 2 Identification of disease resistance of detached peanut pods indoors

[0045]

[0046]

[0047] From Table 2 and Figure 1 It can be seen that Yuhua 22 and Yuhua 11 are highly susceptible varieties, Shanyouhong 2 and Nongdahua 108 are highly resistant varieties, which is consistent with the above field identification results.

[0048] Determination of luteolin content:

[0049] Peanut shell samples were ground into powder, 0.4 g of sample was weighed, 5 mL of petroleum ether was added, and ultrasonication was performed for 20 min. The petroleum ether was discarded, and the sample was defatted twice and blown to dryness with nitrogen. 10 mL of 60% ethanol solution was added and extracted at 80 °C for 2 h. The supernatant was collected by centrifugation and extracted twice. The supernatants were combined and the volume was adjusted to 10 mL with nitrogen blown. The sample was filtered with a syringe filter and then detected by high performance liquid chromatography.

[0050] Liquid chromatography analysis conditions:

[0051] Chromatograph: Agilent 1100 high performance liquid chromatograph; wavelength: 360 nm;

[0052] Chromatographic column: Compass C18 (2) reverse phase column (250 mm * 4.6 mm, 5 μm);

[0053] Column temperature: 30°C;

[0054] Flow rate: 1.0 mL / min, injection volume: 5 μL;

[0055] Mobile phase: 0.1% phosphoric acid aqueous solution: methanol = 70:30 (V / V) 0-8 min, 35:65 (V / V) 15-25 min. Figure 2 shown.

[0056] Depend on Figure 2 It can be seen that there are significant differences in the luteolin content between highly resistant and highly susceptible varieties, and the luteolin content in highly resistant varieties is significantly higher than that in highly susceptible varieties.

[0057] Effects of luteolin on disease resistance of peanuts:

[0058] The pods of highly susceptible peanuts (Yuhua 22) were soaked in a series of luteolin concentrations (0, 0.1, 0.5, 1, 10, 50 mg / L) for 30 seconds, taken out and placed in a 9 cm petri dish covered with two layers of moist filter paper. After drying, two 5 mm Fusarium solani cakes were inoculated on the peanut pods. After 7 days, the disease index was counted and calculated using the same calculation method as above. Sterile water was used as a blank control. Each treatment contained 5 replicate petri dishes, with 5 peanut pods placed in each dish. The results are as follows: Figure 3 shown.

[0059] Depend on Figure 3 It can be seen that luteolin soaking can significantly inhibit the disease index of fruit rot, with the strongest inhibition in the 0.1 mg / L, 0.5 mg / L and 1 mg / L concentration groups, and the inhibition rate decreases starting from 10 mg / L.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of luteolin in identifying peanut varieties resistant to fruit rot.

2. A method for identifying peanut varieties resistant to fruit rot using the luteolin according to claim 1, characterized in that: The following steps are involved: S1. Determine the content of luteolin in the shell of the peanut variety to be tested; S2. Determine the resistance level of peanuts to fruit rot based on a comparison result of the luteolin content determined in S1 with a preset threshold value, wherein the luteolin content is positively correlated with the resistance to fruit rot.

3. The method according to claim 2, characterized in that In S1, the method for determining the luteolin content comprises the following steps: After defatting, the peanut shell sample was extracted with 60% ethanol solution at 80℃ for 2h. The extracts were combined, concentrated to volume and filtered. The product was separated by reverse phase chromatography using Compass C18 column with 0.1% phosphoric acid aqueous solution and methanol as mobile phases for gradient elution at a flow rate of 1.0mL / min and detection wavelength of 360nm.

4. The method according to claim 2, characterized in that In S2, the method for determining the preset threshold comprises the following steps: The luteolin content in the fruit shells of known highly resistant and highly susceptible varieties was determined, and the lower limit of the luteolin content in the highly resistant varieties was used as the high-resistance threshold, while the upper limit of the luteolin content in the highly susceptible varieties was used as the high-susceptibility threshold.

5. The method according to claim 2, characterized in that The fruit rot disease is peanut fruit rot disease caused by Fusarium.

6. Application of luteolin in the preparation of identification reagents or kits for peanut varieties resistant to fruit rot.

7. The application according to claim 6, characterized in that The identification reagent or kit is used for quantitatively detecting the luteolin content in peanut shells, and judging the resistance level of peanuts to fruit rot based on the content.

8. A detection kit for peanut varieties resistant to fruit rot using the luteolin as claimed in claim 6 as a biomarker.

9. Application of luteolin in the preparation of preparations for preventing and treating peanut fruit rot.

10. A preparation for preventing and treating peanut fruit rot, characterized in that: The preparation contains the luteolin according to claim 9.

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