Method for improving black spot resistance of crops by utilizing raffinose

Abstract

The invention discloses a method for improving black spot resistance of crops by raffinose, which comprises the following steps: selecting healthy and consistent seedling-stage plants of the crops, and culturing; raffinose solution preparation: preparing raffinose solutions with different concentrations, respectively spraying the raffinose solutions on the plants in the step 1, spraying the raffinose solutions once every 24 hours in a laboratory scene, spraying the raffinose solutions twice in total, spraying the raffinose solutions once every 5 days in a field scene, spraying the raffinose solutions three times in total, and spraying clear water in a contrast manner; after spraying in the step 2 is finished for 24 hours, inoculating alternaria alternata to the control plants and the treated plants, comparing disease phenotypes under different raffinose concentrations, and screening the concentration with the optimal effect; 3, according to the result of the step 3, determining that the spraying effect of the raffinose solution with the concentration of 2.4% is optimal, selecting the raffinose solution with the concentration to be sprayed on the plants, the spraying method is the same as that of the step 2, performing artificial inoculation 24 hours after the last time of spraying is finished, observing the disease phenotype, and counting the disease state. After being treated by the 2.4% raffinose solution, the disease spot areas of the tea chrysanthemum, the apples and the tomatoes are obviously reduced.

Description

Technical Field

[0001] This invention relates to a method for improving crop resistance to black spot disease, and more particularly to a method for improving crop resistance to black spot disease using raffinose. Background Technology

[0002] Alternaria is a genus of fungi that is widely distributed globally and is an important plant pathogen. This genus is diverse, has a wide host range, and is highly adaptable. Most fungi adopt a saprophytic lifestyle and reside in their hosts. It is a typical airborne disease that can cause severe black spot disease (also known as brown spot or leaf spot) in more than 400 types of economic crops and horticultural plants. The lesions on most infected plants are round or nearly round, but irregular in shape; brown to dark brown; often accompanied by light yellow concentric rings; in severe cases, a grayish-white mold layer appears on the lesions. The occurrence, spread, and prevalence of Alternaria leaf diseases have a significant impact on agricultural production and agricultural product storage worldwide, causing serious economic losses.

[0003] Chrysanthemum black spot is a fungal disease caused by *Alternaria spp.*, which causes spots and scorching on chrysanthemum leaves and wilting of flower buds. The disease is particularly severe and spreads rapidly in warm, humid climates, leading to widespread leaf wilting and plant death, seriously harming the healthy production of chrysanthemums for tea. In addition, apple black spot (also known as leaf spot disease) caused by *Alternaria* is one of the three major diseases in apple production. It is widespread in my country, primarily affecting leaves, but also young branches and fruits. In severe cases, it causes premature leaf drop of up to 80%, weakens the tree, and reduces fruit quality and yield. Tomato black spot is also a common tomato disease, mainly affecting fruits, leaves, and stems. Currently, traditional control of crop black spot mainly relies on pesticide spraying. However, improper application can easily lead to pesticide overuse, resulting in pesticide residues, environmental pollution, and pathogen resistance. Therefore, developing an environmentally friendly and highly effective new black spot control agent is crucial. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to propose a method for improving crop resistance to black spot disease using raffinose. This method is low-cost, simple to operate, and highly practical, and can be widely used for the green prevention and control of crop black spot disease.

[0005] Technical solution: This invention includes:

[0006] S1: Select healthy and uniform crop seedlings for cultivation;

[0007] S2: Preparation of raffinose solution: Prepare raffinose solutions of different concentrations and spray them on the plants in step 1. In the laboratory setting, spray once every 24 hours for a total of 2 times. In the field setting, spray once every 5 days for a total of 3 times. The control is sprayed with water.

[0008] S3: 24 h after the spraying in step 2, both control and treatment plants were inoculated with Alternaria. The disease phenotypes under different raffinose concentrations were compared to screen the concentration with the best effect.

[0009] S4: Based on the results of step 3, it was determined that the 2.4% concentration of raffinose solution was the most effective for spraying. This concentration of raffinose solution was selected and sprayed onto the plants using the same method as in step 2. Artificial inoculation was carried out 24 hours after the last spraying, and the disease phenotype was observed and the disease severity was recorded.

[0010] The cultivation process in S1 involves selecting healthy and uniform plant cuttings for propagation, and then transferring them to a long-day greenhouse for cultivation after rooting to obtain seedlings with uniform growth.

[0011] In S2, the standard for spraying is to moisten the entire plant's leaves until they are dripping with moisture.

[0012] In S3, strain F20, which has been cultured for 5 days, is taken. A 6 mm diameter mycelial block is taken from the edge of the colony and placed in 50 mL of PDB medium. It is then cultured at 28°C and 200 rpm for 24 h with shaking. After grinding into a mycelial slurry, it is cultured with shaking for another 48 h to prepare a high-concentration mycelial suspension for later use.

[0013] In step S3, when inoculating Alternaria, take 2 mL of mycelial solution and inoculate it on the back of the same leaf position of the chrysanthemum. Inoculate at least two points on each leaf. Cover the inoculated leaves with a self-sealing bag to keep them moist and place them in an incubator.

[0014] In step S4, the area of ​​lesions on plant leaves is counted, and the differences in plant disease resistance are evaluated by comparing the size of the lesion areas.

[0015] The leaf lesion area was statistically analyzed using ImageJ.

[0016] When used in field settings, select cuttings of plants with uniform growth and plant them in the experimental field. After 30 days of growth, start spraying with cottonseed sugar.

[0017] This method can be used for tea chrysanthemum, apple, tomato or other species.

[0018] When used on apples or tomatoes, use the detached leaf inoculation method, and take 2 mL of mycelial solution to inoculate on the underside of the same leaf position on the test leaf.

[0019] Beneficial effects: The present invention has the following advantages:

[0020] Significant control effect: After treatment with 2.4% raffinose solution, the area of ​​lesions on tea chrysanthemum, apple and tomato lesions decreased significantly, and the control effect was better than that of traditional chemical pesticides;

[0021] Green, environmentally friendly and safe: Raffinose is a natural oligosaccharide that is non-toxic and leaves no residue. After spraying, it will not pollute the soil, water and other ecological environments. It is safe and harmless to crops, humans and beneficial organisms, and meets the requirements of green agricultural development.

[0022] Simple and easy to operate: No complicated equipment is required. Simply dissolve the raffinose and spray it on the leaves. Growers can quickly master the operation method. It is suitable for laboratory verification and large-scale field application.

[0023] Significant cost advantages: Rapeseed sugar raw materials are readily available and affordable, and no additional investment is required during the spraying process. The comprehensive control cost is more than 30% lower than that of chemical pesticides, significantly reducing the production costs for growers.

[0024] Wide range of applications: Verified in multiple species including tea chrysanthemum (4 varieties), apple, and tomato, it has shown significant resistance enhancement effects and can be extended to other economic crops affected by Alternaria, with broad application prospects. Attached Figure Description

[0025] Figure 1 The effect of different concentrations of raffinose spraying on the resistance of four tea chrysanthemum species to black spot disease: a) the incidence of black spot disease in the four tea chrysanthemum species after spraying with different concentrations of raffinose, b) the statistical results of lesion area;

[0026] Figure 2 To determine the black spot disease resistance phenotype of 'Dayangju' tea chrysanthemum seedlings after spraying with 2.4% raffinose: a) Black spot disease incidence of 'Dayangju' tea chrysanthemum after spraying with 2.4% raffinose; b) Statistical results of lesion area.

[0027] Figure 3 The effect of applying 2.4% raffinose on the control of black spot disease in the main production area of ​​'Fubaiju' chrysanthemum for tea in Macheng, Hubei Province;

[0028] Figure 4 A comparative chart showing the resistance of tomatoes and apples to black spot disease after spraying with different concentrations of raffinose. Detailed Implementation

[0029] The invention will now be further described with reference to the accompanying drawings.

[0030] Example 1

[0031] The method for improving crop resistance to black spot disease using raffinose in this embodiment includes the following steps:

[0032] Step 1: Crop Seedling Preparation

[0033] Healthy and uniform tea chrysanthemum cuttings of 'Dayangju', 'Fubaiju', 'Chuju', and 'Jinsihuangju' were selected for propagation. After rooting (about 15 days), the cuttings were transferred to a long-day greenhouse for cultivation at a temperature of 25±2℃ and a relative humidity of 70%. The cuttings were allowed to continue growing for about 20 days to obtain seedlings with uniform growth, which could then be used for subsequent experiments.

[0034] Step 2: Preparation of raffinose solution: Prepare raffinose solutions of different concentrations and spray them on the plants from Step 1. Spray once every 24 hours for a total of 2 times. Use water as a control.

[0035] Raffinose concentration screening: The experiment included spraying treatments with raffinose solutions at concentrations of 0.2%, 0.6%, 1.2%, and 2.4%, with water spraying serving as a control. Spraying was performed twice, 24 hours apart. 24 hours after the second spraying, the plant was artificially inoculated with the pathogen *Alternaria*. Each treatment consisted of 12 individual chrysanthemum plants. The raffinose spraying was applied until the entire leaf surface was moistened, with a fine mist appearing to drip.

[0036] Step 3: 24 h after the spraying in step 2, both control and treatment plants were inoculated with Alternaria. The disease phenotypes under different raffinose concentrations were compared to screen for the optimal concentration.

[0037] The experimental strain was isolated from a diseased plant of the tea chrysanthemum 'Fubaiju' and preserved by the Chrysanthemum Genetics and Breeding Laboratory of Nanjing Agricultural University. The strain number was F20. After 5 days of culture, a 6 mm diameter mycelial block was taken from the edge of the colony of strain F20 and placed in 50 mL of PDB (potato dextrose broth) medium. The mixture was cultured at 28℃ and 200 rpm for 24 h with shaking. After grinding into a concentrated mycelial slurry, the mixture was cultured with shaking for another 48 h to prepare a high-concentration mycelial suspension for later use.

[0038] Two mL of mycelial solution was inoculated onto the underside of two leaves at the same position on each chrysanthemum leaf. The inoculated leaves were covered with a No. 3 self-sealing bag (7 cm × 10 cm) to retain moisture and placed in an incubator at 28℃ with a relative humidity of 80%. A control was sprayed with water. Each treatment consisted of 12 plants, and the experiment was repeated three times. After 48 hours, the disease phenotype was observed, diseased leaves were removed, photographed, and the disease severity was recorded.

[0039] Step 4: Based on the results of Step 3, a 2.4% raffinose solution was determined to be the most effective spray. This concentration of raffinose solution was used to spray the 'Daphne' plants, following the same spraying method as in Step 2. Artificial inoculation was performed 24 hours after the final spray, and the disease phenotype was observed and the disease severity was recorded. ImageJ software was used to count the area of ​​lesions on the leaves, and the differences in disease resistance among plants were evaluated by comparing the size of the lesion areas.

[0040] The method of using raffinose to improve crop resistance to black spot disease in this embodiment can be used for field control trials. 'Fubaiju' cuttings with uniform growth were selected and planted in the experimental field. After 30 days of growth, raffinose was sprayed, once every 5 days, for a total of three sprays. A treatment group (2.4% raffinose) and a control group (water) were set up.

[0041] The method of using raffinose to improve crop resistance to black spot disease in this embodiment can be used not only for chrysanthemum tea mentioned above, but also for other species such as apples and tomatoes. In the experiment, apples and tomatoes were used as materials, and the inoculation method was adopted by detached leaves. 2 mL of mycelial liquid was inoculated on the back of the same leaf position of the test leaf. The inoculation environment was the same as that for chrysanthemum tea to verify the effect of spraying raffinose on improving crop resistance to black spot disease.

[0042] Example 2

[0043] Screening test of different concentrations of raffinose for resistance to black spot disease in tea chrysanthemum

[0044] 1) Experimental Materials and Design

[0045] The experimental materials included chrysanthemum varieties for tea cultivation: 'Dayangju', 'Fubaiju', 'Chuju', and 'Jinsihuangju', provided by the China Chrysanthemum Germplasm Resource Center. The experimental strain was *Alternaria alternata*, F20, isolated from diseased plants from the 'Fubaiju' production area in Macheng, Hubei Province, and preserved by the Chrysanthemum Genetics and Breeding Laboratory of Nanjing Agricultural University. Raffinose powder was purchased from Sigma-Aldrich and dissolved in water to prepare a solution.

[0046] The experiment was designed to spray raffinose solutions of different concentrations (0.2%, 0.6%, 1.2%, and 2.4%) onto the plants, with water serving as the control. Each treatment consisted of 12 individual chrysanthemum plants. Spraying was performed until the entire plant's leaves were moistened to the point of dripping.

[0047] 2) Preparation of mycelial solution

[0048] For strain F20, which has been grown on PDA (potato dextrose agar) for 5 days, several 6 mm diameter mycelial blocks were cut from the edge of the colony and placed in 50 mL of PDB medium. After culturing at 28°C and 200 rpm for 24 h with shaking, the mycelial blocks were ground into a mycelial slurry and then cultured with shaking for another 48 h to obtain a high concentration of mycelial solution.

[0049] 3) Pathogen inoculation treatment

[0050] Four tea chrysanthemum varieties with consistent growth status—'Dayangju', 'Fubaiju', 'Chuju', and 'Jinsihuangju'—were selected as inoculation materials. One leaf was inoculated from each plant, with the leaf position being the same. 2 mL of mycelial solution was inoculated onto the underside of the leaf. The inoculated leaf was covered with a No. 3 self-sealing bag (7cm×10cm) to retain moisture. After inoculation, the experimental plants were placed in a long-day (16 h daytime, 8 h nighttime) light incubator at 28℃ and 80% relative humidity. After 48 h, the disease phenotype was observed and recorded. Diseased leaves were removed and placed in a photographic box with a scale bar, and photographs were taken for recording.

[0051] 4) Statistics and Analysis

[0052] ImageJ software was used to count the area of ​​lesions, while Prism 10.1.2 software was used for descriptive statistics, analysis of variance, and plotting. Plant disease resistance was determined by the size of the lesion area.

[0053] 5) Concentration screening results

[0054] The results are as follows Figure 1 As shown, compared with spraying with plain water, spraying with 0.6%, 1.2%, and 2.4% raffinose solutions reduced the lesion area by 34.8%, 51.0%, and 95.2% respectively compared with the control group, indicating a significant enhancement in plant disease resistance. Among these, the 2.4% concentration showed the best disease resistance effect.

[0055] Based on the above concentration screening results, 2.4% raffinose was applied to seedlings of 'Daphne odora' for verification. The results are as follows: Figure 2 As shown, spraying with 2.4% raffinose significantly improved the black spot disease resistance of tea chrysanthemum seedlings.

[0056] Example 3: Control test of 2.4% raffinose on black spot disease in tea chrysanthemums in the field.

[0057] Experimental location: The main production area of ​​'Fubai Chrysanthemum' in Macheng, Hubei Province, which has been continuously cropped for many years;

[0058] Experimental design: 'Fubai Chrysanthemum' plants that had been planted for 30 days were selected and divided into a treatment group (2.4% raffinose solution) and a control group (water).

[0059] Spraying treatment: The treatment group was sprayed once every 5 days for a total of 3 times, while the control group was sprayed with water at the same time;

[0060] Effect monitoring: After spraying, normal field management was carried out. The disease incidence was investigated 30 days after the last spraying, and the disease rate and disease index were counted.

[0061] Result: As Figure 3 As shown, the disease incidence rate in the treatment group was lower than that in the control group, the field control effect was stable, and 'Fubaiju' was growing well with no phytotoxicity.

[0062] Example 4: Screening test for resistance to black spot disease in apples and tomatoes at different concentrations of raffinose

[0063] 1) Experimental Materials and Design

[0064] Apples and tomatoes were used as experimental materials; the experimental strain was *Alternaria alternata*, number F20, isolated from diseased plants in the 'Fubaiju' production area of ​​Macheng, Hubei Province, and preserved by the Chrysanthemum Genetics and Breeding Laboratory of Nanjing Agricultural University. Raffinose powder was purchased from Sigma-Aldrich and dissolved in water to prepare a solution.

[0065] The experiment was designed to spray 0.6%, 1.2%, and 2.4% raffinose solutions, with water serving as a control. Twelve chrysanthemum plants were included in each treatment. Spraying was performed until the entire plant's leaves were moistened to the point of dripping.

[0066] 2) Preparation of mycelial solution

[0067] For strain F20, which has been grown on PDA (potato dextrose agar) for 5 days, several 6 mm diameter mycelial blocks were cut from the edge of the colony and placed in 50 mL of PDB medium. After culturing at 28°C and 200 rpm for 24 h with shaking, the mycelial blocks were ground into a mycelial slurry and then cultured with shaking for another 48 h to obtain a high concentration of mycelial solution.

[0068] 3) Pathogen inoculation treatment

[0069] Apple and tomato plants with similar growth status were selected as inoculation materials. One leaf was inoculated on each plant, with the leaf position being the same. 2 mL of mycelial solution was inoculated on the back of the leaf. The inoculated leaf was covered with a No. 3 self-sealing bag (7 cm × 10 cm) to keep it moist. After inoculation, the experimental plants were placed in a long-day (16 h daytime, 8 h nighttime) light incubator at 28℃ and 80% relative humidity. After 48 h, the disease phenotype was observed and the diseased leaves were removed and placed in a photography box with a scale bar for photographic recording.

[0070] 4) Statistics and Analysis

[0071] ImageJ software was used to count the area of ​​lesions, while Prism 10.1.2 software was used for descriptive statistics, analysis of variance, and plotting. Plant disease resistance was determined by the size of the lesion area.

[0072] 5) Concentration screening results

[0073] The results are as follows Figure 4As shown, compared with spraying with plain water, spraying with 0.6%, 1.2%, and 2.4% raffinose solutions reduced the lesion area by 34.8%, 51.0%, and 95.2% respectively compared with the control group, indicating a significant enhancement in plant disease resistance. Among these, the 2.4% concentration showed the best disease resistance effect.

[0074] Artificial inoculation experiments and field verification revealed that exogenous spraying of a green, harmless, low-cost, and readily available raffinose solution significantly improved the resistance of crops such as tea chrysanthemum, apple, and tomato to black spot disease. Specifically, the lesion area of ​​tea chrysanthemum plants treated with a 2.4% concentration decreased by 98.5% compared to the control group (p < 0.01). This method is simple to operate, highly effective, and has a lower overall cost than conventional chemical control. It can replace high-residue pesticides and is suitable for large-scale green control of black spot disease in tea chrysanthemum, fruit trees, and vegetables by enterprises and growers, providing a safe and efficient solution for sustainable agricultural development.

Claims

1. A method for improving crop resistance to black spot disease using raffinose, characterized in that, include: S1: Select healthy and uniform crop seedlings for cultivation; S2: Preparation of raffinose solution: Prepare raffinose solutions of different concentrations and spray them on the plants in step 1. In the laboratory setting, spray once every 24 hours for a total of 2 times. In the field setting, spray once every 5 days for a total of 3 times. The control is sprayed with water. S3: 24 h after the spraying in step 2, both control and treatment plants were inoculated with Alternaria. The disease phenotypes under different raffinose concentrations were compared to screen the concentration with the best effect. S4: Based on the results of step 3, it was determined that the 2.4% concentration of raffinose solution was the most effective for spraying. This concentration of raffinose solution was selected and sprayed onto the plants using the same method as in step 2. Artificial inoculation was carried out 24 hours after the last spraying, and the disease phenotype was observed and the disease severity was recorded.

2. The method for improving crop resistance to black spot disease using raffinose according to claim 1, characterized in that, The cultivation process in S1 involves selecting healthy and uniform plant cuttings for propagation, and then transferring them to a long-day greenhouse for cultivation after rooting to obtain seedlings with uniform growth.

3. The method for improving crop resistance to black spot disease using raffinose according to claim 1, characterized in that, In S2, the standard for spraying is to moisten the entire plant's leaves until they are dripping with moisture.

4. The method for improving crop resistance to black spot disease using raffinose according to claim 1, characterized in that, In S3, strain F20, which has been cultured for 5 days, is taken. A 6 mm diameter mycelial block is taken from the edge of the colony and placed in 50 mL of PDB medium. It is then cultured at 28°C and 200 rpm for 24 h with shaking. After grinding into a mycelial slurry, it is cultured with shaking for another 48 h to prepare a high-concentration mycelial suspension for later use.

5. The method for improving crop resistance to black spot disease using raffinose according to claim 4, characterized in that, In step S3, when inoculating Alternaria, take 2 mL of mycelial solution and inoculate it on the back of the same leaf position of the chrysanthemum. Inoculate at least two points on each leaf. Cover the inoculated leaves with a self-sealing bag to keep them moist and place them in an incubator.

6. The method for improving crop resistance to black spot disease using raffinose according to claim 1, characterized in that, In step S4, the area of ​​lesions on plant leaves is counted, and the differences in plant disease resistance are evaluated by comparing the size of the lesion areas.

7. The method for improving crop resistance to black spot disease using raffinose according to claim 6, characterized in that, The leaf lesion area was statistically analyzed using ImageJ.

8. The method for improving crop resistance to black spot disease using raffinose according to claim 1, characterized in that, When used in field settings, select cuttings of plants with uniform growth and plant them in the experimental field. After 30 days of growth, start spraying with cottonseed sugar.

9. The method for improving crop resistance to black spot disease using raffinose according to claim 1, characterized in that, This method can be used for tea chrysanthemum, apple, tomato, or other species.

10. The method for improving crop resistance to black spot disease using raffinose according to claim 9, characterized in that, When used on apples or tomatoes, use the detached leaf inoculation method, and take 2 mL of mycelial solution to inoculate on the underside of the same leaf position on the test leaf.

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