Soybean anthracnose inoculation method and application
The simplified inoculation method using soybean sprouts as the inoculation target solves the problems of long identification cycle and unstable fungicide determination in traditional soybean anthracnose, and realizes rapid and accurate resistance identification and control effect determination.
Patent Information
- Application Number
- CN202511364810.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-23
- Publication Date
- 2026-01-13
AI Technical Summary
Traditional methods for identifying soybean anthracnose resistance are time-consuming, require large areas of arable land, have high environmental requirements, and the effectiveness of fungicides is unstable, consuming a lot of manpower and resources.
Soybean sprouts were used as the inoculation target. The inoculation process was simplified and resistance was quickly identified by aseptic seedling raising, anthracnose inoculation and lesion area statistics. ImageJ software was used to count the disease level.
The soybean anthracnose inoculation method has a short cycle, is simple to operate, highly accurate, and widely applicable. It can quickly identify the resistance of soybean varieties and determine the effectiveness of fungicides, saving labor and resources.
Smart Images
Figure CN121320488A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of crop disease prevention and treatment, in particular to a soybean anthracnose inoculation method and its application in soybean anthracnose resistance identification and determination of the effect of fungicides on soybean anthracnose prevention and treatment. BACKGROUND
[0002] Soybean contains 8 essential amino acids for the human body, and is an important food crop, oil crop and feed crop, occupying a very important position in China's agricultural production and social and economic life, and is the core source of human plant protein.
[0003] Soybean anthracnose caused by Colletotrichum truncatum (Schwein.) Andr. et Moore is one of the important diseases of soybean, which occurs widely in all soybean producing areas in China. Soybean anthracnose mainly harms pods, stems and seedlings, causing seedling death, stem death, pod dryness and no grain formation, etc. In severe fields, the rate of diseased pods can reach more than 30%. Soybean anthracnose fungus is mainly spread by wind and rain in the field, and rain splash is the main factor for short-distance transmission, while seed transportation with the fungus is the main factor for long-distance transmission. Screening and using disease-resistant varieties is the safest and most effective measure to prevent and control soybean anthracnose, and timely application of fungicides is the most efficient and most widely applicable measure. Crop variety resistance identification is an important means to obtain resistant germplasm resources and is a necessary process for disease-resistant breeding. Traditional soybean anthracnose resistance identification is to artificially spray inoculation with soybean anthracnose spore suspension at the initial pod stage of soybean, to create environmental conditions conducive to soybean disease, and to distinguish the disease resistance of soybean varieties by investigating the soybean pod disease. The traditional initial pod stage spray inoculation method for soybean anthracnose resistance identification has the limitations of long cycle, large occupation of cultivated land area, high requirements for environmental conditions such as temperature and humidity, and complicated steps. In addition, due to the different growth periods of soybean varieties, the initial pod stage and pod swelling stage of different soybean varieties differ greatly, and repeated artificial spray inoculation and disease investigation steps are required, which consumes a lot of manpower and material resources. Determination of the control effect of new compounds or fungicides usually uses the in vitro pod inoculation method, but the in vitro pod is often affected by natural aging, which leads to unstable disease, and cannot truly reflect the control effect of fungicides. Therefore, it is of great significance to establish a simple, rapid and accurate inoculation method for soybean anthracnose resistance identification and new fungicide screening. SUMMARY
[0004] The primary purpose of the present application is to address the problem that the occurrence of soybean anthracnose hinders the healthy development of the soybean industry, and to provide a soybean anthracnose inoculation method.
[0005] The objective of this invention is achieved through the following technical solution:
[0006] S1. Soybean sprout cultivation: Sow soybean seeds in sterile seedling substrate, sprinkle water until the sterile seedling substrate is moist, place at 23-28°C, RH below 80%, and cultivate in the dark for 4 days. Select soybean sprouts with uniform growth, cut off the roots, and wash them with sterile water.
[0007] S2. Cultivation of soybean anthracnose fungus: Soybean anthracnose fungus was cultured on PDA medium at 28°C in the dark for 5 days, and mycelial cakes with a diameter of 1 mm were obtained by sterilizing the punch.
[0008] S3. Inoculation with soybean anthracnose fungus: Place a sterile defatted cotton ball at the bottom of a centrifuge tube and moisten it with sterile water; use a sterile needle to inoculate the soybean anthracnose fungus cake onto the soybean sprout stem, prick the inoculation site with a sterile needle, cover the fungus cake with a moist sterile defatted cotton strip and wrap it to keep it moist, place the inoculated soybean sprout stem base down into the centrifuge tube, and place the soybean sprout stem base on the defatted cotton ball, cover the centrifuge tube and rotate it half a turn, and incubate in the dark at 25°C for 3 days.
[0009] In step S1, the volume ratio of peat moss to vermiculite in the sterile seedling substrate is 3:1, and it is sterilized by moist heat at 121℃ for 30 minutes.
[0010] In step S1, the soybean seeds are sown in a sterile seedling substrate with a row spacing and plant spacing of 3 cm.
[0011] In step S3, a 15 mL centrifuge tube is used. 2 g of sterile defatted cotton balls are placed at the bottom of the centrifuge tube, and 2 mL of sterile water is added.
[0012] The second objective of this invention is to provide a method for statistically analyzing the incidence of soybean anthracnose. Soybean sprouts are obtained using the above-mentioned soybean anthracnose inoculation method, and the disease level is determined based on the percentage of soybean sprout lesions to the total area of the sprout stem.
[0013] The disease level is determined based on the percentage of soybean sprout lesions relative to the total area of the sprout stem, as follows:
[0014] Grade 0: Asymptomatic; Grade 1: Lesions cover less than 10% of the stem area; Grade 3: Lesions cover 10% to 25% of the stem area; Grade 5: Lesions cover 25% to 50% of the stem area; Grade 7: Lesions cover 50% to 75% of the stem area; Grade 9: Lesions cover 75% to 85% of the stem area.
[0015] The incidence of diseases was statistically analyzed using ImageJ software.
[0016] The soybean sprouts were photographed at a fixed height using a camera tripod, and the disease incidence was statistically analyzed based on the area of lesions using ImageJ software.
[0017] The third objective of this invention is to provide an application of soybean anthracnose inoculation method in soybean anthracnose resistance identification.
[0018] The fourth objective of this invention is to provide an application of a soybean anthracnose inoculation method in determining the control efficacy of fungicides or compounds.
[0019] Compared with existing technologies, the advantages of this invention are: the soybean anthracnose inoculation method has a short cycle, simple operation, high accuracy, and strong applicability. Using soybean sprouts as the inoculation target, the method takes only 7 days from sprout cultivation and inoculation to disease incidence survey, saving more than 60 days compared to traditional inoculation methods. This method does not occupy arable land and is not limited by field planting temperature and humidity, or differences in the growth stages of different soybean varieties, saving labor and being simple and easy to operate. The method is highly accurate; by isolating inoculated plants, mutual interference can be avoided, and the resistance of different soybean varieties to anthracnose can be effectively distinguished. Furthermore, the method is highly applicable and can be used for various purposes such as soybean anthracnose resistance identification, compound control efficacy testing, and fungicide control efficacy testing, showing promising application prospects. Attached Figure Description
[0020] Figure 1 This is a schematic diagram illustrating the inoculation process for soybean anthracnose. Note: A and B represent the growth of soybean sprouts in 15 mL centrifuge tubes after inoculation; C and D represent the disease development of soybean sprouts 4 days after inoculation.
[0021] Figure 2 Grading standards for diseases in soybean sprouts after inoculation with anthracnose.
[0022] Figure 3 This section describes the application of soybean anthracnose inoculation methods in the identification of soybean anthracnose resistance. Note: A represents the disease incidence of Xiangchundou 26; B represents the disease incidence of Maodou 3.
[0023] Figure 4 This study examines the application of inoculation methods for soybean anthracnose in determining the control efficacy of fungicides. Note: A is a blank control; B shows the disease incidence after treatment with 75 μg / mL difenoconazole; C shows the disease incidence after treatment with 100 μg / mL difenoconazole; D shows the disease incidence after inoculation with *Anthracnose spp.* Detailed Implementation
[0024] The present invention will now be described in detail with reference to the accompanying drawings and embodiments:
[0025] Example 1: Inoculation method for soybean anthracnose
[0026] 1. Sterilization of experimental materials
[0027] Place the experimental materials, including 15 mL centrifuge tubes, absorbent cotton, tweezers, scalpel, needle, and seedling substrate (peat moss and vermiculite in a volume ratio of 3:1), into a cloth bag. Place purified water into a 300 mL Erlenmeyer flask and sterilize by moist heat at 121℃ for 30 min.
[0028] 2. Culture of soybean anthracnose pathogen
[0029] The soybean anthracnose fungus (Colletotrichum truncatum) was cultured on a PDA plate at 28°C for 5 days. Mycelial cakes were then collected from the colonies using a 1 mm diameter punch for later use.
[0030] 3. Soybean sprout cultivation
[0031] The soybean variety used in the experiment was Zhexian 24. A sterile seedling substrate (peat moss and vermiculite in a 3:1 volume ratio) was spread in the seedling trays to a thickness of approximately 2 cm. After removing shriveled and broken soybean seeds, they were sown in the seedling substrate with a row and plant spacing of 3 cm. After sowing, the seeds were covered with approximately 1 cm of sterile seedling substrate, and watered until the substrate was moist. The trays were then placed in the dark at 23-28°C and a relative humidity not exceeding 80% for 4 days. Once the soybean sprouts reached 3-4 cm in length, they were harvested, and sprouts with uniform growth were selected. The roots were removed, and the sprouts were washed with sterile water before use.
[0032] 4. Inoculation with soybean anthracnose fungus
[0033] Take approximately 2 g of sterile defatted cotton, roll it into a ball, and place it at the bottom of a 15 mL centrifuge tube. Add 2 mL of sterile water. Using a sterile needle, inoculate the soybean anthracnose fungal cake onto the stem of the soybean sprout (approximately 1 cm from the leaf). Create a small wound at the inoculation site using the sterile needle. Cover the fungal cake with a moistened defatted cotton strip and wrap it to retain moisture. The experiment was conducted in triplicate, with 30 soybean sprouts inoculated in each replicate. A sterile agar block inoculated with the fungal fungus served as a blank control. Place the inoculated soybean sprouts root-and-stem side down in the centrifuge tube, ensuring the base of the sprouts is in contact with the defatted cotton ball. Cap the centrifuge tube and rotate it half a turn. Incubate the inoculated centrifuge tubes at 25°C in the dark for 3 days.
[0034] 5. Survey on the incidence of soybean anthracnose
[0035] ImageJ software was used to statistically analyze disease incidence. The ImageJ method involved taking photos of soybean sprouts at a fixed height using a camera tripod, and then using ImageJ software to statistically analyze disease incidence based on the area of lesions.
[0036] Example 2: Application of soybean anthracnose inoculation method in the identification of anthracnose resistance in soybean varieties.
[0037] 1. Sterilization of experimental materials
[0038] Place the experimental materials, including 15 mL centrifuge tubes, absorbent cotton, tweezers, scalpel, needle, and seedling substrate (peat moss and vermiculite in a volume ratio of 3:1), into a cloth bag. Place purified water into a 300 mL Erlenmeyer flask and sterilize by moist heat at 121℃ for 30 min.
[0039] 2. Culture of soybean anthracnose pathogen
[0040] The soybean anthracnose fungus (Colletotrichum truncatum) was cultured on PDA plates at 28°C for 5 days. Mycelial cakes were then collected from the colonies using a 1 mm diameter punch for later use.
[0041] 3. Sprout cultivation of the tested soybean varieties
[0042] The soybean varieties tested were Maodou No. 3, Zhexian 39, Liaoxiandou 31, Zhenong 160, Tianlong No. 1, and Xiangchundou 26. Shriveled and broken seeds were removed and the seeds were set aside. Seeds of the six soybean varieties were sown in sterile seedling substrate (approximately 2 cm thick) in seedling trays, with a row and plant spacing of 3 cm. 100 seeds were sown per tray. After sowing, the trays were covered with approximately 1 cm of sterile seedling substrate, and watered until the substrate was moist. The trays were then placed in the dark at 23-28°C and a relative humidity not exceeding 80% for 4 days. Once the soybean sprouts reached 3-4 cm in length, they were harvested. Sprouts with uniform growth were selected, and their roots were removed. The sprouts were then washed with sterile water and set aside.
[0043] 4. Inoculation with soybean anthracnose fungus
[0044] Take about 2 g of defatted cotton, roll it into a ball, and place it at the bottom of a 15 mL centrifuge tube. Add 2 mL of sterile water to each tube and set aside. Use a sterile needle to prick a small wound on the stem of a soybean sprout (about 1 cm from the leaf). Inoculate the wound with soybean anthracnose fungal cake using the sterile needle. Cover the fungal cake with a moist defatted cotton strip and wrap it to retain moisture. Perform three replicates for each variety, with 30 soybean sprouts inoculated in each replicate. Use a sterile agar block as a blank control. Place the inoculated soybean sprouts root-and-stem side down in the centrifuge tube, ensuring the base of the sprouts is in contact with the defatted cotton ball. Cover the centrifuge tube and rotate it half a turn. Incubate the inoculated centrifuge tubes at 25°C in the dark for 3 days.
[0045] 5. Disease survey and soybean variety resistance evaluation
[0046] Three days after inoculation, a disease index survey was conducted based on the extent of lesion expansion, and the variety's disease resistance was evaluated. The disease grading standards were: Grade 0, no symptoms; Grade 1, lesion area less than 10% of stem area; Grade 3, lesion area greater than or equal to 10% and less than 25% of stem area; Grade 5, lesion area greater than or equal to 25% and less than 50% of stem area; Grade 7, lesion area greater than or equal to 50% and less than 75% of stem area; Grade 9, lesion area greater than or equal to 75% of stem area. The disease index calculation formula was: Disease Index = [∑(Number of diseased plants at each grade × Relative grade value) ÷ (Total number of plants surveyed × 9)] × 100. The soybean variety anthracnose resistance evaluation standards were: Highly resistant, disease index = 0; resistant, 0 < disease index ≤ 10; moderately resistant, 10 < disease index ≤ 20; moderately susceptible, 20 < disease index ≤ 40; susceptible, 40 < disease index ≤ 60; highly susceptible, 60 < disease index.
[0047] 6. Evaluation of resistance in tested soybean varieties
[0048] Table 1. Evaluation of the resistance of tested soybean varieties to anthracnose.
[0049]
[0050] As shown in Table 1, the disease index of the susceptible control variety Maodou No. 3 was 60.14, indicating it was highly susceptible. The disease index of the resistant control variety Tianlong No. 1 was 8.19, indicating it was resistant. This demonstrates that the identification results of this method are accurate and reliable. The tested varieties Zhexian 39 had a disease index of 18.13, indicating moderate resistance; Zhenong 160 had a disease index of 32.18, indicating moderate susceptibility; Liaoxiandou 31 had a disease index of 22.50, indicating moderate susceptibility; and Xiangchundou 26 had a disease index of 41.38, indicating susceptibility.
[0051] Example 3: Application of soybean anthracnose inoculation method in determining the control effect of fungicides.
[0052] 1. Sterilization of experimental materials
[0053] Place the experimental materials, including 15 mL centrifuge tubes, absorbent cotton, tweezers, scalpel, needle, and seedling substrate (peat moss and vermiculite in a volume ratio of 3:1), into a cloth bag. Place purified water into a 300 mL Erlenmeyer flask and sterilize by moist heat at 121℃ for 30 min.
[0054] 2. Culture of soybean anthracnose pathogen
[0055] The soybean anthracnose fungus (Colletotrichum truncatum) was cultured on a PDA plate at 28°C for 5 days. Mycelial cakes were then collected from the colonies using a 1 mm diameter punch for later use.
[0056] 3. Sprout cultivation of the tested soybean varieties
[0057] Remove shriveled and broken seeds from the tested soybean variety Zhexian 24. Sow the selected seeds in a sterile seedling substrate (about 2 cm thick) with a row spacing and plant spacing of 3 cm. After sowing, cover with about 1 cm of sterile seedling substrate and water until the seedling substrate is moist. Place in the dark for 4 days under conditions of 23-28°C and relative humidity not exceeding 80%. When the soybean sprouts grow to 3-4 cm, pull out the soybean sprouts, select those with uniform growth and cut off the roots. Wash them with sterile water and set aside.
[0058] 4. Preparation and application of fungicides
[0059] 95% difenoconazole technical grade was prepared into a 1×10 solution using dimethyl sulfoxide. 4 The solution was prepared by adding sterile water to difenoconazole at concentrations of 75 μg / mL and 100 μg / mL. Soybean sprouts were soaked in the difenoconazole solutions at concentrations of 75 μg / mL and 100 μg / mL for 10 min, respectively. Thirty soybean sprouts were treated with each agent, and the experiment was repeated three times. The sterile water soaking treatment served as a blank control and an inoculation control. After drying, the sprouts were inoculated with soybean anthracnose pathogens.
[0060] 5. Inoculation with soybean anthracnose pathogen
[0061] Take approximately 2 g of sterile defatted cotton, roll it into a ball, and place it at the bottom of a 15 mL centrifuge tube. Add 2 mL of sterile water. Using a sterile needle, inoculate the soybean anthracnose fungal cake onto the soybean sprout stem (approximately 1 cm from the leaf). Simultaneously, use the sterile needle to create a small wound. Cover the fungal cake with a moistened defatted cotton strip and wrap it to retain moisture. For the control treatment, inoculate with a sterile agar block and soybean anthracnose fungal cake. Place the inoculated soybean sprout root-stem side down in the centrifuge tube, ensuring the base of the sprout is in contact with the defatted cotton ball. Cover the centrifuge tube and rotate it half a turn. Incubate the inoculated centrifuge tube at 25°C in the dark for 3 days.
[0062] 5. Disease survey and evaluation of fungicide efficacy
[0063] Three days after inoculation, a disease index survey was conducted based on the extent of lesion expansion. The disease grading criteria were as follows: Grade 0, asymptomatic; Grade 1, lesion area less than 10% of stem area; Grade 3, lesion area greater than or equal to 10% and less than 25% of stem area; Grade 5, lesion area greater than or equal to 25% and less than 50% of stem area; Grade 7, lesion area greater than or equal to 50% and less than 75% of stem area; Grade 9, lesion area greater than or equal to 75% of stem area. The disease index was calculated using the formula: Disease Index = [∑(Number of diseased plants at each grade × Relative grade value) ÷ (Total number of plants surveyed × 9)] × 100. The control effect was calculated using the formula: Control Effect (%) = [(Disease index of blank control area - Disease index of treated area) ÷ Disease index of blank control area] × 100.
[0064] 6. Evaluation of the control effect of fungicides
[0065] Table 2. Control efficacy of tested fungicides against soybean anthracnose
[0066]
[0067] The experimental results showed that the control efficacy of difenoconazole at 75 μg / mL and 100 μg / mL against soybean anthracnose was 72.35% and 81.58%, respectively. The difference in efficacy was significant at both the 5% and 1% significance levels, indicating that this inoculation method can effectively determine the preventive effect of the compound against soybean anthracnose.
Claims
1. A method for inoculating soybeans with anthracnose, characterized in that, Includes the following steps: S1. Soybean sprout cultivation: Sow soybean seeds in sterile seedling substrate, sprinkle water until the sterile seedling substrate is moist, place at 23-28°C, RH below 80%, and cultivate in the dark for 4 days. Select soybean sprouts with uniform growth, cut off the roots, and wash them with sterile water. S2. Cultivation of soybean anthracnose fungus: Soybean anthracnose fungus was cultured on PDA medium at 28°C in the dark for 5 days, and mycelial cakes with a diameter of 1 mm were obtained by sterilizing the punch. S3. Inoculation with soybean anthracnose fungus: Place a sterile defatted cotton ball at the bottom of a centrifuge tube and moisten it with sterile water; use a sterile needle to inoculate the soybean anthracnose fungus cake onto the soybean sprout stem, prick the inoculation site with a sterile needle, cover the fungus cake with a moist sterile defatted cotton strip and wrap it to keep it moist, place the inoculated soybean sprout stem base down into the centrifuge tube, and place the soybean sprout stem base on the defatted cotton ball, cover the centrifuge tube and rotate it half a turn, and incubate in the dark at 25°C for 3 days.
2. The soybean anthracnose inoculation method according to claim 1, characterized in that: In step S1, the volume ratio of peat moss to vermiculite in the sterile seedling substrate is 3:1, and it is sterilized by moist heat at 121℃ for 30 min.
3. The soybean anthracnose inoculation method according to claim 2, characterized in that: In step S1, the soybean seeds are sown in a sterile seedling substrate with a row spacing and plant spacing of 3 cm.
4. The soybean anthracnose inoculation method according to claim 1, characterized in that: In step S3, a 15 mL centrifuge tube is used. 2 g of sterile defatted cotton balls are placed at the bottom of the centrifuge tube, and 2 mL of sterile water is added.
5. A method for statistically analyzing the incidence of soybean anthracnose, wherein soybean sprouts are obtained using the inoculation method described in claim 1, characterized in that, The disease level is determined by the percentage of soybean sprout lesions relative to the total area of the soybean sprout stem.
6. A method for statistically analyzing the incidence of soybean anthracnose according to claim 5, characterized in that: The disease level is determined based on the percentage of soybean sprout lesions relative to the total area of the sprout stem, as follows: Grade 0: Asymptomatic; Grade 1: Lesions cover less than 10% of the stem area; Grade 3: Lesions cover 10% to 25% of the stem area; Grade 5: Lesions cover 25% to 50% of the stem area; Grade 7: Lesions cover 50% to 75% of the stem area; Grade 9: Lesions cover 75% to 85% of the stem area.
7. A method for statistically analyzing the incidence of soybean anthracnose according to claim 5, characterized in that: The incidence of diseases was statistically analyzed using ImageJ software.
8. A method for statistically analyzing the incidence of soybean anthracnose according to claim 7, characterized in that: The soybean sprouts were photographed at a fixed height using a camera tripod, and the disease incidence was statistically analyzed based on the area of lesions using ImageJ software.
9. The application of the soybean anthracnose inoculation method as described in claim 1 in the identification of soybean anthracnose resistance.
10. The application of the soybean anthracnose inoculation method as described in claim 1 in determining the control effect of fungicides or compounds.