A method for inoculating peanut sclerotinia blight resistance identification under field conditions

Abstract

The application discloses a kind of field conditions under peanut white thread disease resistance identification inoculation method, belong to the field of agricultural biotechnology.The method includes: activated Sclerotium rolfsii is inoculated to sterilization treated oat grain, and culture obtains bacterial oat grain;Vermiculite is mixed with nutrient soil according to mass ratio (1.4-1.6):1, and mixed matrix is obtained;Bacterial oat grain is premixed with mixed matrix according to mass ratio (0.3-0.5):(5-7), is cultivated under the condition of moisture 1.5-2.5 days, and obtains activation inoculum;In peanut flowering lower needle period, activation inoculum is applied to the soil surface around the base of plant stem, and the soil water content is controlled to be 65%-75% after inoculation.The inoculation method provided by the application realizes the standardization of peanut white thread disease resistance identification under field conditions, repeatable operation, significantly improves the uniformity of disease occurrence and the reliability of identification results, and is suitable for large-scale peanut germplasm resistance screening.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to an inoculation method for identifying peanut resistance to white mold disease under field conditions. Background Technology

[0002] Peanut white mold is a soil-borne disease that seriously damages peanut yield. Because the sclerotium overwinters as sclerotia in diseased plant debris and the soil layer, it can survive in the soil for many years. Chemical control is difficult and easily causes environmental pollution, and agricultural measures such as crop rotation have limited effectiveness. Therefore, planting disease-resistant varieties is the most economical and effective measure to control this disease, and establishing stable and reliable resistance identification techniques is a prerequisite for screening and breeding disease-resistant varieties.

[0003] Currently, the identification of peanut white mold resistance mainly relies on field inoculation. Existing field inoculation methods include spot inoculation, root drenching, and broadcasting. Spot inoculation involves placing infected wheat grains, mycelial blocks, or mycelial discs directly at the base of the peanut stem or attaching them to the stem; root drenching involves applying mycelial suspension or spore suspension to the rhizosphere soil through an irrigation system; broadcasting involves directly broadcasting infected grains (such as wheat grains, oat grains, sorghum grains, etc.) onto the soil surface around the base of the plant stem. Among these methods, Chinese patent CN107135828B discloses a greenhouse seedling inoculation method for peanut white mold, which preferentially uses the "infected oat grain surface spreading method." This involves directly spreading infected oat grains onto the soil surface at the base of the peanut seedling stem under greenhouse conditions, followed by daily watering to achieve good disease control. This method is simple to operate and results in rapid disease development, providing an effective means for screening resistance in greenhouse seedlings.

[0004] However, the aforementioned existing methods have significant technical limitations in practical applications. Spot inoculation easily leads to uneven disease development among individual plants, forming obvious disease centers, and the inoculation effect is greatly affected by the operator's technique, resulting in low standardization. Root drenching is affected by the heterogeneity of soil texture, irrigation method, and water distribution, leading to uneven pathogen diffusion in the soil and large fluctuations in disease severity among different replicates of the same variety, resulting in poor reproducibility of experimental results. While broadcast application is relatively simple, existing technologies (such as CN107135828B) are explicitly limited to greenhouse seedling conditions and do not address applications in field environments. Directly transplanting greenhouse methods to the field often results in decreased pathogen survival rates and unstable infection due to various uncontrollable factors such as temperature fluctuations, light variations, rain erosion, and competition among soil microorganisms, making it difficult to achieve uniform disease occurrence and reproducible results.

[0005] Therefore, there is an urgent need in this field to establish a standardized, repeatable, and accurate field inoculation method for peanut white mold disease that can simulate natural disease conditions, so as to provide reliable technical support for the identification of peanut variety resistance to white mold disease. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide an inoculation method for identifying peanut resistance to white mold disease under field conditions.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, this invention provides an inoculation method for identifying peanut white mold resistance under field conditions, comprising the following steps: (1) Inoculate activated neat sclerotium into sterilized oat grains and culture until the mycelium covers the surface of the oat grains to obtain oat grains with bacteria; (2) Mix vermiculite and nutrient soil at a mass ratio of (1.4-1.6):1, sterilize, and obtain a mixed substrate; (3) The bacteria-infected oat grains obtained in step (1) and the mixed substrate obtained in step (2) are premixed at a mass ratio of (0.3-0.5): (5-7). The mixture is then cultured under moist conditions for 1.5-2.5 days, and stirred once every 4 hours to ensure that the mycelium is evenly distributed and that the mycelium spreads from the oat grains to the mixed substrate to obtain activated inoculum. (4) During the flowering and pegging stage of peanuts, the activated inoculum obtained in step (3) is applied to the soil surface around the base of the peanut plant stem. After inoculation, the soil moisture content is controlled to be 65%-75%.

[0008] In step (1), the sterilization process is a secondary high-pressure steam sterilization; the conditions for the secondary high-pressure steam sterilization are: temperature 121℃, time 20-30 minutes, repeated once every 24 hours.

[0009] In step (1), the conditions for cultivating the mycelium to cover the surface of the oat grains are: 25°C and 5-7 days in the dark.

[0010] In step (2), the mass ratio of vermiculite to nutrient soil is 1.5:1; in step (3), the mass ratio of bacteria-carrying oat grains to mixed substrate is 0.4g:6g / plant, and the moisturizing culture time is 2 days.

[0011] In step (4), the application is to evenly spread the soil around the base of the peanut stem within a radius of 3-7 cm.

[0012] In step (4), the soil moisture content is controlled by drip irrigation.

[0013] In a second aspect, the present invention provides the application of the above method in the identification of peanut varieties' resistance to white mold or the screening of germplasm resistant to white mold.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention precisely defines key parameters such as inoculum preparation (secondary sterilization, culture conditions), mixed substrate ratio (vermiculite: nutrient soil = 1.4~1.6:1), pre-activation conditions (moisture-controlled culture for 2 days), inoculum amount (0.3~0.5g: 5~7g / plant), and soil moisture (65%~75%), forming a standardized process that can be operated in an assembly line, greatly reducing human error and improving the comparability of test results from different batches and locations.

[0015] 2. This invention uses a composite inoculum of "infected oat grains + mixed substrate," which undergoes pre-activation treatment to allow the mycelium to pre-spread into the substrate. During application, the inoculum contacts the rhizosphere soil of the plants in a "surface" rather than "point" manner, simulating the ecological process of natural diffusion and gradual infection of pathogens in the soil in the field, thus achieving uniform disease occurrence within the experimental plots. Results from the examples show that the coefficient of variation of the disease index among different replicates of the same variety is significantly lower than that of existing methods.

[0016] 3. This invention, through standardized inoculum preparation and uniform inoculation operations, combined with precise soil moisture control, ensures the stability of disease conditions across different batches of trials. In the examples, resistance identification was performed on 10 peanut varieties. The disease index distribution was continuous, and there was a clear distinction between susceptible and resistant varieties, indicating that this method can generate stable and clearly differentiated disease pressure.

[0017] 4. The operation process of this invention is clear, easy to master and batch operation, and is particularly suitable for breeding projects that require large-scale resistance screening of dozens or even hundreds of varieties or lines, which can significantly improve identification efficiency and throughput.

[0018] 5. This invention employs a two-stage sterilization process, effectively ensuring the purity of the inoculum and preventing contamination by other microorganisms. The dried, bacteria-laden oat grains are easy to store and transport for long periods. Before use, they are mixed with the substrate to maintain moisture and pre-activate, which is equivalent to activation and propagation, ensuring that the mycelium is in a highly active state at the time of inoculation, resulting in strong and consistent infectivity. Attached Figure Description

[0019] Figure 1 : Activated, neatly arranged sclerotium; Figure 2 Operational procedures; Figure 3 Comparison of anti-infection materials 21 days after vaccination; Figure 4 : Mycelial growth at the base of the germplasm stem 21 days after inoculation. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0022] Example 1: Establishment and Operation Flow of the Method of the Invention (1) Activation of microbial strains Take out the neatly preserved *Sclerotium sclerotiorum* strain stored at -80℃ and thaw at room temperature. Pick out the sclerotia, sterilize them, absorb the surface moisture, and inoculate them in the center of a PDA plate. Incubate at 25℃ in the dark for 3-4 days until the mycelium covers the surface of the plate, and the activated strain is obtained.

[0023] (2) Preparation of bacteria-infected oat grains Weigh 500g of oat grains and place them in a 2L Erlenmeyer flask. Add distilled water and soak for 4 hours, then drain off excess water. Place the flask in an autoclave and sterilize at 121℃ for 30 minutes. Remove and cool to room temperature. After 24 hours, sterilize again at 121℃ for 30 minutes (double sterilization). When the oat grains have cooled to approximately 40℃, in a clean bench, use sterile forceps to take activated mycelial blocks (approximately 1cm in diameter) from a PDA plate and inoculate them into the oat grains, shaking well. Incubate the flask in the dark at 25℃ for 6 days, shaking three times daily, until all oat grains are covered with white mycelium. Remove the mycelium-covered oat grains and dry them at 40℃ until constant weight to obtain dried, mycelium-infected oat grains. Seal and store in a 4℃ refrigerator for later use.

[0024] (3) Preparation of mixed matrix Vermiculite and nutrient soil were sterilized at 121℃ for 30 minutes at a mass ratio of 1.5:1 (i.e., 3 kg of vermiculite and 2 kg of nutrient soil) and then placed in a mixer and thoroughly mixed to obtain a mixed matrix.

[0025] (4) Pre-activation of inoculum Take 40g of the bacteria-infected oat grains obtained in step (2) and 600g of the mixed substrate obtained in step (3) (equivalent to 0.4g of each bacteria-infected oat grain and 6g of the mixed substrate, for a total of 100 plants). Mix the two thoroughly and spray with an appropriate amount of sterile water to make the substrate moisture content 60% (it can be formed into a clump when squeezed in the hand, but crumbles when dropped). Spread the mixture evenly in a sterilized plastic tray, cover it with a moisturizing film (with holes for ventilation), and place it in a constant temperature room at 25℃ for 2 days for moisturizing cultivation. After the cultivation is completed, white mycelia can be seen to spread obviously from the oat grains to the surrounding substrate, forming a mycelial network, thus obtaining activated inoculum.

[0026] (5) Field planting Experimental site: Agronomic Experiment Station, Shandong Agricultural University, Tai'an City, Shandong Province. The soil was brown soil with moderate fertility. Peanut varieties P001-P010 were sown on May 10, 2025, with a row spacing of 40 cm and a plant spacing of 20 cm. Single seeds were sown per hill, with four replicate plots of each variety, each containing 20 plants, arranged in a randomized block design. Field inoculation was carried out at the flowering and pegging stage (approximately 60 days after sowing). Before inoculation, the soil was thoroughly irrigated using drip irrigation to moisten the topsoil.

[0027] Following the ratio of 0.4g of infected oat grains and 6g of mixed substrate per plant as described in step (4) of Example 1, calculate the required total amount based on the total number of plants in the plot, weigh it, and evenly spread it on the soil surface with a radius of 5cm around the base of the peanut stem. After spreading, start the drip irrigation system to control the soil moisture content at 65%~75% (monitored with a handheld soil moisture meter at a depth of 0~10cm). Monitor the soil moisture daily, and promptly replenish water through drip irrigation when it falls below 65%, and continue this management for 4 weeks.

[0028] (6) Disease investigation and resistance identification Surveys began on day 7 post-inoculation and continued every 7 days for a total of 4 surveys (days 7, 14, 21, and 28). Each plant in each plot was surveyed, and the disease severity was recorded. The disease grading criteria are as follows: Level 0: Asymptomatic, healthy plant; Grade 1: Disease spots only on the stem; Level 2: Less than 25% of the entire plant is wilted and dead; Grade 3: 26% to 50% of the branches of the entire plant wilt and die; Level 4: 50% to 75% of the plant's branches wilt and die; Level 5: ≥76% of the entire plant shows signs of wilting and death.

[0029] The Disease Index (DI) is calculated as follows: I = {[∑(each level of diseased plants × corresponding level value)] / (total number of plants investigated × highest disease level 5)}×100.

[0030] Variety resistance was evaluated using the Relative Disease Index (RDI). The disease index of the variety with the most severe disease outbreak in this trial was used as the baseline (set at 1.0). The RDI for the remaining varieties was calculated using the following formula: RDI = DI Most susceptible varieties DI variety.

[0031] Resistance grading standards: High resistance (HR): RDI ≤ 10; Resistant (R): 10 < RDI ≤ 20; MR (Moderately resistant): 20 < RDI ≤ 40; MS (Moderately susceptible): 40 < RDI ≤ 60; Susceptible (S): 60 < RDI ≤ 80; Highly susceptible (HS): RDI > 80.

[0032] Example 2: Resistance identification results of different peanut varieties Using the method of Example 1, resistance identification was carried out on 10 commercially available peanut varieties (lines) P001 - P010. At the same time, an un-inoculated control was set (10 plants for each variety, without inoculation, and the rest of the management was the same). The un-inoculated control did not show any disease during the entire test period, indicating that the natural disease occurrence conditions in the field did not interfere with the test.

[0033] The survey results on the 21st day after inoculation are shown in Table 1. [[ID=IO]]

[0034] Table 1 Disease index and resistance evaluation of different peanut varieties after inoculation with Sclerotium rolfsii As can be seen from Table 1, the method of the present invention produced obvious resistance differences among different varieties. Among them, variety P003 had the highest disease index and was used as the most susceptible variety to calculate RDI. The disease index of variety P005 was only 4.4 and was evaluated as a highly resistant (HR) variety.

[0035] Repeatability analysis: The standard deviation of the disease index of 3 replicates of the same variety was between 0.25 - 0.5, and the coefficient of variation (CV%) was between 1.16 - 5.63, indicating that the method of the present invention has excellent repeatability and stability and can provide reliable disease occurrence conditions for resistance identification.

[0036] Uniformity observation: Field observation found that the distribution of diseased plants in each plot was uniform, without obvious disease centers, and the degree of disease occurrence was highly correlated with the variety resistance, indicating that the method of the present invention can simulate the natural infection process and achieve uniform disease occurrence.

[0037] Example 3: Comparative test of the method of the present invention with existing methods To verify the superiority of the method of the present invention, the following control treatments were set and compared with the method of the present invention. The test was carried out in the same test field at the same time. The tested varieties were the susceptible variety P003 (with the highest disease index in Example 2) and the resistant variety P005 (highly resistant). There were 4 replicates for each treatment, and 20 plants for each replicate.

[0038] 1. Control setting Treatment A (method of the present invention): carried out according to the steps of Example 1, namely, secondary sterilization of bacteria-carrying oat grains + mixed substrate + pre-activation for 2 days + application + humidity control of 65%~75%.

[0039] Treatment B (direct application of bacteria-infected oat grains): Refer to the "oat grain application method" in CN107135828B, apply bacteria-infected oat grains prepared by sterilization (121℃, 20min) directly to the soil surface at the base of the peanut stem, 4 grains per plant, water once a day (do not control precise humidity, do not mix with substrate, do not pre-activate), and other management is the same as treatment A.

[0040] Treatment C (infected oat grains + mixed substrate without pre-activation): The sterilized oat grains were mixed with the mixed substrate (1.5:1) at a ratio of 0.4g:6g / plant. The mixture was applied immediately after application (without 2 days of moisture retention). The humidity control after application was the same as that for treatment A.

[0041] Treatment D (blank control): No vaccination, other management is the same as treatment A.

[0042] 2. Survey Results The disease index was investigated on the 21st day after vaccination, and the results are shown in Table 2.

[0043] Table 2. Effects of different inoculation methods on the disease index of susceptible variety P003 and resistant variety P005. Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0044] Based on the test results: (1) Disease intensity: The disease index of treatment A in this invention is significantly higher than that of other treatments, indicating that the disease pressure generated by the method of this invention is stronger and more conducive to distinguishing resistant varieties.

[0045] (2) Distinguishing effect of disease-resistant varieties: The disease index of disease-resistant varieties under other treatments is lower and the difference between resistance and susceptibility is smaller, indicating that the distinguishing effect between resistance and susceptibility is not as good as the method of the present invention.

[0046] (3) Field uniformity observation: In treatment B (direct application), obvious disease centers were visible in each plot. Plants in areas with concentrated inoculum were more severely affected, while those far from the inoculum were less affected, resulting in extremely uneven distribution. Treatment C (no pre-activation) showed some improvement in uniformity, but there were still some areas with milder disease. Treatment A (this invention) showed uniform disease distribution throughout the entire plot with no obvious disease centers, indicating that the pre-activation step significantly improved the uniformity of inoculum distribution in the soil.

[0047] Conclusion: The method of the present invention has significant advantages in terms of disease intensity, resistance-susceptibility differentiation and field uniformity, and in particular solves the technical problems of uneven disease occurrence and poor result repeatability in field inoculation.

[0048] Example 4: Effect of different pre-activation times on inoculation efficacy To further optimize the pre-activation conditions, different moisturizing incubation times were set (0 days, 1 day, 2 days, 3 days, and 4 days), with other steps the same as in Example 1. The tested variety was the susceptible variety P002, with 4 replicates per treatment and 20 plants per replicate. The disease index was investigated on day 21 after inoculation, and the results are shown in Table 3.

[0049] Table 3. Effects of different pre-activation times on the disease index of susceptible variety P002. Results analysis: When the pre-activation time was 0 days (i.e., used immediately after mixing), the disease index was the lowest and the coefficient of variation was the highest, indicating that the hyphae had not yet spread to the substrate and were prone to inactivation or uneven distribution in the field environment after inoculation. After 1 day of pre-activation, the disease index increased to 6.54, and the coefficient of variation decreased to 2.5. After 3 days of pre-activation, the disease index reached its highest level and the coefficient of variation was the lowest, with the hyphae spreading evenly into the substrate and forming a good hyphal network. After 4 days of pre-activation, the disease index was not significantly different from that after 2 days, but the disease index decreased slightly, and hyphal aging and sclerotium formation were observed, which may affect the long-term stability of the inoculum. Therefore, a pre-activation time of 3 days is preferred.

[0050] Example 5: Effect of different soil moisture on inoculation effect Different soil moisture content treatments were set up (50%~55%, 60%~65%, 65%~70%, 70%~75%, 75%~80%), with other steps the same as in Example 1. The tested variety was the susceptible variety P002, with 4 replicates per treatment and 20 plants per replicate. The disease index was investigated on day 21 after inoculation, and the results are shown in Table 4.

[0051] Table 4. Effects of different soil moisture contents on the disease index of susceptible variety P002 Results analysis: When soil moisture content is below 60%, the disease index decreases significantly, and the coefficient of variation increases, indicating that insufficient moisture limits mycelial growth and infection. The optimal moisture range is 65%–75%, where the disease index is highest and the coefficient of variation is lowest. At excessively high moisture content (75%–80%), although the disease index remains high, the coefficient of variation increases, possibly due to decreased soil aeration and reduced root vitality caused by excessive humidity. Therefore, a soil moisture content of 65%–75% is preferred.

[0052] In summary, this invention provides a standardized, reproducible, and accurately simulates natural disease conditions for field inoculation of peanut white mold. The method has a clear operational procedure, well-defined key parameters, and the inoculum is easy to preserve and transport, making it suitable for large-scale screening of peanut germplasm resources for resistance and breeding of disease-resistant varieties. Using this method, uniform and stable disease occurrence can be achieved under field conditions, significantly improving the accuracy and reproducibility of resistance identification, and it has good prospects for widespread application.

[0053] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted. Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. An inoculation method for identifying peanut white mold disease resistance under field conditions, characterized in that, Includes the following steps: (1) Inoculate activated and neatly arranged Sclerotium sclerotium onto sterilized oat grains and culture until the mycelium covers the surface of the oat grains to obtain oat grains with bacteria; (2) Mix vermiculite and nutrient soil at a mass ratio of (1.4-1.6):1, sterilize, and obtain a mixed substrate; (3) The bacteria-infected oat grains obtained in step (1) and the mixed substrate obtained in step (2) are premixed at a mass ratio of (0.3-0.5): (5-7). The mixture is then cultured under moist conditions for 1.5-2.5 days, and stirred once every 4 hours to ensure that the mycelium is evenly distributed and that the mycelium spreads from the oat grains to the mixed substrate to obtain activated inoculum. (4) During the flowering and pegging stage of peanuts, the activated inoculum obtained in step (3) is applied to the soil surface around the base of the peanut plant stem. After inoculation, the soil moisture content is controlled to be 65%-75%.

2. The method according to claim 1, characterized in that, In step (1), the sterilization process is a secondary high-pressure steam sterilization; the conditions for the secondary high-pressure steam sterilization are: temperature 121℃, time 20-30 minutes, repeated once every 24 hours.

3. The method according to claim 1, characterized in that, In step (1), the conditions for cultivating the mycelium to cover the surface of the oat grains are: 25°C and 5-7 days in the dark.

4. The method according to claim 1, characterized in that, In step (2), the mass ratio of vermiculite to nutrient soil is 1.5:1; in step (3), the mass ratio of bacteria-carrying oat grains to mixed substrate is 0.4g:6g / plant, and the moisturizing culture time is 2 days.

5. The method according to claim 1, characterized in that, In step (4), the application is to evenly spread the soil around the base of the peanut stem within a radius of 3-7 cm on the soil surface.

6. The method according to claim 1, characterized in that, In step (4), the soil moisture content is controlled by drip irrigation.

7. The application of the method according to any one of claims 1-6 in the identification of peanut varieties resistant to white mold or the screening of germplasm resistant to white mold.

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