Plant soil disease control effect evaluation method

Abstract

The invention relates to a plant soil disease control effect evaluation method, which is suitable for soil disease control effect evaluation of phytopathogen infection research, and realizes good growth of peanut root systems and controllability of pathogen infection intensity by optimizing a cultivation device, a matrix and an inoculation method. On the basis, 100 strains of actinomycetes are separated by taking faeces of protaetia brevitarsis larvae as a sample, strains with bacteriostatic activity on sclerotium rolfsii are screened through a plate confrontation experiment, and finally 7 strains of actinomycetes with remarkable antagonism are obtained. Through a soil disease control effect evaluation system, the growth promoting and disease resisting effects of the seven strains on peanuts are determined, and particularly, the X15 strain has the best performance. According to the method established by the research, actinomycetes with an excellent biocontrol function are effectively screened out, and important resources are provided for agricultural disease prevention and control and green biopesticide development.

Description

Technical Field

[0001] The present invention relates to the field of biological control, and in particular to a method for evaluating the control effect of plant and soil diseases. Background Art

[0002] Plant diseases are a major threat to agricultural production, leading to reduced crop yields, decreased quality, and even crop failure. Diseases are categorized as fungal, bacterial, viral, and nematode (Gai YP, Wang HK. 2024. Plant Disease: A Growing Threat to Global Food Security. Agronomy-Basel 14.). Soil-borne diseases are caused by pathogens found in the soil, with common examples including Fusarium, Phytophthora, and Rhizoctonia. These diseases invade plants through their roots, causing symptoms such as root rot, wilting, and damping-off, severely impacting crop growth. Because the soil provides a suitable colonization environment for some pathogens and limits the delivery and action of fungicides, the prevention and control of soil-borne diseases has become a difficult point in agricultural production (Bakker P, Berendsen RL, Van PeltJA, Vismans G, Yu K, Li E, Van Bentum S, Poppeliers SWM, Sanchez Gil JJ, Zhang H, Goossens P, Stringlis IA, Song Y, de Jonge R, Pieterse CMJ. 2020. The Soil-Borne Identity and Microbiome-Assisted Agriculture: Looking Back to the Future. Mol Plant 13: 1394-1401.).

[0003] Screening for effective fungicides is a crucial step in current soil-borne disease control. Unlike aerial diseases such as those affecting leaves, the occurrence of soil-borne diseases, the application of fungicides, and the quantitative assessment of control effectiveness are all difficult due to the barrier nature of soil, which limits the screening and application of fungicides for soil-borne diseases. Therefore, establishing a reproducible and quantifiable screening model for soil-borne disease fungicides against important soil-borne diseases is a crucial requirement for current soil-borne disease control (Compant S, Cassan F, Kostic T, Johnson L, Brader G, Trognitz F, Sessitsch A. 2025. Harnessing the plant microbiome for sustainable crop production. Nat Rev Microbiol 23:9-23.).

[0004] Screening for beneficial agricultural microorganisms that can colonize soil and rhizosphere for soil-borne disease control is an effective strategy with advantages such as environmental protection and sustainability (Ma M, Taylor PWJ, Chen D, Vaghefi N, He JZ. 2023. Major Soilborne Pathogens of Field Processing Tomatoes and Management Strategies. Microorganisms 11.). For example, Bacillus velezensis inhibits the growth of pathogens by secreting small molecule metabolites such as lipopeptides (Zhao TX, Zhang LD, Qi CP, Bing H, Ling L, Cai Y, Guo LF, Wang XJ, Zhao JW, Xiang WS. 2023. A seed-endophytic bacterium NEAU-242-2: Isolation, identification, and potential as a biocontrol agent against. Biological Control 185.). Trichoderma inhibits the growth of pathogens by competing for nutrients and space and secreting antibiotics and cell wall-degrading enzymes (Singh S, Singh AK, Pradhan B, Tripathi S, Kumar KS, Chand S, Rout PR, Shahid MK. 2024. Harnessing Trichoderma Mycoparasitism as a Tool in the Management of Soil Dwelling Plant Pathogens. Microbial Ecology 87.). Bacillus subtilis can induce systemic resistance in peanuts, enhancing resistance to white rot (Zou L, Wang Q, Wu R, Zhang Y, Wu Q, Li M, Ye K, Dai W, Huang J. 2022. Biocontrol and plant growth promotion potential of endophytic Bacillus subtilis JY-7-2L on Aconitum carmichaelii Debx. Front Microbiol 13: 1059549.).Bio-organic fertilizers containing Trichoderma can improve soil microbial communities and effectively inhibit peanut white rot (Meena PN, Meena AK, Tiwari RK, Lal MK, Kumar R. 2024. Biological Control of Stem Rot of Groundnut Induced by Sclerotium rolfsii sacc. Pathogens 13.). Actinomycetes are widely distributed in the natural environment and are the dominant group of soil microorganisms that can effectively colonize the soil. Actinomycetes can produce a variety of metabolites, including insecticides and fungicides, and are widely used in human health, animal husbandry, and agricultural pest control. Currently, more than 70% of antibiotics are produced by actinomycetes (Rey T, Dumas B. 2017. Plenty Is No Plague: Streptomyces Symbiosis with Crops. Trends Plant Sci 22: 30-37.). Actinomycetes hold great potential as green pest control tools. Their metabolites can inhibit pathogens and pests, activate plant resistance mechanisms, and reduce reliance on chemical pesticides. Their ability to decompose organic matter, improve soil structure, and degrade pollutants can also restore ecosystems, reduce agricultural non-point source pollution, and achieve environmentally friendly and sustainable agricultural development. Leveraging the highly effective bactericidal and disease-resistant properties of actinomycetes, as well as their ability to colonize in soil, is a viable approach for developing highly effective soil-borne disease control products. Currently, Streptomyces lydicus, launched by Novozymes in the United States, has achieved success. Novozymes BioAg Inc.).

[0005] Soil-borne diseases are one of the main factors affecting current crop yields, and screening for highly effective fungicides is the main strategy to solve this problem. Peanut is an important crop. Due to its characteristic of pod formation in the soil, soil-borne diseases pose a more serious threat to peanut production (Zhou Y, Yang Z, Liu J, Li X, Wang X, Dai C, Zhang T, Carrion VJ, Wei Z, Cao F, Delgado-Baquerizo M, Li X. 2023. Crop rotation and native microbiomeinoculation restore soil capacity to suppress a root disease. Nat Commun 14:8126.; Wang S, Wang Y, Shi X, Herrera-Balandrano DD, Chen X, Liu F, Laborda P. 2024. Application and antagonistic mechanisms of atoxigenic Aspergillus strains for the management of fungal plant diseases. Appl Environ Microbiol 90:e0108524.; Ojiewo CO, Janila P, Bhatnagar-Mathur P, Pandey MK, Desmae H,OkoriP,Mwololo J,Ajeigbe H,Njuguna-Mungai E,Muricho G,Akpo E,Gichohi-Wainaina WN,Variath MT,Radhakrishnan T,Dobariya KL,Bera SK,Rathnakumar AL,Manivannan N,Vasanthi RP,Kumar MVN,VarshneyRK. 2020.Advances in Crop Improvement and Delivery Research for Nutritional Quality and Health Benefits of Groundnut (Arachis hypogaea L.). Front Plant Sci 11:29.).The existing method for evaluating the effectiveness of soil disease control is generally the plate confrontation method. However, since the effect between the strain and the pathogen is evaluated on the plate, it is completely different from the actual plant growth environment. Therefore, the plate confrontation method can only be used as a preliminary screening. To screen out strains with better effects, it is necessary to seek better control effectiveness evaluation methods. Summary of the Invention

[0006] In response to the needs in the above-mentioned fields, the present invention provides a method for evaluating the effectiveness of plant soil disease prevention. The laboratory simulates the peanut growth environment, which can more realistically demonstrate the actual prevention and control effects of the strains to be screened, and is of great significance for more effective screening of efficient fungicides for soil-borne diseases.

[0007] A method for evaluating plant soil disease prevention effectiveness comprises the following steps:

[0008] (1) Take a 100ml sterilized centrifuge tube and make a slit: make a 1mm×2cm slit at the bottom of the centrifuge tube, cut a round hole with a diameter of 6-8mm at a distance of 5-6cm from the bottom on the side, put the mixed culture matrix, organic fertilizer and spores of the disease-resistant strain to be tested at the bottom, then select full and disease-free plant seeds, gently bury the tip downward into the mixed matrix, so that the top of the plant seed is 1cm away from the centrifuge tube mouth, and then cover the plant seed with the mixed matrix. Immerse the bottom of the sown centrifuge tube in water to maintain humidity and light cultivation.

[0009] (2) When the lateral roots of the plants grow and touch the tube wall, sorghum grains treated with soil-borne pathogens are inoculated from the side holes and cultured for 12-18 days;

[0010] (3) Evaluate the efficacy of the disease-resistant strain to be tested: a) observe the root lesions, or calculate the ratio of the root lesion area to the total root area, b) the fresh weight of the underground roots, c) the dry weight of the underground roots; the higher the fresh weight and dry weight of the underground roots, the better the growth promotion effect of the disease-resistant strain to be tested on the plant seedlings; the smaller the root lesion area or the lower the ratio of the root lesion area to the total root area, the higher the dry weight of the underground roots, the higher the resistance of the disease-resistant strain to soil-borne diseases.

[0011] The step (1) further includes a blank control culture of the disease-resistant strain to be tested, in which no spores of the disease-resistant strain to be tested are added to the mixed matrix; the step (3) further includes a comparison of the root lesion area between the treatment group and the control group.

[0012] The culture in step (1) is a 16L:8D culture at 26°C with a constant photoperiod.

[0013] The humidity maintenance in step (1) is to insert the centrifuge tube into the culture rack, and place the culture rack into a culture tray containing water, and regularly add water into the culture tray to maintain the culture humidity.

[0014] The dosage of culture medium, organic fertilizer and spores of the disease-resistant strain to be tested in the mixed matrix is: 100ml: 4g: 2×10 13 CFU spores.

[0015] The culture medium is vermiculite, and the organic fertilizer is fermented mushroom residue.

[0016] The inoculation time is 10-16 days after the plant seeds are planted, and the evaluation sampling time is 28 days after the plant seeds are planted.

[0017] The plant is peanut.

[0018] The pathogen is Sclerotium rolfsii Sacc., and the disease-resistant bacteria to be tested are actinomycetes.

[0019] The actinomycetes are derived from the feces of white-spotted flower beetle larvae, and the mixed matrix also includes sterilized white-spotted flower beetle feces.

[0020] The ratio of root lesion area to total root area was calculated by photographing the roots using an Epson Perfection V500 Photo scanner. Using ImageJ software, a threshold of 70 was set to automatically identify and mark the lesion area; a threshold of 220 was set to mark the total root area. The software's "Analyze Particles" function was used to calculate the pixel area of ​​the lesion area and the total root area. The percentage of lesion area was calculated using the formula: (Pixel area of ​​lesion area / Pixel area of ​​total root area) × 100%.

[0021] This application uses peanuts and the important soil-borne pathogen Sclerotium sclerotiorum as the research objects, and establishes a precise and controllable cultivation system to simulate the pathogen infection environment, quantify the root lesions and evaluate the fungicide screening technology for the prevention and control effect. Using this method, actinomycetes that are resistant to peanuts and promote growth were effectively obtained. This research will provide acceptable and applicable suggestions for fungicide development companies to quantitatively compare candidate products for plant root diseases, which is of great significance for the prevention and control of soil-borne diseases. Although the cultivation system is established for peanuts, because it only involves young plants, the system is also applicable to other plants involved in soil-borne diseases.

[0022] This application successfully established a soil disease prevention and control effectiveness evaluation system suitable for the study of peanut pathogen infection. By optimizing the cultivation equipment, substrate and inoculation method, it achieved good growth of the peanut root system and controllable pathogen infection intensity, making it an effective model for screening highly effective fungicides for soil-borne diseases. The data from this study showed that there was a big difference between the antibacterial performance of microbial strains in confrontation culture and their performance in protecting plants in the soil. This application screened out the best antibacterial effect of strain X3 using the plate confrontation method. However, using the soil disease prevention and control effectiveness evaluation method established by this application, it was found that the two actinomycetes X4 and X5 had better activity through testing of various physiological indicators of peanuts. These results show that this application is of great significance for more effective screening of highly effective fungicides for soil-borne diseases.

[0023] When establishing the potted model, it was discovered that for peanuts, the cultivation space of a 100mL centrifuge tube was sufficient for normal peanut growth, while also allowing the roots to adhere to the wall due to spatial limitations. This phenomenon is crucial for inoculating pathogens through the window openings in the side walls of the centrifuge tube. Therefore, for different plants, the culture container needs to be appropriately adjusted to allow the roots to adhere to the wall due to spatial limitations, ensuring effective pathogen inoculation. Furthermore, in this study, fermented mushroom residue was added as organic matter to the vermiculite mineral to simulate a soil environment. First, vermiculite is a natural, inorganic, and non-toxic mineral. Using it as a support medium for soilless cultivation can provide crops with naturally occurring elements such as K, Mg, Ca, Fe, and trace amounts of Mn, Cu, and Zn (Wang Z, Hou S, Liao B, Yao Z, Zhu Y, Liu H, Feng J. 2025. Improving Lunar Soil Simulant for Plant Cultivation: Earthworm-Mediated Organic Waste Integration and Plant-Microbe Interactions. Plants (Basel) 14). It also acts as a buffer, inhibiting rapid pH changes and ensuring a stable root environment for plants. Furthermore, the addition of organic matter is essential. Data from this study showed that the addition of organic matter significantly limited the growth of S. rolfsii. Peanuts grew well in cultivation media containing 4% to 8% organic fertilizer, while roots were severely infected in media containing 4% organic fertilizer, and plant stem height was significantly affected. Therefore, adjusting the amount of organic matter added so that the infection intensity of the root system is within the range of infection controllable by fungicides (Wei P, LiY, Lai D, Geng L, Liu C, Zhang J, Shu C, Liu R. 2020. Protaetia brevitarsis larvaecan feed on and convert spent mushroom substrate from Auricularia auriculaand Lentinula edodes cultivation. Waste Manag 114: 234-239.) is very important for evaluating the control effect of different fungicides.

[0024] The results of this application will play an important role in further screening highly active actinomycete resources that can be used for the development of soil-borne disease control products. At the same time, the discovered X5 strain has become a valuable resource for new drug development and agricultural disease control, and is expected to play a huge role in soil disease control. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This shows the disease status of peanut roots inoculated with pathogens. DETAILED DESCRIPTION

[0026] The biological materials used below are all preserved in this laboratory and can be distributed publicly.

[0027] 1. Methods

[0028] 1.1 Activation and inoculation of Sclerotium rolfsii

[0029] Sclerotium rolfsii was obtained from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. Sclerotium rolfsii was inoculated into PDA medium (200 g / L potato, 20 g / L glucose, 20 g / L agar) and cultured at 26°C for 5 days. 100 g of commercially available sorghum grains were washed and boiled in boiling water for 10 minutes. Excess water was removed from the boiled sorghum grains, placed in a 500 ml Erlenmeyer flask, and sterilized at 121°C for 15 minutes. Five Sclerotium rolfsii cakes were taken from the edge of the PDA plate using a 5mm hole puncher, placed in a sterilized sorghum grain Erlenmeyer flask, and cultured at 26°C. Shake the flask once a day to loosen the sorghum grains to prevent them from clumping or deteriorating due to local high temperatures. After 9 days of culture, shake well and set aside for inoculation with peanuts.

[0030] 1.2 Peanut planting, pathogen inoculation and sampling

[0031] Take a 100ml sterile centrifuge tube and slit it: make a 1mm x 2cm slit in the bottom (either horizontal or vertical), and cut a 6-8mm diameter hole in the side 5.5cm from the bottom. Mix 60g of dried white star beetle excrement with 3000ml of vermiculite. Pour 90ml of the mixture into the treated 100ml sterile centrifuge tube. Select plump peanut kernels (variety: Hy22) weighing approximately 1.0g and gently bury them, tip down, into the mixed substrate, leaving the top of the peanut 1cm from the top of the centrifuge tube. Add 10ml of the mixed substrate to cover the peanuts. Place the seeded centrifuge tubes in a culture rack, each rack holding 8 tubes. Place the culture rack in a water-filled culture tray and incubate at 26°C under a constant light cycle (16L:8D). Add 1L of water to the culture tray every 3 days to maintain humidity. Starting on the 4th day after planting, sample 5 seedlings every 3 days for physiological measurements. When the peanut lateral roots grew and touched the tube wall, five sorghum grains treated with pathogens were inoculated from the side holes. After 28 days of culture, samples were taken for physiological index measurement.

[0032] 1.3 Determination of various physiological indicators of peanuts

[0033] Peanut plants were carefully removed from the culture tubes, their roots rinsed clean with water, and the root surface moisture was blotted dry with absorbent paper. The plants were photographed, and various physiological parameters were measured. Stem length: The distance from the plant base to the stem tip was measured using a vernier caliper. Plants were cut at the base of the main stem and separated into the aboveground and underground parts (roots). The fresh weight of the aboveground and roots was measured using a precision electronic balance. The samples were placed in an 80°C oven. After 24 hours, the dry weight of the aboveground and roots was measured using a precision electronic balance. For diseased plants, the roots were photographed using an Epson Perfection V500 Photo scanner. Using ImageJ software, a threshold of 70 was set to automatically identify and mark the lesion area, and a threshold of 220 was set to mark the total root area. The "Analyze Particles" function in the software was used to calculate the pixel area of ​​the lesion area and the total root area. The percentage of lesion area was calculated using the formula: (pixel area of ​​lesion area / pixel area of ​​total root area) × 100%.

[0034] 1.4 Preparation of insect feces culture medium

[0035] The larval rearing of white-spotted flower beetles is described in the literature (Zhang L, Zhao T, Geng L, Zhang C, Xiang W, Zhang J, Wang X, Shu C. 2024. Characterization and evaluation of actinomycete from the Protaetia brevitarsis Larva Frass. Front Microbiol 15: 1385734.). Wheat straw was used as feed. Well-developed second-instar PB larvae were selected, washed with sterile water, and placed in an incubator at 25°C. Feces were collected after 1 hour. After drying the feces, impurities such as undigested feed were removed using an electric sieve. 135 g of feces were added to 1 L of 0.33 mol / L potassium hydroxide and heated at 100°C for 1 hour. The mixture was then centrifuged at 4000 × g for 20 min. The supernatant was collected and the pH was adjusted to 7.2-7.4 to prepare the feces extract. Take 20 ml of insect feces extract, add 80 ml of water and 2 g of agar, sterilize at 121°C for 20 minutes, and prepare insect feces extract culture medium.

[0036] 1.5 Isolation of actinomycetes

[0037] Grind 5g of fresh insect feces and add it to a conical flask. Then add 50ml of sterile deionized water and an appropriate amount of glass beads. After shaking at 250rpm for 30 minutes, let it stand for 10 minutes. Take the insect feces stock solution in the conical flask, perform a gradient dilution, and evenly spread it on the insect feces extract culture medium. After culturing at 30℃ for 7 days, observe the colony morphology and select 100 dry, compact, and wrinkled clones. Transfer them to the insect feces extract culture medium. Use a sterile spatula to scrape the spores from the surface of the culture medium, add 30% glycerol solution, and store at -80℃.

[0038] 1.6 Flat plate confrontation experiment

[0039] Activate the actinomycetes isolated from insect feces and inoculate them on the outer circle of the PDA plate. Use a 9mm punch to take a solid culture of pathogenic fungi that has been cultured for 5 days and inoculate it in the center of the PDA plate and culture it at 30°C. When the pathogenic fungi on the control plate without actinomycetes grow to the edge of the plate, observe the inhibition zone of other plates. Select the actinomycetes with obvious inhibition zones, measure the fungal diameter with a vernier caliper, and express the inhibition rate as [(pathogenic fungus diameter - inhibition zone diameter) / pathogenic fungus diameter] × 100%. Perform genome sequencing on active actinomycetes.

[0040] Analysis of growth-promoting and disease-resistance abilities of actinomycetes

[0041] When planting peanuts, add 4g of organic fertilizer and 100ml of water to 100ml of cultivation medium. Add 2×10 13 Different actinomycetes (X11-X17) with CFU spores and 2g sterilized insect feces. Two control groups were set up: (1) no actinomycetes, only 2g sterilized insect feces (PBF) were added; (2) no actinomycetes and insect feces were added (CK). 6 seedlings were planted in each treatment. Samples were taken on the 28th day after peanut planting, and various physiological indicators were analyzed. In the disease resistance experiment, the planting method was the same as above. However, 5 sorghum grains infected with S. rolfsii were added to the side holes of each cultivation tube 16 days after planting. Three control groups were set up: (1) no actinomycetes were added, only 2g sterilized insect feces (PBF) were added; (2) no actinomycetes and insect feces were added, only pathogenic bacteria were added (PO); (3) no actinomycetes, insect feces and pathogenic bacteria were added (CK). 24 seedlings were planted in each treatment. Samples were taken on the 12th day after pathogen infection, and various physiological indicators were analyzed.

[0042] 1.7 Statistical analysis

[0043] All data were independently replicated at least three times. Data were processed using GraphPad Prism software. Statistical analysis was performed using the Duncan test function in SPSS (version 19.0).

[0044] 2. Results

[0045] 2.1 Establishment and optimization of peanut pathogen control efficacy evaluation system

[0046] 2.1.1 Optimization of peanut cultivation equipment

[0047] To achieve a dense distribution of plant roots within the cultivation device, thus facilitating pathogen infection, the cultivation device was optimized. Using peanuts as the test plant, when the size of the cultivation device was not restricted, the peanut lateral roots extended and grew in opposite directions. However, when peanuts were planted in a 100mL centrifuge tube filled with vermiculite, the lateral roots were able to contact the walls of the cultivation tube and grow downward along them by the 10th day of cultivation. By the 13th day, the roots had reached a dense distribution. This suggests that the cultivation space of a 100mL centrifuge tube can meet the needs of normal peanut growth, while also allowing the roots to adhere to the wall due to space constraints.

[0048] To ensure water absorption, minimize substrate loss, and prevent outward root growth, 100mL centrifuge tubes were designed with a slit at the bottom. Peanuts were planted in these slit-bottomed tubes filled with vermiculite. The slits were 1mm wide and 2cm long. Shaking or vibrating the tubes prevented the vermiculite from falling out of the bottom slit. After 28 days of cultivation, the peanut roots were unable to grow from the bottom slit to the outside of the tube. However, when the slit was 2mm wide, the peanut roots would grow out of the slit, affecting the accuracy of subsequent root measurements. These results demonstrate that 100mL centrifuge tubes with a 1mm x 2cm bottom slit are suitable for peanut cultivation.

[0049] The inoculation position of the pathogen was further explored. In order to allow the pathogen to directly contact the roots of peanuts, two circular holes with a diameter of 6 to 8 mm were cut at different heights on the side wall of the cultivation tube, 9 cm and 5.5 cm away from the bottom respectively, called the upper hole and the middle hole. The peanuts were planted in a cultivation tube filled with vermiculite. When the lateral roots touched the tube wall, the same amount of pathogens were inoculated from each hole. After 28 days of cultivation, it was found that compared with the control without pathogen inoculation, the base of the peanut stem was infected when the pathogen was inoculated at the upper hole position. When the pathogen was inoculated at the middle hole position, the peanut roots were evenly infected, such as Figure 1 These results indicate that the mesopore position is more suitable for inoculating pathogens.

[0050] 2.1.2 Determination of peanut growth cycle and pathogen inoculation time

[0051] To determine the optimal time for pathogen inoculation and peanut sampling during peanut growth, the peanut growth cycle was analyzed. Peanuts were planted in slotted tubes filled with vermiculite, with one peanut seed planted in each tube at a depth of 1 cm from the surface of the tube substrate. The tubes were placed in tube racks, which were then placed in water-filled trays. The trays were placed in an incubator at 26°C with a constant light cycle (16 L:8 D). Water was then added to the trays every three days to ensure the substrate remained moist and aerated. Starting on the fourth day, five seedlings were collected every three days, washed, and photographed. Peanut physiological parameters, including stem length, aboveground fresh weight, aboveground dry weight, root fresh weight, and root dry weight, were measured. Results showed that stem length increased with increasing culture time. By day 10, the average stem length was 12.7 cm (Table 1), functional leaves had emerged, and lateral roots were able to contact the walls of the cultivation tubes and grow downward along them. By day 28, the average stem length was 35.66 cm. Aboveground fresh weight also increased with increasing culture time, but increased insignificantly after day 19 (Table 1). Aboveground dry weight decreased with increasing culture time, reaching a minimum of 0.692 g on day 13. It then slowly increased with increasing culture time, with no significant difference between days 13 and 22, but a significant difference compared to day 10. Both root fresh and dry weights increased with increasing culture time. Root fresh weight decreased slightly after day 19, with no significant difference in the days following day 13; root dry weight decreased slightly after day 16, with no significant difference in the days following day 10 (Table 1). Based on various physiological indicators of peanuts, the best time to inoculate with pathogens is between the 10th and 16th day of cultivation. Sampling is suitable 28 days before planting.

[0052] Table 1 Table 1 shows the growth parameters during peanut cultivation (p < 0.05).

[0053]

[0054] 2.1.3 Effects of different cultivation media on plant growth and pathogen infection

[0055] In order to clarify the controllable range of pathogen infection intensity and meet the nutritional needs of peanut growth, vermiculite was selected as a mineral and fermented mushroom residue as an organic fertilizer. The two were mixed as a cultivation matrix to simulate the natural soil microecological environment and explore the effects of different ratios of organic fertilizer on pathogens. The matrix of the control group (CK) was only vermiculite without adding organic fertilizer, that is, the organic fertilizer content was 0. In the matrix of the four experimental groups, organic fertilizer accounted for 1%, 2%, 4% and 8% of the total volume of the cultivation matrix. After mixing vermiculite with organic fertilizer, 30cm 3Place in a culture dish with a diameter of 9 cm and a height of 3 cm. Inoculate a sorghum grain that has been infected with Sclerotium rolfsii for 10 days in the center of the culture dish. Add 15 mL of sterile distilled water to the culture dish, culture at 26°C for 5 days, and observe and measure the diameter of S.rolfsii hyphae. The results showed (Table 2) that compared with the control, the addition of different concentrations of organic fertilizer significantly reduced the hyphae diameter. When the organic fertilizer concentration was 1, 2, and 4%, there was no significant difference in the hyphae diameter. When the organic matter concentration was 8%, the hyphae diameter decreased significantly. This shows that the addition of organic fertilizer significantly limits the growth of S.rolfsii. By adjusting the content of organic fertilizer, the intensity of pathogen infection can be further controlled, which can be used to evaluate the effect of chemical control.

[0056] Table 2 shows the effect of organic fertilizer concentration on the mycelium of peanut Sclerotium rolfsii pathogen (p<0.05).

[0057]

[0058]

[0059] The effects of 4% and 8% organic fertilizer content on the growth of peanut roots, stems and leaves were explored. The control group (CK) had only vermiculite as the substrate, without the addition of organic fertilizer. In the substrates of the two experimental groups, organic fertilizer accounted for 4% and 8% of the total volume of the cultivation substrate. 3 The mixed cultivation medium was placed in a cultivation tube, and a single peanut seed was planted 1 cm from the surface of the cultivation medium and incubated at a constant temperature. The results (Table 3) showed that on days 14 and 28 of cultivation, there were no significant differences in physiological parameters (stem length, root fresh weight, aboveground fresh weight, root dry weight, and aboveground dry weight) between peanuts cultured in the organic-supplemented medium and the control. At day 28 of cultivation, there was no significant difference in root length between peanuts and the control (Table 3). These results indicate that peanuts grow well in cultivation medium containing 4% to 8% organic fertilizer.

[0060] Table 3 shows the effect of organic fertilizer concentration on the growth of peanut seedlings (p<0.05)

[0061]

[0062] A comprehensive evaluation of peanut growth and pathogen infection was further conducted. The control group (CK) had only vermiculite as the substrate, with no organic fertilizer added. In the substrates of the two experimental groups, organic fertilizer accounted for 4% and 8% of the total volume of the cultivation substrate. The mixed cultivation substrate was placed in a cultivation tube for the cultivation of peanut seeds. When the stem height of the peanut seedlings was about 14 cm, plants with consistent growth were selected for pathogen inoculation. Five sorghum grains infected with S. rolfsii were added to the middle hole of each cultivation tube. The culture was continued until the 28th day, when samples were taken to measure the stem height of the peanuts and observe the disease status of the roots. The results showed (Table 4) that the degree of peanut root disease was lower in the substrate containing 8% organic fertilizer. In the substrate containing 4% organic fertilizer, the roots were severely infected, and the stem height of the plants was also significantly affected. This shows that the substrate with 4% organic fertilizer is suitable for peanut growth and is conducive to the analysis of the control effect of biocontrol bacteria.

[0063] Table 4 is a comparison of pathogen infection on peanut roots under different organic fertilizer concentrations (p < 0.05)

[0064]

[0065] 2.2 Analysis of the growth-promoting and disease-resistant effects of actinomycetes on plants

[0066] Using the plate standoff method, we compared the antibacterial effects of seven actinomycete strains and found that strain X3 had the highest inhibition rate, indicating that strain X3 was the most effective (see Table 5).

[0067] Table 5 shows the inhibition rate of 7 actinomycetes against Sclerotium sclerotiorum (p<0.05)

[0068]

[0069] The peanut pathogen prevention and control evaluation system established above was used to test the growth-promoting ability of the actinomycete strains screened by plate confrontation in the early stage. 13 cfu of actinomycete spores were mixed with 2g of insect feces, stirred with 100mL of vermiculite and 4% organic fertilizer mixture, and placed in peanut planting containers, with one peanut seed in each container. Samples without actinomycete spores and insect feces were used as controls. 28 days after peanut planting, samples were taken to measure underground fresh weight and underground dry weight. For peanut crops, due to their underground fruiting characteristics, the health of the roots has an important impact on their growth and yield. The dry weight and fresh weight of the roots are usually used as indicators to evaluate the growth and development of plant roots. The experimental results showed that compared with the blank control without the addition of actinomycetes and insect feces, the mixed application of X2, X3 and X5 strains and insect feces significantly increased the underground fresh weight of peanut roots (Table 6);

[0070] The combined application of strains X1, X2, X3, X5, X6, and X7 with insect manure significantly increased the underground dry weight of peanut roots (Table 6). Notably, the addition of insect manure alone also increased the underground fresh and dry weights of peanut roots, and the addition of actinomycetes further enhanced the growth-promoting effect. Strain X5 performed best in increasing both underground fresh and dry weights, indicating that strain X5 possesses the best growth-promoting effect.

[0071] Table 6 shows the growth promotion experiment of 7 actinomycetes

[0072]

[0073] To clarify the role of the seven strains in plant disease resistance, a peanut disease resistance test was conducted. Similar to the peanut growth promotion experiment, the actinomycete preparation was first mixed with insect feces, then stirred with vermiculite, and placed in peanut planting containers. Sixteen days after planting, when the peanut seedlings were fully grown, five sorghum kernels treated with Sclerotium sclerotiorum were added through a side hole. Twenty-eight days after planting, samples were taken and the underground dry weight was measured, and the ratio of lesion area to total root area was calculated. When peanuts are affected by diseases, roots may necrotize, which restricts root growth and ultimately affects plant growth and yield. Therefore, using lesion area and root dry weight as indicators can help assess plant root health. The experimental results (Table 7) show that compared with the control treated with pathogens alone, samples supplemented with strains X2, X3, X4, X5, X6, and X7 showed a significant decrease in lesion area ratio (Table 7). Samples supplemented with strains X1, X2, X3, X4, X5, and X7 showed a significant increase in underground dry weight (Table 7). This suggests that the addition of actinomycetes reduced disease severity, but the effects varied among different strains. Interestingly, samples supplemented with only insect feces and pathogenic bacteria had a lower lesion area ratio than the blank control, suggesting that sterilized insect feces itself may have a positive impact on plant health. The combination of insect feces and actinomycetes further enhanced crop disease resistance. Strain X4 performed best in reducing lesion area, but showed no significant difference from X5. Strain X5, on the other hand, outperformed the control in increasing underground dry weight while maintaining a smaller lesion area. These results indicate that strain X5 performs best in promoting growth and resisting disease in peanuts.

[0074] Table 7 is the disease resistance test of 7 actinomycetes

[0075]

Claims

1. A method for evaluating plant soil disease control effectiveness, comprising the following steps: (1) Take a 100ml sterilized centrifuge tube and make a slit: make a 1mm×2cm slit at the bottom of the centrifuge tube, cut a round hole with a diameter of 6-8mm at a distance of 5-6cm from the bottom on the side, put the mixed culture matrix, organic fertilizer and spores of the disease-resistant strain to be tested at the bottom, then select full and disease-free plant seeds, gently bury the tip downward into the mixed matrix, so that the top of the plant seed is 1cm away from the centrifuge tube mouth, and then cover the plant seed with the mixed matrix. Immerse the bottom of the sown centrifuge tube in water to maintain humidity and light cultivation. (2) When the lateral roots of the plants grow and touch the tube wall, sorghum grains treated with soil-borne pathogens are inoculated from the side holes and cultured for 12-18 days; (3) Evaluate the efficacy of the disease-resistant strain to be tested: a) observe the root lesions, or calculate the ratio of the root lesion area to the total root area, b) the fresh weight of the underground roots, c) the dry weight of the underground roots; the higher the fresh weight and dry weight of the underground roots, the better the growth promotion effect of the disease-resistant strain to be tested on the plant seedlings; the smaller the root lesion area or the lower the ratio of the root lesion area to the total root area, the higher the dry weight of the underground roots, the higher the resistance of the disease-resistant strain to soil-borne diseases.

2. The method according to claim 1, wherein step (1) further comprises a blank control culture of the disease-resistant strain to be tested, in which spores of the disease-resistant strain to be tested are not added to the mixed matrix; and step (3) further comprises a comparison of the root lesion area between the treatment group and the control group.

3. The method according to claim 1, wherein the culture in step (1) is a 16L:8D culture at 26°C with a constant photoperiod.

4. The method according to claim 1, wherein the humidity is maintained in step (1) by inserting the centrifuge tube into a culture rack, placing the culture rack into a culture tray containing water, and regularly adding water to the culture tray to maintain the culture humidity.

5. The method according to claim 1, wherein the amount of culture medium, organic fertilizer and spores of the disease-resistant strain to be tested in the mixed matrix is: 100 ml: 4 g: 2×10 13 CFU spores.

6. The method according to claim 5, wherein the culture medium is vermiculite and the organic fertilizer is fermented mushroom residue.

7. The method according to claim 6, wherein the inoculation time is 10-16 days after the plant seeds are planted, and the evaluation sampling time is 28 days after the plant seeds are planted.

8. The method according to claim 7, wherein the pathogen is Sclerotium rolfsii Sacc., the disease-resistant bacteria to be tested are actinomycetes, and the plant is peanut.

9. The method according to claim 8, wherein the actinomycetes are derived from feces of white-spotted flower beetle larvae, and the mixed matrix further comprises sterilized white-spotted flower beetle feces.

10. The method according to claim 1, wherein the ratio of the root lesion area to the total root area is calculated by: photographing the root system using an Epson Perfection V500 Photo scanner, and using ImageJ software with a threshold set to 70 to automatically identify and mark the lesion area; Set the threshold to 220 and mark the total root area. Use the "Analyze Particles" function of the software to calculate the pixel areas of the lesion area and the total root area. The percentage of lesion area was calculated by the formula: (pixel area of ​​lesion area / pixel area of ​​total root area) × 100%.

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