Microbial strain streptomyces microflavus satc2601 and its use

Abstract

The application discloses a micro-white Streptomyces avermitilis strain SaTC2601 and application thereof, and belongs to the field of biological control. The micro-white Streptomyces avermitilis strain SaTC2601 is isolated from the excrement of white star flower chafer (WSFC) larvae, and has a preservation number of CGMCC No.33741. The strain has a good prevention and treatment effect on peanut white thread blight caused by Sclerotium rolfsii on peanut roots, and also has a prevention and treatment effect on soil-borne pathogenic bacteria such as Fusarium graminearum, Fusarium solani, Fusarium moniliforme, Fusarium oxysporum, Rhizoctonia solani and Streptomyces scabiei. Further, the strain SaTC2601 also has a good growth promotion effect. The strain becomes a valuable resource for new drug development and agricultural disease prevention and control, and is expected to play a huge role in soil disease prevention and control.

Description

Technical Field

[0001] This invention relates to the field of biological control, and in particular to a strain of Streptomyces microleucovorum SaTC2601 and its application in promoting growth and controlling soil-borne pathogens. Background Technology

[0002] Plant diseases are one of the major threats to agricultural production, leading to reduced crop yields, decreased quality, and even total crop failure. Diseases are classified into fungal, bacterial, viral, and nematode types (Gai YP, Wang HK. 2024. Plant Disease: A Growing Threat to Global Food Security. Agronomy-Basel 14.). Soil-borne diseases are caused by pathogens in the soil, commonly including Fusarium, Phytophthora, and Rhizoctonia. These diseases invade plants through the root system, causing symptoms such as root rot, wilting, and damping-off, severely impacting crop growth. Because soil provides a suitable colonization environment for some pathogens and limits the delivery and action of fungicides, the control of soil-borne diseases has become a challenge in agricultural production (Bakker P, Berendsen RL, Van Pelt JA, 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. MolPlant 13:1394-1401.).

[0003] Screening effective fungicides is a crucial step in the current control of soil-borne diseases. Unlike diseases affecting foliage and other aerial tissues, the occurrence of soil-borne diseases, the application of fungicides, and the quantitative assessment of their control effects are all challenging due to soil barriers, limiting the screening and application of fungicides specifically for soil-borne diseases. Therefore, establishing reproducible and quantifiable screening models for soil-borne fungicides is a vital requirement for current soil-borne disease control efforts (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 beneficial agricultural microorganisms that can colonize in soil and rhizosphere for soil-borne disease control is an effective strategy with advantages such as environmental friendliness 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 belye inhibits pathogen growth 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 pathogen growth 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 their resistance to white mold (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 Aconitumcarmichaelii Debx. Front Microbiol 13:1059549.).Bio-organic fertilizer containing Trichoderma can improve soil microbial communities and effectively inhibit peanut white mold disease (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, capable of effectively colonizing the soil. Actinomycetes can produce a variety of metabolites, including insecticides and fungicides, which are widely used in human health, animal husbandry, and agricultural pest and disease 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.). Developing highly effective soil-borne disease control products by leveraging the high bactericidal and disease-resistant properties of actinomycetes and their ability to colonize in soil is a feasible approach. Actinomycetes hold immense potential as green 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. Currently, Novozymes' *Streptomyces lydicus* has achieved success (Actinovate). (Novozymes BioAg Inc.)

[0005] Peanuts are an important crop. Due to their characteristic of forming pods 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 microbiome inoculation 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, Okori P, Mwololo J, Ajeigbe H, Njuguna-Mungai E, Muricho G, Akpo E, Gichohi-Wainaina WN, Variath MT, Radhakrishnan T, Dobariya KL, Bera SK, Rathnakumar AL, Manivannanan N, Vasanthi RP, Kumar MVN, Varshney RK. 2020. Advances in Crop Improvement and Delivery Research for Nutritional Quality and Health Benefits of Groundnut (Arachis hypogaea L.). Front Plant Sci 11:29. In recent years, white mold disease of peanut caused by Sclerotium rolfsii has posed a great challenge to peanut production. Summary of the Invention

[0006] This invention provides a Streptomyces albidoflavus strain, SaTC2601, which has a good control effect on peanut white mold disease caused by Sclerotium rolfsii on peanut roots. It also has a control effect on soil-borne pathogens such as Fusarium graminearum, Fusarium solani, Fusarium moniliforme, Fusarium oxysporum, Rhizoctonia solani, and Streptomyces scabies. Furthermore, this strain SaTC2601 also has a good growth-promoting effect.

[0007] Streptomyces albidoflavus strain SaTC2601, with accession number CGMCC No. 33741.

[0008] Application of strain SaTC2601 in promoting plant growth and controlling soil-borne pathogens.

[0009] The soil-borne pathogens mentioned are Sclerotium rolfsii, Fusarium graminearum, Fusarium solani, Fusarium moniliforme, Fusarium oxysporum, Rhizoctonia solani, and Streptomyces scabies.

[0010] The application described involves adding spores of strain SaTC2601 to plant organic fertilizer and applying it to the plant roots.

[0011] The plant organic fertilizer contains sterilized excrement from the white star flower beetle.

[0012] The plant in question is a peanut.

[0013] This application focuses on peanuts and the important soil-borne pathogen *Sclerotium guillezei*, and utilizes a precise and controllable cultivation system to effectively obtain actinomycetes that promote disease resistance and growth in peanuts.

[0014] This application utilizes the excrement of white-spotted flower beetle larvae as a sample to isolate actinomycetes that can colonize straw and its derived soil organic matter for further fungicide development, representing a novel and acceptable strategy for controlling soil-borne diseases (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. FrontMicrobiol 15:1385734.). White-spotted flower beetle larvae feed on fermented and decaying straw, producing excrement rich in actinomycetes and humic substances similar to soil humic acid. Strains obtained from white-spotted flower beetle larval excrement have a higher chance of colonizing and functioning in the soil; therefore, using excrement as the source of strain isolation in this application is a wise choice.

[0015] Using the soil disease control efficacy evaluation system established in this application, genes with biocontrol functions were obtained from seven active bacterial strains. Their genomes were submitted to the antiSMASH website for functional annotation of the strain genome drafts. The results showed that 16 secondary metabolite gene clusters with a similarity greater than 70% were predicted in the seven strains. Strain SaTC2601 contained the most secondary metabolite gene clusters, followed by strain X4. These secondary metabolites exhibit broad-spectrum antibacterial and antifungal activities, suggesting that the antibacterial activity of these strains is contributed by the products synthesized from the aforementioned gene clusters. Pot tests showed that strain SaTC2601 had the best activity. The genome of strain SaTC2601 contained TRPI, which is related to tryptophan synthesis; tam and tamB, which are closely related to IAA synthesis; and ahpF, which is related to peroxidase—genes not found in the other six strains. The presence of more disease resistance and growth-promoting genes in the SaTC2601 genome may explain its superior activity.

[0016] This application plays an important role in further screening highly active actinomycete resources that can be used for the development of products for the control of soil-borne diseases. At the same time, the SaTC2601 strain discovered in this application has become a valuable resource for new drug development and agricultural disease control, and is expected to play a significant role in the control of soil diseases.

[0017] Preservation information for Streptomyces albidoflavus strain SaTC2601:

[0018] Strain classification and nomenclature: Streptomyces albidoflavus

[0019] Strain accession number: CGMCC No. 33741

[0020] Address: General Microbiology Center, China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China

[0021] Deposit date: March 7, 2025 Attached Figure Description

[0022] Figure 1 Phylogenetic analysis of seven actinomycete strains (X5 being strain SaTC2601). Detailed Implementation

[0023] The biological materials used below are all stored in our laboratory and can be publicly distributed.

[0024] 1. Method

[0025] 1.1 Activation and inoculation of Sclerotium rolfsii

[0026] Sclerotium rolfsii was obtained from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. Sclerotium rolfsii was inoculated into PDA medium (potato 200 g / L, glucose 20 g / L, agar 20 g / L) and incubated at 26°C for 5 days. 100 g of commercially available sorghum grains were taken, washed, and boiled in boiling water for 10 minutes. Excess water was removed from the boiled sorghum grains, and they were placed in 500 ml Erlenmeyer flasks and autoclaved at 121°C for 15 minutes. Five Sclerotium rolfsii mycelial cakes were taken from the edge of a PDA plate using a 5 mm punch and placed into the sterilized sorghum grain Erlenmeyer flasks. The flasks were incubated statically at 26°C. The flasks were shaken once daily to loosen the sorghum grains and prevent clumping or spoilage due to localized high temperatures. After 9 days of incubation, the mixture was shaken well and ready for inoculation of peanuts.

[0027] 1.2 Preparation of insect excrement culture medium

[0028] The rearing of white-spotted flower beetle larvae 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.), with wheat straw as the feed. Well-developed second-instar PB larvae were selected, washed with sterile water, and placed in an incubator at 25℃. Feces were collected after 1 hour. The feces were dried and undigested feed and other impurities were removed using an electric sieve. 135g of feces was added to 1L of 0.33mol / L potassium hydroxide and heated at 100℃ for 1 hour. The mixture was then centrifuged at 4000×g for 20min, and the supernatant was collected. The pH was adjusted to 7.2-7.4 to prepare the feces extract. Take 20ml of insect excrement extract, add 80ml of water and 2g of agar, sterilize at 121℃ for 20 minutes to prepare the adult insect excrement extract culture medium.

[0029] 1.3 Isolation of Actinomycetes

[0030] Grind 5g of fresh insect excrement into a fine powder and add it to an Erlenmeyer flask. Add 50ml of sterile deionized water and an appropriate amount of glass beads. Shake at 250rpm for 30 minutes, then let stand for 10 minutes. Take the undiluted insect excrement from the Erlenmeyer flask and perform serial dilutions, then spread it evenly on insect excrement extract medium. After incubating at 30℃ for 7 days, observe the colony morphology. Select 100 dry, tightly packed, and wrinkled clones and transfer them to insect excrement extract medium. Spores on the surface of the medium are scraped off with a sterile spatula, and 30% glycerol solution is added. Store at -80℃.

[0031] 1.4 Flat Plate Standoff Experiment

[0032] Activated actinomycetes isolated from insect excrement were inoculated onto the outer ring of a PDA plate. Solid cultures of pathogenic fungi cultured for 5 days were taken using a 9mm punch and inoculated into the center of the PDA plate, then incubated at 30°C. When the pathogenic fungi on the control plate (without actinomycetes) grew to the edge of the plate, the inhibition zones on other plates were observed. Actinomycetes with clearly defined inhibition zones were selected, and their diameter was measured using calipers. The inhibition rate was expressed as [(pathogenic fungal diameter - inhibition zone diameter) / pathogenic fungal diameter] × 100%. The genomes of the viable actinomycetes were sequenced.

[0033] 1.5 Peanut planting, pathogen inoculation and sampling

[0034] Prepare 100ml sterile centrifuge tubes by making a 1mm x 2cm slit at the bottom and a 6-8mm diameter hole cut on the side 5.5cm from the bottom. Mix 60g of dried white-spotted beetle excrement with 3000ml of vermiculite, and transfer 90ml of this mixture to the prepared 100ml sterile centrifuge tube. Select a plump peanut kernel (variety: Hy22) weighing approximately 1g, gently bury it tip-down in the mixed substrate, ensuring the top of the peanut is 1cm from the centrifuge tube opening. Add another 10ml of mixed substrate to cover the peanut. Place the centrifuge tubes in a culture rack, holding 8 tubes per rack. Place the rack in a water-containing culture tray and incubate at 26℃ with a constant photoperiod (16L:8D). Add 1L of water to the culture tray every 3 days to maintain humidity. Starting from the 4th day after peanut planting, take 5 seedlings every 3 days for physiological parameter measurements. When the peanut lateral roots grow to touch the tube wall, inoculate 5 sorghum grains treated with pathogens through the side hole. After 28 days of cultivation, take samples for physiological index measurement.

[0035] 1.6 Analysis of Actinomycete Growth Promotion and Disease Resistance Ability

[0036] Peanut planting methods are described in section 1.5. When planting, add 4g of organic fertilizer and 100ml of water to 100ml of cultivation substrate. Add 2×10 13 Different actinomycetes from CFU spores and 2g of sterilized insect excrement. Two control groups were set up: (1) no actinomycetes, only 2g of sterilized insect excrement (PBF); (2) no actinomycetes and insect excrement (CK). Six 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 grains of sorghum infected by S. rolfsii were added to the side hole of each cultivation tube 16 days after planting. Three control groups were set up: (1) no actinomycetes, only 2g of sterilized insect excrement (PBF); (2) no actinomycetes and insect excrement, only pathogenic bacteria (PO); (3) no actinomycetes, insect excrement, and pathogenic bacteria (CK). Eighteen seedlings were planted in each treatment. Samples were taken on the 12th day after pathogen infection, and various physiological indicators were analyzed.

[0037] 1.7 Determination of various physiological indicators of peanuts

[0038] Carefully remove the peanut plants from the culture tubes, rinse the roots with water, and dry the surface moisture with absorbent paper. Photograph the plants and measure various physiological indicators. Stem length: Measure the distance from the base of the plant to the top of the stem using calipers. Cut the plant at the base of the main stem, separating it into the above-ground part and the underground part (roots). Measure the fresh weight of the above-ground part and the fresh weight of the roots using a precision electronic balance. Place the samples in an 80℃ oven for 24 hours, then measure the dry weight of the above-ground part and the dry weight of the roots using a precision electronic balance. For diseased plants, photograph the root system using an Epson Perfection V500 Photo scanner. Using ImageJ software, set the threshold to 70 to automatically identify and mark the lesion area; set the threshold to 220 to mark the total root area. Use the software's "Analyze Particles" function to calculate the pixel area of ​​the lesion area and the total root area, respectively. Calculate the percentage of lesion area using the formula: (pixel area of ​​lesion area / pixel area of ​​total root area) × 100%.

[0039] 1.8 Statistical Analysis

[0040] All data were replicated at least three times. Data processing was performed using GraphPad Prism software. Statistical analysis was performed using Duncan's test function in SPSS software (version 19.0).

[0041] 2. Results

[0042] 2.1 Isolation and Identification of Actinomycetes with Biocontrol Characteristics

[0043] One hundred actinomycetes were isolated from the feces of white-spotted flower beetle (WSFC) larvae. To target peanut white mold disease, this application used the plate confrontation method to screen for strains active against *Sclerotium rolfsii*. The results showed that seven strains exhibited antagonistic activity. The inhibition rate results (Table 1) showed that the inhibition rates of the seven strains against *S. rolfsii* ranged from 31.28% to 44.56%, with strain X3 showing the highest inhibition rate at 44.56%. Statistical analysis indicated that the inhibition rate of strain X3 was significantly different from the other strains. This demonstrates that all seven strains exhibited some antibacterial activity against *S. rolfsii*, with X3 showing the best performance.

[0044] Table 1 shows the inhibition rates of 7 actinomycetes against *Sclerotium riberi* (p<0.05).

[0045]

[0046] Genome sequencing and phylogenetic analysis were performed on the seven viable strains. Based on the amino acid sequence-based tree constructed using CVTree, they were found to belong to five different branches. Six strains (X1, X3, X4, X5, X6, X7) belonged to *Streptomyces albidoflavus* and related species, while one strain (X2) belonged to *Nocardiopsis alba* and related species. See [link to relevant documentation]. Figure 1 As shown.

[0047] 2.2 Analysis of the effects of actinomycetes on plant growth promotion and disease resistance

[0048] To clarify the effects of the seven selected bacterial strains on plant growth and their biocontrol function in practical applications, the established peanut pathogen efficacy evaluation system was used to test the growth-promoting ability of these strains on peanuts. (2×10⁶) 13 CFU of actinomycete spores were mixed with 2g of insect excrement, then stirred with 100mL of vermiculite and a 4% organic fertilizer mixture, and placed in peanut planting containers, with one peanut seed in each container. Samples without actinomycete spores and insect excrement served as controls. Sampling and underground fresh and dry weights were measured 28 days after peanut planting. For peanut crops, due to their underground seed-bearing characteristics, root health has a significant impact on their growth and yield. Root dry and fresh weights are commonly used as indicators to assess plant root growth and development. Experimental results showed that, compared to the blank control without actinomycete spores and insect excrement, the mixed application of strains X2, X3, and X5 with insect excrement significantly increased the underground fresh weight of peanut roots (Table 2); the mixed application of strains X1, X2, X3, X5, X6, and X7 with insect excrement significantly increased the underground dry weight of peanut roots (Table 2). It is worth noting that adding insect excrement alone also promoted the underground fresh weight and dry weight of peanut roots. However, the growth-promoting effect was more obvious after adding actinomycetes. Among them, strain X5 performed best in promoting underground fresh weight and dry weight, indicating that strain X5 has the best growth-promoting effect.

[0049] Table 2 shows the growth-promoting experiments of 7 actinomycete strains.

[0050]

[0051] To clarify the function of the seven bacterial strains in enhancing plant disease resistance, a peanut disease resistance experiment was conducted. Similar to the peanut growth-promoting experiment, the actinomycete preparation and insect excrement were mixed, then stirred with vermiculite, and placed in the peanut planting container. Seventeen days after planting, when the peanut seedlings were uniform, five uniform sorghum grains treated with *Sclerotium sclerotium* were added through a side hole. Twenty-eight days after planting, samples were taken to measure the underground dry weight, and the ratio of lesion area to total root area was calculated. When peanuts are affected by disease, root necrosis may occur, which will restrict root growth and ultimately affect plant growth and yield. Therefore, using lesion area and root dry weight as indicators can help assess the health of the plant root system. The experimental results (Table 3) showed that compared with the control group that only added pathogenic bacteria, the samples with the addition of strains X2, X3, X4, X5, X6, and X7 all showed a significantly reduced lesion area ratio (Table 3); the samples with the addition of strains X1, X2, X3, X4, X5, and X7 all showed a significantly increased underground dry weight (Table 3). This indicates that the addition of actinomycetes reduced the severity of the disease, but the effects varied among different strains. Interestingly, the lesion area ratio of the sample with only insect excrement and pathogens was lower than that of the control, suggesting that sterilized insect excrement itself may have a positive impact on plant health. The combination of insect excrement and actinomycetes further enhanced crop disease resistance. Strain X4 performed best in reducing lesion area, but there was no significant difference compared to X5; while strain X5 was superior to the control in both increasing underground dry weight and maintaining a smaller lesion area. These results suggest that strain X5 performed best in promoting growth and disease resistance in peanuts.

[0052] Table 3 shows the disease resistance experiments of 7 actinomycete strains.

[0053]

[0054] Further investigation was conducted on the broad-spectrum activity of strain X5. The antagonistic effect of strain X5 against various plant pathogens was tested using a plate confrontation experiment. The results (Table 4) showed that strain X5 exhibited antagonistic effects against pathogens such as *Fusarium graminearum*, *Fusarium solani*, *Fusarium moniliforme*, *Fusarium oxysporum*, *Rhizoctonia solani*, and *Streptomyces cabie*. The inhibition rates were all above 25%, indicating that strain X5 has good application prospects for controlling plant diseases caused by these pathogens. Strain X5 was named *Streptomyces albidoflavus* strain SaTC2601, with accession number CGMCC No. 33741.

[0055] Table 4 shows the inhibition rate (%) of strain SaTC2601 against different plant pathogens (p<0.05).

[0056]

Claims

1. Streptomyces leucovorum ( Streptomyces albidoflavus The strain SaTC2601, with accession number CGMCC No. 33741, is used.

2. The application of the *Streptomyces microcyticus* strain SaTC2601 according to claim 1 in promoting plant growth and controlling soil-borne pathogens, wherein the plant is peanut; and the soil-borne pathogen is *Sclerotium sclerotiorum* (…). Sclerotium rolfsii Fusarium graminearum ( ), Fusarium graminearum ( Fusarium graminearum Fusarium solani () Fusarium solani Fusarium moniliforme () Fusarium moniliforme Fusarium oxysporum ( Fusarium oxysporum Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani ) and Streptomyces scabies ( Streptomyces scabies ).

3. The application according to claim 2 is to add the spores of Streptomyces microcyticus strain SaTC2601 to plant organic fertilizer and apply it to the roots of plants.

4. The application according to claim 3, wherein the plant organic fertilizer contains sterilized excrement of the white star beetle.

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