Streptomyces albidoflavus strain SaTC2601 and application thereof

Abstract

The invention discloses a streptomyces albidoflavus strain SaTC2601 and application thereof, and belongs to the field of biological control. According to the invention, a streptomyces albidoflavus strain SaTC2601 is separated from faeces of larvae of WSFC, and the preservation number of the streptomyces albidoflavus strain SaTC2601 is CGMCC (China General Microbiological Culture Collection Center) No.33741. The strain SaTC2601 has a very good prevention and treatment effect on peanut southern blight caused by sclerotium rolfsii at peanut roots, 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 scabies, and further, the strain SaTC2601 also has a very good growth promoting effect. The strain becomes a precious resource for new drug development and agricultural disease prevention and control, and is expected to play a huge role in the aspect of soil disease prevention and control.

Description

Technical Field

[0001] The present invention relates to the field of biological control, in particular to a Streptomyces albiflorus strain SaTC2601 and its application in promoting growth and controlling soil-borne pathogens. 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.). Leveraging the highly effective bactericidal and disease-resistant properties of actinomycetes, as well as their ability to colonize in soil, is a viable approach to developing highly effective soil-borne disease control products. 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. Currently, Streptomyces lydicus, launched by Novozymes in the United States, has achieved success (Actinovate lydicus). , Novozymes BioAg Inc.).

[0005] 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, 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 rot caused by Sclerotium rolfsii has posed a great challenge to peanut production. Summary of the Invention

[0006] The present invention provides a Streptomyces albidoflavus strain SaTC2601, which has a good control effect on peanut white rot caused by Sclerotium rolfsii on the roots of peanuts, and 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, the strain SaTC2601 has a good growth-promoting effect.

[0007] Streptomyces albidoflavus strain SaTC2601, whose deposit number is CGMCC No.33741.

[0008] The strain SaTC2601 is used in promoting plant growth and preventing and controlling soil-borne pathogens.

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

[0010] The application is to add the spores of the strain SaTC2601 into plant organic fertilizer and apply it to the roots of plants.

[0011] The plant organic fertilizer contains sterilized feces of white-spotted scarab beetles.

[0012] The plant is peanut.

[0013] This application takes peanuts and the important soil-borne pathogen Sclerotium uniformis as the objects, and uses a precise and controllable cultivation system to effectively obtain actinomycetes that are disease-resistant and growth-promoting for peanuts.

[0014] This application uses the feces 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, which is a new 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 actinomycetes from the Protaetia brevitarsis Larva Frass. Front Microbiol 15:1385734.). White-spotted flower beetle larvae feed on fermented and rotten straw, and the insect feces they produce are rich in actinomycetes and humus similar to soil humic acid. Strains obtained from white-spotted flower beetle larvae feces have a higher chance of colonizing and exerting their effects in the soil. Therefore, using insect feces as a source for strain isolation in this application is a wise choice.

[0015] Using the soil disease prevention and control effectiveness evaluation system established in this application, genes with biocontrol functions were obtained for 7 active strains, and their genomes were submitted to the antiSMASH website for functional annotation of the draft genomes of the strains. The results showed that 16 secondary metabolite gene clusters with a similarity greater than 70% were predicted in the 7 strains. The SaTC2601 strain contained the most secondary metabolite gene clusters, followed by the X4 strain. These secondary metabolites have a wide range of antibacterial and antifungal activities, and it is speculated that the antibacterial activity of these strains is contributed by the products synthesized by the above-mentioned gene clusters. Pot tests showed that the SaTC2601 strain had the best activity. TRPI related to tryptophan synthesis, tam and tamB closely related to IAA synthesis, and ahpF related to peroxidase were found in the genome of the SaTC2601 strain, which are not found in the other 6 strains. The SaTC2601 genome contains more genes related to disease resistance and growth promotion, which may be the reason for its best activity.

[0016] This application plays 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 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 huge role in soil disease control.

[0017] Deposit information of Streptomyces albidoflavus strain SaTC2601:

[0018] Strain classification and nomenclature: Streptomyces albidoflavus

[0019] Strain deposit number: CGMCC No.33741

[0020] Deposit address: Center for General Microbiology, China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China

[0021] Deposit date: March 7, 2025 BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Phylogenetic analysis of seven actinomycete strains. (X5 is the SaTC2601 strain) DETAILED DESCRIPTION

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

[0024] 1. Methods

[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 (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.

[0027] 1.2 Preparation of insect feces culture medium

[0028] Larval rearing of white-spotted flower beetles was carried out as 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 the 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.

[0029] 1.3 Isolation of actinomycetes

[0030] 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℃.

[0031] 1.4 Flat plate confrontation experiment

[0032] 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.

[0033] 1.5 Peanut planting, pathogen inoculation and sampling

[0034] Prepare a 100ml sterile centrifuge tube by slitting it: create a 1mm x 2cm slit in the bottom of the tube, and cut a 6-8mm diameter hole in the side 5.5cm from the bottom. Mix 60g of dried white-star beetle feces with 3000ml of vermiculite, and transfer 90ml of the mixture to the treated 100ml sterile centrifuge tube. Select plump peanut kernels (Hy22 variety), approximately 1.0g in weight, and gently bury them, tip down, into the mixed substrate, leaving the top of the peanut 1cm from the tube opening. Add 10ml of the mixed substrate to cover the peanuts. Place the seeded centrifuge tubes in a culture rack, each holding 8 tubes. Place the 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 tray every 3 days to maintain humidity. Starting on the fourth day of 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.

[0035] 1.6 Analysis of growth promotion and disease resistance of actinomycetes

[0036] See 1.5 for peanut planting method. When planting, add 4g organic fertilizer and 100ml water to 100ml of cultivation medium. Add 2×10 13 CFU spores of different actinomycetes 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 pathogens were added (PO); (3) no actinomycetes, insect feces and pathogens were added (CK). 18 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] Peanut plants were carefully removed from the culture tubes, their roots rinsed with water, and the surface moisture of the roots 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 infected plants, 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; 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%.

[0039] 1.8 Statistical analysis

[0040] 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).

[0041] 2. Results

[0042] 2.1 Isolation and identification of actinomycetes with biocontrol properties

[0043] 100 strains of actinomycetes were isolated from the feces of the larvae of the white star flower beetle (WSFC). For peanut white rot, this application used the plate confrontation method to screen strains that were active against Sclerotium rolfsii. The results showed that 7 strains produced antagonistic effects. The inhibition rate results showed (Table 1) that the inhibition rates of the 7 strains against S.rolfsii ranged from 31.28% to 44.56%, and X3 had the highest inhibition rate, reaching 44.56%. Statistical analysis showed that the inhibition rate of the X3 strain was significantly different from that of the other strains. This shows that these 7 strains all showed certain antibacterial activity against S.rolfsii, and X3 performed best.

[0044] Table 1 shows the inhibition rate of 7 actinomycetes against Sclerotium sclerotiorum (p<0.05)

[0045]

[0046] The genome sequencing and phylogenetic tree analysis of these seven strains with good activity were performed. According to the results of the amino acid sequence-based tree construction on the CVTree website, they were found to be divided into five different branches. Among them, six strains (strains X1, X3, X4, X5, X6, and X7) were Streptomyces albidoflavus and related species, and one strain (strain X2) was Nocardiopsis alba and related species. Figure 1 shown.

[0047] 2.2 Analysis of the growth-promoting and disease-resistant effects of actinomycetes on plants

[0048] In order to clarify the effects of the 7 strains screened on plant growth and their biocontrol functions in actual applications, the peanut pathogen control efficacy evaluation system established above was used to test the growth-promoting ability of these strains on peanuts. 13 Actinomycete spores containing 100 cfu (1000 micrograms) of actinomycete spores were mixed with 2 g of insect excrement and then stirred with 100 mL of a mixture of vermiculite and 4% organic fertilizer. The mixture was then placed in peanut planting containers, with one peanut seed placed in each container. Samples without actinomycete spores or insect excrement served as controls. Peanut samples were collected 28 days after planting to measure underground fresh and dry weights. For peanut crops, root health has a significant impact on their growth and yield due to their underground fruiting nature. Root dry and fresh weights are commonly used as indicators of plant root growth and development. The results showed that, compared to the blank control without actinomycete and insect excrement, the combination of strains X2, X3, and X5 with insect excrement significantly increased the underground fresh weight of peanut roots (Table 2). The combination 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 the addition of insect feces alone also promoted the underground fresh weight and dry weight of peanut roots. After adding actinomycetes, the growth-promoting effect was more obvious. Among them, the X5 strain performed best in promoting underground fresh weight and dry weight, indicating that the X5 strain has the best growth-promoting effect.

[0049] Table 2 shows the growth promotion experiments of 7 actinomycetes.

[0050]

[0051] 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. Seventeen 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 3) show that compared with the control treated with pathogens alone, the lesion area ratio was significantly reduced in samples treated with strains X2, X3, X4, X5, X6, and X7 (Table 3). The underground dry weight of samples treated with strains X1, X2, X3, X4, X5, and X7 was significantly increased (Table 3). 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.

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

[0053]

[0054] The broad-spectrum activity of the X5 strain was further studied. The antagonistic effect of the X5 strain on a variety of plant pathogens was tested through a plate standoff experiment. The results showed (Table 4) that the X5 strain had antagonistic effects on pathogens such as Fusarium graminearum, Fusarium solani, Fusarium moniliforme, Fusarium oxysporum, Rhizoctonia solani, and Streptomyces scabies. The inhibition rates were all above 25%, indicating that the X5 strain has good application prospects in preventing and controlling plant diseases caused by these pathogens. The X5 strain was named Streptomyces albidoflavus strain SaTC2601, and its deposit number is CGMCC No. 33741.

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

[0056]

Claims

1. Streptomyces albidoflavus strain SaTC2601, whose deposit number is CGMCC No.33741.

2. Use of the strain SaTC2601 according to claim 1 in promoting plant growth and controlling soil-borne pathogens.

3. The use according to claim 2, wherein the soil-borne pathogens are Sclerotium rolfsii Sacc., Fusarium graminearum, Fusarium solani, Fusarium moniliforme, Fusarium oxysporum, Rhizoctonia solani and Streptomyces scabies.

4. The method according to claim 2, wherein the spores of the bacterial strain SaTC2601 are added to plant organic fertilizer and applied to plant roots.

5. The use according to claim 2, wherein the plant organic fertilizer comprises sterilized feces of the white-spotted flower beetle.

6. The use according to claim 2, wherein the plant is peanut.

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