Application of Gliocladium mucilaginosum RS44 and composite flora of Gliocladium mucilaginosum RS44 and Rahnella RS86 in prevention and treatment of corn root rot
By using a complex microbial community of *Aureobasidium* RS44 and *Laenia* RS86 to synergistically inhibit *Fusarium graminearum*, the problems of drug resistance and environmental pollution associated with chemical control of maize root rot have been solved, achieving effective control and growth promotion of maize root rot.
Patent Information
- Application Number
- CN202610533640.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-22
- Publication Date
- 2026-05-19
AI Technical Summary
Existing chemical agents for controlling maize root rot have problems such as drug resistance, environmental pollution, and damage to soil microbial structure, making it difficult to meet the requirements for safe and efficient control. Existing biocontrol strains have poor efficacy in controlling maize root rot or cannot promote maize growth.
A compound microbial community of *Fusarium graminearum* RS44 and *Larnella gracilis* RS86 was used to inhibit the growth of *Fusarium graminearum* and promote maize growth through synergistic effects. The concentration of *Fusarium graminearum* RS44 spores in the compound microbial agent was ≥1×106 spores/mL, and the number of viable *Larnella gracilis* RS86 cells was ≥1×106 CFU/mL.
It significantly reduced the incidence index of maize root rot by 26.32%, promoted maize growth, significantly increased the fresh weight of the aboveground parts and roots, and the complex microbial community was stable in the soil, with better effects than a single strain.
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Figure CN122060602A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biocontrol technology, and in particular to the application of *Cladosporium spp.* RS44 and its complex with *Laenia spp.* RS86 in the control of maize root rot. Background Technology
[0002] In corn production, root rot is a widespread and serious soil-borne disease, mainly caused by Fusarium graminearum (…). Fusarium graminearum This disease is caused by various pathogenic fungi, including [list of fungi]. It typically occurs from the seedling stage to the grain-filling stage of corn. Affected plants exhibit browning and rotting of the roots, a reduction in secondary roots, and stunted growth; in severe cases, it can lead to lodging and even death. Root rot not only directly reduces corn yield but also lowers grain quality and may be accompanied by fungal toxin contamination.
[0003] Currently, the control of maize root rot still relies primarily on chemical treatments, such as seed dressing or root drenching with fungicides. However, long-term, irrational use of chemical pesticides can easily lead to drug resistance in pathogens, resulting in a gradual decline in control effectiveness. Simultaneously, chemical residues in the soil may inhibit non-target microorganisms, disrupting the soil microbial community structure and consequently affecting soil health and crop growth. Furthermore, some chemical pesticides pose environmental pollution and food safety risks. Therefore, relying solely on chemical control methods is insufficient to meet the current practical needs of maize root rot prevention and control, necessitating the exploration of safer, more efficient, and environmentally friendly alternative technologies.
[0004] Biological control, as a strategy that utilizes beneficial microorganisms and their metabolites to inhibit plant pathogens and enhance plant resistance, has attracted widespread attention due to its environmental friendliness and diverse mechanisms of action. In recent years, various bacterial and fungal biocontrol microorganisms have been reported to have potential applications in controlling soil-borne diseases of maize, such as inhibiting pathogen infection through competition for nutrients and ecological niches, production of antagonistic substances, and induction of systemic resistance in plants. However, existing strains have shown poor efficacy in controlling maize root rot or can only control the disease without promoting maize growth. Summary of the Invention
[0005] To address the aforementioned problems, this invention provides the application of *Strombyx mori* RS44 and its complex with *Laenia natans* RS86 in the control of maize root rot. The *Strombyx mori* RS44 provided by this invention not only exhibits good control effects against maize root rot but also promotes maize growth.
[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a strain of *Cladosporium* (…). Clonostachys sp.) RS44, accession number CGMCCNo.42448.
[0007] This invention provides a biocontrol agent, the active ingredient of which includes the *Stipa oryzae* RS44 described in the above technical solution.
[0008] Preferably, the spore concentration of *Strombyx mori* RS44 in the biocontrol agent is ≥1×10⁻⁶. 6 per mL.
[0009] This invention provides a complex microbial community, including Larynella ( Rahnella The above-mentioned technical solution describes both *R. sp.* RS86 and *R. 44*, with the preservation number of *R. sp.* RS86 being CCTCC NO: M 20242043.
[0010] Preferably, the live spores of *Laenia lancifolium* RS86 and *Synthia spp.* RS44 are 1-2 CFU: 1-2 spores.
[0011] This invention provides a compound microbial agent, the effective components of which include the compound microbial community described in the above technical solution.
[0012] Preferably, the spore concentration of *Strombyx mori* RS44 in the compound microbial agent is ≥1×10⁻⁶. 6 The viable count of *Raenella esculenta* RS86 is ≥1×10⁻⁶ / mL. 6 CFU / mL.
[0013] This invention provides the application of the above-described *Schynostemma* RS44, the above-described biocontrol agent, the above-described compound microbial community, or the above-described compound microbial agent in the prevention and control of plant root rot and / or the promotion of plant growth, wherein the pathogen of the root rot includes *Fusarium graminearum*.
[0014] Preferably, the plant includes corn.
[0015] Preferably, the spore concentration of *Sclerotium spp.* RS44 in the soil is ≥10⁻⁶. 7 The concentration of *Laenia natans* RS86 in the soil of the compound microbial community or compound microbial agent is ≥10. 7 CFU / g soil.
[0016] Beneficial effects: The *Rhizopus oryzae* RS44 provided by this invention was isolated from the roots of maize plants that were not infected with root rot in Baicheng City, Jilin Province. It has the characteristics of producing iron carriers and fixing nitrogen, and has a good antagonistic effect on the pathogen *Fusarium graminearum*, with an inhibition rate of 26.39%. It has a good control effect on maize root rot and can promote maize growth.
[0017] Furthermore, the composite microbial community composed of *Aureobasidium* and *R. lanatula* RS86 provided by this invention can effectively inhibit the growth of *Fusarium graminearum*, has a good control effect on maize root rot (the disease index is reduced by 26.32%), and can promote maize growth.
[0018] Biological Preservation Instructions Broom mold RS44, classified and named Broom mold Clonostachys sp. was deposited on January 30, 2026, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42448.
[0019] Larvae RS86, classified as Rahnella sp. RS86 was deposited on September 23, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M 20242043. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.
[0021] Figure 1 The results of cultivating *Sclerotium spp.* RS44 on PDA plates; Figure 2 Phylogenetic tree of *Sclerotium spp.* RS44; Figure 3 The results show the inhibitory effect of *Fusarium graminearum* RS44 on *Fusarium graminearum*. Figure 4 The results of the determination of the siderogenic carrier of *Sclerotium globosum* RS44; Figure 5 The results show the nitrogen-fixing ability of *Sclerotium spp.* RS44. Figure 6 The results of an experiment on the combined control of maize root rot by *Cladosporium argentea* RS44 and *Larnella lataniae* RS86. Detailed Implementation
[0022] This invention provides a strain of *Sclerotium spp.* RS44, with accession number CGMCC No. 42448.
[0023] The *Rhizopus oryzae* RS44 provided by this invention was isolated from the rhizosphere of healthy maize in Tongyu County, Baicheng City, Jilin Province. It appears white on PDA plates, initially as white, loose, cotton-like mycelia. As it grows, the colony turns blue-green to olive-green due to the production of a large number of spores. It has the characteristics of producing iron carriers and fixing nitrogen, and has a good antagonistic effect on the pathogen *Fusarium graminearum*, with an inhibition rate of 26.39%. It has a good control effect on maize root rot and can promote maize growth.
[0024] This invention provides a biocontrol agent, the active ingredient of which includes *Rhizopus oryzae* RS44 as described in the above-mentioned technical solution. As one embodiment, the spore concentration of *Rhizopus oryzae* RS44 in the biocontrol agent is ≥1×10⁻⁶. 6 per mL.
[0025] This invention provides a composite microbial community, comprising *Laenia natans* RS86 and *Pseudomonas aeruginosa* RS44 as described in the above-mentioned technical solution. The preservation number of *Laenia natans* RS86 is CCTCC NO: M 20242043. As one embodiment, the live spores of *Laenia natans* RS86 and *Pseudomonas aeruginosa* RS44 are 1-2 CFU:1-2 spores. Existing research mostly focuses on the disease control effects of single strains, which often suffer from unstable colonization and large fluctuations in efficacy in complex soil environments. The composite microbial community provided by this invention, through the synergistic effect between different functional microorganisms, can exert superior effects compared to single strains in pathogen inhibition, rhizosphere colonization, and plant growth promotion. It can effectively control maize root rot and promote maize growth, possessing significant research value and application prospects.
[0026] This invention provides a compound microbial agent, the effective components of which include the compound microbial community described in the above technical solution.
[0027] As one embodiment, the spore concentration of *Strombyx mori* RS44 in the compound microbial agent is ≥1×10⁻⁶. 6 The viable count of *Raenella esculenta* RS86 is ≥1×10⁻⁶ / mL. 6 CFU / mL.
[0028] This invention provides the application of the above-described *Schynostemma* RS44, the above-described biocontrol agent, the above-described compound microbial community, or the above-described compound microbial agent in the prevention and control of plant root rot and / or the promotion of plant growth, wherein the pathogen of the root rot includes *Fusarium graminearum*.
[0029] In one embodiment, the plant includes corn. In another embodiment, the spore concentration of *Strombyx mori* RS44 in the soil is ≥10⁻⁶. 7 The concentration of *Laenia natans* RS86 in the soil of the compound microbial community or compound microbial agent is ≥10. 7CFU / g soil.
[0030] To further illustrate the present invention, the application of *Cladosporium spp.* RS44 and its complex with *Laenia spp.* RS86 in the prevention and control of maize root rot is described in detail below with reference to embodiments and accompanying drawings. However, these descriptions should not be construed as limiting the scope of protection of the present invention.
[0031] The pathogen Fusarium graminearum used in the embodiments of the present invention is a commercially available strain purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (product code BMZ097952).
[0032] Example 1 1. Isolation and molecular biological identification of endophytic fungus RS44 Rhizosphere samples were taken from healthy maize roots in Tongyu County, Baicheng City, Jilin Province. Root tissue was cut, washed with deionized water, cleaned with 75% ethanol, soaked in 1% sodium hypochlorite solution for 10 minutes, rinsed 3-5 times with sterile water, and then dried. The root tissue was cut into small pieces with a sterile blade and directly inoculated onto PDA medium. The culture was carried out at 25℃ for 5 days, and the growth of endophytic fungal colonies was observed. Once the endophytic fungi had fully grown, they were purified using an inoculation needle until uniformly morphological colonies appeared. Classification was based on the color and morphology of the hyphae and colonies. The morphological characteristics of RS44: It appears white on PDA plates, initially consisting of white, loose, cottony hyphae (…). Figure 1 As the colony grows, it turns from blue-green to olive-green due to the production of numerous spores. PDA medium: 200g potato, 20g glucose, 15g agar, and 1L water.
[0033] DNA molecular identification of RS44 strain: Using RS44 strain DNA as a template, the ITS gene sequence was amplified using universal primers ITS1 and ITS4. The primer sequences are as follows: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO. 1); ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO. 2); PCR reaction system (50 μL): primer ITS1 2 μL, primer ITS4 2 μL, template DNA 2 μL, 2×SanTaqPCR Master Mix 25 μL, sterile water 19 μL.
[0034] PCR reaction program: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ extension for 10 min.
[0035] The amplified products were detected by agarose gel electrophoresis and then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO.3, as follows: 5'--3'.
[0036] Sequencing results were aligned to the GenBank database using the Blast algorithm to construct a phylogenetic tree. The results showed that strain RS44 clustered with *Pseudomonas* species, and was associated with strains... Clonostachys epichloe The sequence has the highest homology ( Figure 2 Therefore, strain RS44 is classified as belonging to the genus *Synthia*. Strain RS44 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42448.
[0037] 2. Inhibitory effect of *Fusarium graminearum* RS44 on *Fusarium graminearum*. The inhibitory effect of *Rhizopus oryzae* RS44 on the pathogen *Fusarium graminearum* was tested using the plate confrontation method. *Rhizopus oryzae* RS44 and *Fusarium graminearum* were activated by inoculating them separately onto PDA medium. Using a sterile punch with a diameter of 5 mm, mycelial blocks of the same age were collected from the edge of the *Rhizopus oryzae* RS44 and *Fusarium graminearum* colonies and placed symmetrically on both sides of the PDA medium with mycelium facing down. After incubation at 25℃ for 5 days, mycelial growth was evaluated, and a blank control was included for comparison. The inhibition rate was calculated as follows: Inhibition rate = (control colony radius - confrontation culture colony radius) / control colony radius × 100%.
[0038] The results showed that *Fusarium graminearum* RS44 had a good antagonistic effect on the pathogen *Fusarium graminearum*, with an inhibition rate of 26.39%. Figure 3 ).
[0039] 3. Determination of the probiotic properties of *Sclerotium rosenbergii* RS44 (1) Characteristics of the strain producing iron carriers Inoculate *Cladosporium* RS44 onto CAS detection medium, incubate for 7 days, and observe whether red colonies form. Figure 4 CAS detection medium formula: Chromium azurite S (CAS) 60.5mg, hexadecyltrimethylammonium bromide (HDTMA) 72.9mg, FeCl3·6H2O 2.645mg, Agar 9000mg, NaH2PO4·2H2O 295.25mg, Na2HPO4·12H2O 1213.5mg, NH4Cl 125mg, KH2PO4 37.5mg, NaCl 62.5mg, and sterile water 1L.
[0040] (2) Determination of nitrogen fixation capacity of strains Inoculate *Sclerotium gloeosporioides* RS44 onto Ashby medium and incubate for 5 days, then observe whether colonies grow. Figure 5 The Ashube culture medium formula is as follows: Mannitol 15.0g, CaCl2·2H2O 0.2g, K2HPO4 0.2g, MgSO4·7H2O 0.2g, MoO 0.001g, FeCl3 0.005g, Agar 15.0g and sterile water 1L.
[0041] The measurement results are shown in Table 1: Table 1. Results of strain characteristic determination of *Sterculia lycopersicola* RS44
[0042] Note: "+" indicates that the strain has this characteristic.
[0043] Example 2 1. Preparation of microbial inoculants for *Schynostemma pentaphyllum* RS44, *Larnella granatum* RS86, and *Fusarium graminearum* (Fg). (1) Preparation of Fusarium graminearum spore suspension: Fusarium graminearum mycelium was inoculated onto PDA medium and grown in a fungal incubator at 25℃ for 5-7 days; then, Fusarium graminearum mycelium blocks were inoculated onto liquid PDA medium using a sterile punch and cultured at 25℃ with shaking at 180 rpm for 5-7 days. The mycelium was filtered through 4 layers of sterile gauze, and the spores were precipitated by centrifugation at 4000 rpm for 10 min, and washed twice with sterile water. The spores were counted using a hemocytometer and the concentration was adjusted to 1×10⁻⁶. 6 Spores / mL. PDA medium: 200g potato, 20g glucose, 15g agar (liquid PDA does not require addition), 1L distilled water.
[0044] (2) Preparation of Raenella RS86 bacterial culture: Raenella RS86 with preservation number CCTCC NO: M 20242043 was streaked on LB solid medium and incubated at 30℃ for 2 days; single colonies of the cultured strain were picked and transferred to LB liquid medium and cultured at 30℃ and 180 rpm in a shaker until the logarithmic growth phase was reached; centrifuged at 4℃ and 4000 rpm for 10 min, washed 2-3 times with sterile water, and finally resuspended in an appropriate amount of sterile water and adjusted to OD. 600 =1. LB medium: 10g peptone, 5g yeast extract, 5g sodium chloride, 15g agar (not added to liquid LB), 1L distilled water.
[0045] (3) Preparation of RS44 spore suspension: The mycelia of RS44 isolated in Example 1 were inoculated into PDA medium and grown in a fungal incubator at 25°C for 7-10 days; then, the RS44 mycelia were inoculated into liquid PDA medium using a sterile punch and cultured at 25°C with shaking at 180 rpm for 7 days. The mycelia were filtered through 4 layers of sterile gauze, and the spores were precipitated by centrifugation at 4000 rpm for 10 min and washed twice with sterile water. The concentration was adjusted to 1×10⁻⁶ using a hemocytometer. 6 Spores / mL.
[0046] 2. Experiment on the combined control of maize root rot by *Cladosporium argentea* RS44 and *Larnella lataniae* RS86. The maize seeds were Zhengdan 958, which were sterilized and then germinated at 25℃. Seedlings with uniform growth were selected and divided into 5 groups of 4 seedlings each: blank control group, control group 1 (Fg), treatment group 1 (RS86), treatment group 2 (RS44), and treatment group 3 (RS86+RS44). The specific treatments are as follows: Control group: No inoculation with the pathogen Fusarium graminearum Fg group: Inoculated with Fusarium graminearum (Fg), inoculation amount 10 7 spores / g soil; RS86 group: Inoculated with Fusarium graminearum, inoculum size 10 7 Spores / g soil, apply RS86 bacterial solution, application rate 10. 7 CFU / g soil; RS44 group: Inoculated with Fusarium graminearum, inoculum size 10 7 Spores / g soil, apply *Strombyx mori* RS44, inoculum size 10. 7 spores / g soil; RS86+RS44 group: Inoculated with Fusarium graminearum, inoculation amount 10 7 Spores / g soil, S86 application rate is 10 7 CFU / g soil, apply *Cladosporium* RS44, inoculum size 10. 7 spores / g soil; After two weeks of cultivation at 25°C for 16 hours during the day and 22°C for 8 hours at night, photographs were taken, and the aboveground fresh weight, root fresh weight, root length, and disease index were measured to assess the disease suppression and growth promotion effects. The disease index was calculated as follows: Disease incidence index = (length of diseased root / total root length) × 100%.
[0047] See results Figure 6 The results showed that the RS86+RS44 treatment significantly reduced the root disease index of maize. Compared with the control group treated with pathogen Fg alone (disease index of 69.27%), the root disease index of maize treated with RS86+RS44 (51.04%) was significantly reduced by 26.32%, slightly lower than the single treatments of RS86 and RS44, but the difference was not statistically significant. Compared with the control group treated with pathogen Fg alone (disease index of 69.27%), the root disease index of maize treated with RS44 (54.45%) was significantly reduced by 21.40%.
[0048] The RS86+RS44 treatment significantly promoted the growth of both aboveground and root parts of maize. Compared with the single application of Fg pathogen (5.91 g), the aboveground fresh weight of maize treated with RS86+RS44 increased significantly by 64.81%, reaching 9.74 g. Compared with the control (7.47 g), the aboveground fresh weight of maize treated with RS86+RS44 increased significantly by 30.39%. Compared with the RS86 treatment (7.80 g), the aboveground fresh weight of maize treated with RS86+RS44 increased significantly by 24.87%. Compared with the RS44 treatment (8.30 g), the aboveground fresh weight of maize treated with RS86+RS44 increased significantly by 17.35%. Compared with the single application of Fg pathogen (5.91 g), the aboveground fresh weight of maize treated with RS44 increased significantly by 40.44%.
[0049] In maize roots, compared with the Fg-treated maize roots (1.66g), the fresh weight of maize roots treated with RS86+RS44 significantly increased by 92.77%, reaching 3.20g. Compared with the blank control (2.46g), the RS86+RS44 treatment significantly promoted maize root growth, increasing it by 30.08%. Compared with the RS86 treatment (2.29g), the fresh weight of maize roots treated with RS86+RS44 significantly increased by 39.74%. Compared with the RS44 treatment (2.28g), the fresh weight of maize roots treated with RS86+RS44 significantly increased by 40.35%. Compared with the Fg-treated maize roots (1.66g), the fresh weight of maize roots treated with RS44 (2.28g) significantly increased by 37.35%.
[0050] In summary, the *Fusarium graminearum* RS44 and its composite microbial community with RS86 provided by this invention can effectively inhibit the growth of *Fusarium graminearum* and promote corn growth.
[0051] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A broom mold ( Clonostachys sp.)RS44, characterized in that, The accession number is CGMCC No.42448.
2. A biocontrol agent, characterized in that, The active ingredient includes the *Stipa spp.* RS44 as described in claim 1.
3. The biocontrol agent according to claim 2, characterized in that, The spore concentration of *Strombyx mori* RS44 in the biocontrol agent is ≥1×10⁻⁶. 6 per mL.
4. A complex microbial community, characterized in that, Including Larynella ( Rahnella sp.) RS86 and the broom mold RS44 according to claim 1, wherein the preservation number of the broom mold RS86 is CCTCC NO: M 20242043.
5. The complex microbial community according to claim 4, characterized in that, The live spores of *Laenia natans* RS86 and *Synthia spp.* RS44 were 1-2 CFU: 1-2 spores.
6. A compound microbial agent, characterized in that, The active ingredient includes the complex microbial community as described in claim 4 or 5.
7. The compound microbial agent according to claim 6, characterized in that, The spore concentration of *Pseudomonas aeruginosa* RS44 in the compound microbial agent is ≥1×10⁻⁶. 6 The viable count of *Raenella esculenta* RS86 is ≥1×10⁻⁶ / mL. 6 CFU / mL.
8. The application of *Sterculia lycopersicum* RS44 as described in claim 1, or the biocontrol agent as described in claim 2 or 3, or the compound microbial community as described in claim 4 or 5, or the compound microbial agent as described in claim 6 or 7, in the prevention and control of plant root rot and / or the promotion of plant growth, characterized in that, The pathogens causing root rot include Fusarium graminearum (… Fusariumgraminearum ).
9. The application according to claim 8, characterized in that, The plant mentioned includes corn.
10. The application according to claim 8, characterized in that, The spore concentration of *Rhizopus oryzae* RS44 in the soil is ≥10. 7 The concentration of *Laenia natans* RS86 in the soil of the compound microbial community or compound microbial agent is ≥10. 7 CFU / g soil.