Streptomyces spectabilis capable of preventing and treating towel gourd fusarium wilt and application of streptomyces spectabilis

Abstract

The invention relates to streptomyces spectabilis AMC3-4 capable of preventing and treating towel gourd fusarium wilt, which is preserved in China General Microbiological Culture Collection Center (CGMCC) on November 24, 2025, and has a preservation number of CGMCC No.36755. The invention also relates to an application of the streptomyces spectabilis AMC3-4 in preventing and treating towel gourd fusarium wilt. The streptomyces spectabilis AMC3-4 strain disclosed by the invention has a very strong inhibition effect on pathogenic bacteria of towel gourd fusarium wilt, and fermentation liquor of the streptomyces spectabilis AMC3-4 strain contains various metabolites with antibacterial effects. The prevention and treatment effect of the towel gourd potted plant treated by the AMC3-4 fermentation liquor is 47.62%. Compared with a chemical pesticide, the natural strain separated from soil has no toxic or side effect and no residue on soil ecology, so that the strain has potential commercial development and application values in biological prevention and control practice of diseases.

Description

Technical Field

[0001] This invention relates to the field of crop disease control, and more particularly to a Streptomyces spectabilis strain that can control wilt disease in loofah and its applications. Background Technology

[0002] Fusarium wilt of loofah is an important soil-borne disease that has occurred in loofah-growing areas in recent years. It can occur at any stage of loofah growth, with the flowering, vine-growing, and fruiting stages being the most concentrated periods of infection. The proportion of diseased plants in the field is typically between 20% and 40%. Severe cases can lead to the wilting and death of large areas of plants, and may even result in complete crop failure, significantly impacting loofah yield. High-density planting increases the risk of concentrated outbreaks, with symptoms appearing in clusters.

[0003] Currently, chemical pesticides are one of the most important control measures for Fusarium wilt in loofah. They are relatively simple, quick, and inexpensive, with rapid results. However, excessive use of chemical pesticides can lead to pesticide resistance and pesticide residues in the soil, ultimately resulting in poor control effects, harm to ecological security, and damage to human health.

[0004] Biocontrol microorganisms have unique advantages in overcoming plant disease resistance, reducing environmental pollution and ecological damage caused by chemical fungicides, and minimizing chemical pesticide residues in agricultural products. Biopesticides prepared using the secondary metabolites of biocontrol bacteria are characterized by being pollution-free, residue-free, unlikely to induce resistance in harmful organisms, highly compatible with the environment, and safe for humans and animals. Furthermore, research on using the metabolites produced by microorganisms to treat plant pathogens for biological disease control is currently a hot topic.

[0005] Therefore, vigorously carrying out research on biological control is an important way to achieve sustainable agricultural development and has significant practical implications for production. Summary of the Invention

[0006] To address the above problems, this invention provides a Streptomyces spectabilis strain that can prevent and control Fusarium wilt in loofah. It was deposited on November 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36755.

[0007] The present invention also provides the application of the above-mentioned Streptomyces speciosa in the prevention and control of wilt disease in loofah.

[0008] The present invention also provides a method for preventing and controlling wilt disease in loofah, comprising the step of applying the fermentation liquid of the above-mentioned Streptomyces speciosa to the soil in which the crop is planted.

[0009] In one specific implementation, the method includes the following steps:

[0010] S1: Cultivate the Streptomyces speciosa to prepare Streptomyces speciosa fermentation broth or sterile fermentation broth;

[0011] S2: Apply the Streptomyces speciosa fermentation broth or sterile fermentation broth to the soil in which the crop is planted.

[0012] In one specific implementation, in S1, the *Streptomyces speciosa* is cultured in Gao's No. 1 medium.

[0013] In one specific embodiment, the *Streptomyces speciosa* fermentation broth described in S1 is prepared by the following steps:

[0014] S11: Inoculate the Streptomyces speciosa into Gao's No. 1 liquid culture medium and culture it;

[0015] S12: The Streptomyces speciosa fermentation broth was obtained by culturing at 28℃ and 180 rpm for 5-7 days.

[0016] In one specific implementation, the aseptic fermentation broth of Streptomyces spectabilis S1 is obtained by decellularizing the Streptomyces spectabilis fermentation broth.

[0017] In one specific implementation, in S2, the crop is treated with a root irrigation of the Streptomyces speciosa fermentation broth or a sterile fermentation broth.

[0018] The *Streptomyces speciosa* strain AMC3-4 of this invention exhibits strong inhibitory activity against the pathogen causing *Fusarium wilt* in *Luffa cylindrica*, and its fermentation broth contains various metabolites with antibacterial effects. After streak plating, the inhibition rate against *Fusarium wilt* reached 82.37%, and the inhibition rate against *Fusarium wilt* in the aseptic fermentation broth reached 73.64%. (1.0 × 10⁻⁶) 7 Fermentation broth of strain AMC3-4 with spores / mL showed good control of Fusarium wilt, with a control effect of 47.62% on potted loofah plants treated with AMC3-4 fermentation broth. This strain is a natural strain isolated from soil and has no toxic side effects or residues on the soil ecosystem compared to chemical pesticides. Therefore, it has potential commercial development and application value in the biological control of diseases.

[0019] Preservation of biological materials

[0020] The purified Streptomyces speciosa AMC3-4 of this invention was deposited on November 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36755. Attached Figure Description

[0021] Figure 1 Colony morphology of Streptomyces speciosa AMC3-4 cultured for 7 days.

[0022] Figure 2 The aerial hyphae morphology of Streptomyces spectabilis AMC3-4.

[0023] Figure 3 The spore chain morphology of Streptomyces speciosa AMC3-4.

[0024] Figure 4 Streptomyces speciosa AMC3-4 was used to demonstrate the inhibitory effect of Streptomyces speciosa AMC3-4 on Fusarium wilt pathogens.

[0025] Figure 5 The inhibitory effect of aseptic fermentation filtrate of Streptomyces speciosa AMC3-4 added to the culture medium on Fusarium wilt pathogens.

[0026] Figure 6 The effect of fermentation filtrate of Streptomyces speciosa AMC3-4 on the mycelial morphology of Fusarium wilt.

[0027] Figure 7 To investigate the control effect of Streptomyces speciosa AMC3-4 on wilt disease in loofah. Detailed Implementation

[0028] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0029] 1. Identification and preservation of Streptomyces speciosa AMC3-4

[0030] Rhizosphere soil samples were collected from non-agricultural ecosystems such as lawns, bamboo forests, and street trees in four areas of Chongqing: Beibei, Shapingba, Banan, and Nan'an. Multiple strains were isolated and screened, and finally, strain AMC3-4 was identified as having a good control effect on Fusarium wilt after indoor pot inoculation tests on live plants.

[0031] (1) Morphological observation and identification: Strain AMC3-4 was cultured on Gao's No. 1 agar plates at 28℃ for 7 days, and its colony morphology (shape, color, whether the surface is raised or sunken, edge shape, etc.) was observed. Single colony photographs were obtained as follows: Figure 1 As shown, this strain grows well on Gao's No. 1 solid medium. The colonies are initially light pink, and the color gradually deepens in the later stages. The single colony is round in appearance and the surface is dry and opaque. The spore filaments are diverse in morphology, either straight or curled, with obvious branching, and the branches are mostly right angles.

[0032] (2) Observation of hyphal morphology: A pre-sterilized coverslip was inserted at a 45° angle into a plate and incubated upside down in a 28°C incubator for 7 days. Afterward, the coverslip was removed, a small amount of distilled water was added, and the plate was placed on a slide. The strain, hyphae, and spore chains were observed using an optical microscope. Figure 2 and 3 As shown.

[0033] (3) Molecular biological identification: Genomic DNA was extracted from the strain and amplified by PCR using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGACGAC TT-3'). Using the genomic DNA of strain AMC3-4 as a template, the 16S rDNA sequence was obtained by electrophoresis and sequencing, as shown in SEQ ID NO:1. The obtained sequence results were compared with BLAST in NCBI, and the 16S rDNA sequences of related strains were selected. A phylogenetic tree was constructed using MEGA11 software, and combined with morphological characteristics, the species-level taxonomic rank of AMC3-4 was confirmed. The taxonomic position of this bacterium was determined. The results showed that this strain is *Streptomyces spectabilis*.

[0034] The above-mentioned strain was deposited on November 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number: CGMCC No. 36755.

[0035] 2. Preparation of fermentation broth and analysis of metabolites of Streptomyces speciosa AMC3-4

[0036] Two 5 mm diameter AMC3-4 bacterial pellets were inoculated into a 300 mL Erlenmeyer flask containing 100 mL of liquid Gao's No. 1 medium and cultured at 28℃ and 180 r / min for 5 days to obtain the fermentation broth. The fermentation broth was poured into a sterile 50 mL centrifuge tube and centrifuged at 8000 rpm for 15 min. The supernatant was filtered through a 0.22 μm filter to obtain sterile fermentation filtrate. Non-targeted metabolomics identification showed that, compared with the metabolites of a control strain without antagonistic activity, the metabolites of strain AMC3-4 were significantly upregulated in the fermentation filtrate of AMC3-4, including aucubin, xanthocyanidin, kaempferol glucoside, and 2-decarboxylated cauliflower acid. KEGG enrichment analysis revealed that the differentially expressed metabolites were significantly enriched in pathways such as ABC transporter protein, coenzyme biosynthesis, galactose metabolism, citric acid cycle, and nucleotide metabolism.

[0037] 3. *Streptomyces speciosa* AMC3-4 for the prevention and control of pathogens.

[0038] 3.1 Antifungal activity of Streptomyces speciosa AMC3-4 against Fusarium wilt pathogens of loofah

[0039] The effect of *Streptomyces speciosa* AMC3-4 on the mycelial growth of pathogenic fungi was determined using the plate confrontation method. AMC3-4 strain was streaked onto Gao's No. 1 medium and incubated upside down at 28°C for 5 days. Five-mm mycelial discs of AMC3-4 were then collected for further processing. These five-mm mycelial discs were placed on PDA plates approximately 25 mm from the center on both sides and incubated upside down at 28°C for 2 days. Five-mm mycelial discs of *Fusarium oxysporum* were then collected and placed in the center of the plates inoculated with AMC3-4 mycelial discs. For the control group, *Fusarium oxysporum* mycelial discs were inoculated in the center of PDA plates without AMC3-4 mycelial discs. Each treatment was repeated in triplicate. All treatments were incubated at 28°C for 5 days. Afterward, the colony diameter of *Fusarium oxysporum* was measured, and the inhibition rate was calculated.

[0040] The results are as follows Figure 4 The AMC3-4 strain showed good inhibitory effect on the growth of Fusarium wilt, with an inhibition rate of 82.37%.

[0041] 3.2 Inhibitory effect of Streptomyces speciosa AMC3-4 on sclerotium formation in Fusarium wilt pathogens

[0042] Two 5 mm diameter AMC3-4 bacterial pellets were inoculated into a 300 mL Erlenmeyer flask containing 100 mL of liquid Gao's No. 1 medium. The flask was incubated at 28°C and 180 rpm for 5 days to obtain the fermentation broth. The fermentation broth was then poured into a sterile 50 mL centrifuge tube and centrifuged at 8000 rpm for 15 min. The supernatant was filtered through a 0.22 μm filter to obtain sterile fermentation filtrate. 32 mL of melted and cooled PDA medium to 50°C was added to the 50 mL centrifuge tube, followed by 8 mL of fermentation filtrate. After mixing thoroughly, the mixture was evenly poured into three 90 mm sterile petri dishes to prepare a plate containing 20% ​​fermentation filtrate. The control consisted of 32 mL of PDA medium inoculated with 8 mL of Gao's No. 1 liquid medium. The plates were allowed to cool and solidify completely before use. One 5 mm mycelium of *Fusarium wilt* was inoculated into the center of a plate containing 20% ​​fermentation filtrate of strain AMC3-4. The plate was then placed in a 28°C incubator and incubated in the dark for 30 days. The sclerotia were removed from each plate, dried until their weight no longer changed, and then weighed. The dry weight of the sclerotia in each plate was recorded, and the inhibition rate was calculated.

[0043] The results are as follows Figure 5 As shown, the fermentation broth of strain AMC3-4 inhibited the growth of Fusarium wilt pathogens by 73.64%. Scanning electron microscopy revealed that the fermentation filtrate of strain AMC3-4 caused Fusarium wilt mycelia to become shrunken and deformed. Figure 6 ).

[0044] 2.3 Effect of Streptomyces speciosa AMC3-4 fermentation broth on Fusarium wilt of Luffa cylindrica

[0045] A pot experiment was conducted using the needle inoculation method to inoculate loofah. AMC3-4 mycelial cakes were inoculated into Gao's No. 1 liquid culture medium and cultured in a shaker at 28℃ and 180 rpm for 7 days to obtain a concentration of 10%. 7 Fermentation broth of AMC3-4 strain (CFU / mL) was used. Loofah seedlings of similar size were selected and cultured at 25℃ until the plants had fully expanded their leaves (10 days later). Treatment groups: 30 mL of AMC3-4 strain fermentation broth was applied to the roots of each plant via root drenching. The control group received the same amount of water. Each group consisted of 12 plants, and the experiment was repeated three times. Two days after root drenching, Fusarium wilt fungal spores were inoculated into the base of each loofah stem using an inoculation needle. Disease incidence was assessed and the control effect calculated after 7 days.

[0046] The results are as follows Figure 7 As shown, 1.0×10 7 The fermentation broth of AMC3-4 strain with 1 spore / mL has a good control effect on Fusarium wilt. The control effect of AMC3-4 fermentation broth on potted loofah plants was 47.62%.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Streptomyces spectabilis capable of controlling fusarium wilt of Lagenaria leucantha, characterized in that, The Streptomyces spectabilis was preserved in China General Microbiological Culture Collection Center on November 24, 2025, and the preservation number is CGMCC No. 36755.

2. The Streptomyces spectabilis of claim 1 is applied to the prevention and treatment of the fusarium wilt of the luffa.

3. A method for controlling fusarium wilt of luffa, characterized by, The method comprises the step of applying the fermentation liquor of the Streptomyces spectabilis of claim 1 to the soil where crops are planted.

4. The method of claim 3, wherein, The method comprises the following steps: S1: culturing the Streptomyces spectabilis to prepare a Streptomyces spectabilis fermentation liquor or a sterile fermentation liquor; S2: applying the Streptomyces spectabilis fermentation liquor or the sterile fermentation liquor to the soil where crops are planted.

5. The method of claim 4, wherein, In S1, the Streptomyces spectabilis is cultured in Gao's No. 1 medium.

6. The method of claim 5, wherein, The Streptomyces spectabilis fermentation liquor in S1 is prepared by the following steps: S11: inoculating the Streptomyces spectabilis into Gao's No. 1 liquid medium for culture; S12: culturing at 28°C and 180 rpm for 5-7 days to obtain the Streptomyces spectabilis fermentation liquor.

7. The method of claim 6, wherein, The sterile fermentation liquor of the Streptomyces spectabilis in S1 is obtained by decellularization filtration of the Streptomyces spectabilis fermentation liquor.

8. The method of claim 4, wherein, In S2, the Streptomyces spectabilis fermentation liquor or the sterile fermentation liquor is used for root irrigation treatment of the crops.

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