Fungus and actinomycete complex microbial inoculant for preventing and treating root rot and application thereof

Abstract

The invention provides a fungus and actinomycete complex microbial inoculant for preventing and treating root rot. The complex microbial inoculant contains spiropolyspora rosea with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.42332 and streptomyces taenanensis with the preservation number of CGMCC No.36965. The invention further provides a preparation method of the fungus and actinomycete complex microbial inoculant. The invention also provides an application of the fungus and actinomycete complex microbial inoculant. The complex microbial inoculant provided by the invention contains fermentation products of spiropolyspora roseus HLY-1 and streptomyces talanigerum NYC 1-5, the two fermentation products have no inhibition effect, and a synergistic effect can be generated by mixed application of the two fermentation products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a compound preparation of *Polyspora pinkis* strain and *Streptomyces tailanhei* strain, and its application. Background Technology

[0002] Fusarium ( Fusarium Fusarium root rot (S. spp.) is a highly destructive soil-borne disease that seriously harms the production of cash crops such as vegetables, fruits, medicinal herbs, and flowers, causing root rot, plant wilting, and even death, resulting in huge economic losses. Currently, the control of Fusarium root rot in agricultural production mainly relies on chemical fungicides. However, the long-term use of chemical agents has led to increased pathogen resistance, pesticide residues, and environmental pollution, posing certain safety hazards to agricultural production.

[0003] Utilizing biocontrol microorganisms to suppress pathogens is an environmentally friendly and sustainable disease control strategy that is increasingly attracting attention. Some biocontrol bacteria, such as Bacillus, Streptomyces, and Trichoderma, have been extensively studied and have played a crucial role in green disease control. However, current biocontrol practices against Fusarium root rot still face several challenges. First, strains from different sources exhibit significant differences in their antimicrobial and disease-preventing abilities, requiring multi-stage screening—from initial screening to secondary screening, and from greenhouse to field—to obtain a highly effective strain. Second, the stability of biocontrol efficacy needs to be addressed. The complex soil environment, influenced by soil antimicrobial activity and other factors, often leads to unstable field efficacy of biocontrol bacteria. Discovering multifunctional strains that exert their effects through different mechanisms and sites of action can achieve stable biocontrol results. Furthermore, single strains often have limited biocontrol capabilities; combining different types of biocompatible biocontrol bacteria (such as fungi and bacteria) can achieve higher disease control efficacy and complementary effects.

[0004] Pink Spiral Polyporus ( Clonostachys rosea Fusarium root rot has become a hot research topic in the field of biocontrol in recent years. This fungus can parasitize various plant pathogenic fungi such as Fusarium, Sclerotinia sclerotiorum, Rhizoctonia solani, and Botrytis cinerea, and can also inhibit plant diseases through multiple mechanisms, including producing active substances, competition, and inducing plant resistance. To develop stable biocontrol agents and improve disease control efficacy, it is crucial to screen for new biocontrol microbial resources that are highly effective against Fusarium root rot and can promote plant growth and enhance disease resistance. Developing synergistic compound microbial agents will provide an efficient, safe, and green solution for the control of soil-borne diseases such as root rot, which is of great significance for sustainable industrial development and safe agricultural production. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention aims to provide a *Polyporus pulcherrimus* strain and a *Streptomyces tailanhei* strain for preventing and controlling Fusarium root rot, and a compound inoculum composed thereof.

[0006] The present invention also proposes the application of a compound microbial agent containing the aforementioned *Polyspora pinkis* and *Streptomyces tailanensis* in the prevention and control of Fusarium root rot.

[0007] A compound fungal and actinomycete agent for the prevention and control of root rot, wherein the compound fungal agent contains *Polyspora pinki* with accession number CGMCC No. 42332 and *Streptomyces tailanhei* with accession number CGMCC No. 36965.

[0008] This invention provides highly efficient microbial strains for controlling Fusarium root rot. Through initial screening on plates, secondary screening using hydroponic test-tube seedlings, verification in greenhouse pots, and field evaluation, strains *Polyspora pinkis* HLY-1 and *Streptomyces tailanhei* NYC 1-5 were selected. Both fungi and actinomycetes have been deposited with the China General Microbiological Culture Collection Center (CGMCC) under accession numbers CGMCC No. 42332 and CGMCC No. 36965, respectively.

[0009] Currently, several *Streptomyces pinki* products have been registered both domestically and internationally, and are being used in the control of gray mold in fruits and vegetables and seedling diseases. *Streptomyces tailanheense* (… Streptomyces tailanensis This is a new species of Streptomyces discovered and identified by Chinese scientists for the first time in 2020. We found that the Streptomyces tyrannus NYC 1-5 (accession number CGMCC No. 36965) used in this study has broad-spectrum resistance to a variety of plant pathogenic fungi, including Fusarium oxysporum, and can also promote plant growth, showing strong potential for biocontrol applications.

[0010] The compound microbial agent contains fermentation products of *Polyspora pinkis* and *Streptomyces tailanhei*, with a spore ratio of 1:0.5~2.

[0011] Furthermore, the compound microbial agent is in the form of liquid, granules, or powder, wherein the bacterial content of *Polyspora pinki* and *Streptomyces tailanhei* in the liquid is (5~50)×10⁻⁶. 7 Spores / mL.

[0012] A preferred embodiment of the present invention is that the fungal and actinomycete composite agent is prepared by a method comprising the following steps: 1) Plate culture: The *Polyspora pinkis* and *Streptomyces tailanhei* were inoculated onto potato dextrose agar and Gao's No. 1 medium, respectively, and cultured at 26-28℃ for 7-14 days until sporulation. 2) Seed culture: Elute the spores to prepare a spore suspension, or cut the mycelium blocks from the cultured plate and inoculate them into potato dextrose liquid medium and Gao's No. 1 liquid medium, respectively, and culture them in a shaker at 25~28℃ for 48~84 h to obtain the seed culture. 3) Fermentation culture: Inoculate the seed liquid into the liquid fermentation medium and culture for 60-120 h to obtain the fermentation broth; 4) Formulation: Mix the fermentation broth of *Polyspora pinkis* and *Streptomyces tailanhei* obtained in step 3) to obtain a liquid formulation of the fungal and actinomycete compound agent, or add fillers and adjuvants to prepare granules or powders (the fermentation broth of *Polyspora pinkis* and *Streptomyces tailanhei* can be made into solid formulations separately and then mixed; or the fermentation broths can be mixed and then made into solid formulations).

[0013] In step 2), a 3-6×3-6 mm mycelium block can be cut from the cultured plate for inoculation.

[0014] In step 3), the liquid fermentation medium for the fermentation culture of *Polyspora pinkis* is formulated as follows: 10-40 g sucrose, 5-15 g yeast extract, 5-15 g soybean meal powder, 0.5-2 g K₂HPO₄, 0.1-1 g NaCl, 0.1-1 g MgSO₄•7H₂O, 0.05-0.1 g FeSO₄•7H₂O, 0.01-0.1 g ZnSO₄•7H₂O, with water added to 1000 mL and the pH adjusted to 5.5-6.5. Preferably, in step 3), the liquid fermentation medium for the fermentation culture of *Streptomyces tailanhei* is formulated as follows: 20-30 g soluble starch, 5-10 g glucose, 10-20 g soybean flour, 1-5 g peptone, 0.5-2 g (NH4)2SO4, 0.5-2 g K2HPO4, 0.5-2 g NaCl, 0.1-0.5 g MgSO4•7H2O, and 0.5-2 g CaCO3, with water added to 1000 mL and the pH adjusted to 7.0-7.5.

[0015] In step 3), the fermentation culture conditions of *Polyspora pinkis* and *Streptomyces tailanhei* are independent of each other: seed liquid inoculation amount 1~6%, rotation speed 150~220 r / min, culture temperature 25~30℃, and culture time 72~108 h.

[0016] In step 4), operation A or B is performed on the fermentation broth or fermentation broth mixture: A: Add filler, mix and granulate to obtain granules; B: Add fillers and additives, dry and pulverize to obtain powder; The filler is one or more of diatomaceous earth, kaolin, bentonite, talc, starch, dextrin, and peat moss; the auxiliary agent is one or more of sodium carboxymethyl cellulose, chitosan, polyethylene glycol, sodium lignosulfonate, sodium dodecylbenzenesulfonate, amino oligosaccharide, and sodium alginate; the filler is added at 80% to 300% of the fermentation broth mass, and the auxiliary agent is added at 0.1% to 10% of the fermentation broth mass.

[0017] The fungal and actinomycete compound agent described in this invention is used to prevent and control soil-borne diseases of fruits and vegetables such as tomato root rot, pepper root rot, strawberry root rot, and watermelon root rot caused by Fusarium, and to promote plant growth.

[0018] Furthermore, the application method is as follows: the fungal and actinomycete compound inoculant is applied at one or more stages during the seedling stage, transplanting stage, and growth stage, and the application method is seed soaking, seed mixing, root dipping, root irrigation, drip irrigation, or soil irrigation.

[0019] The compound microbial agent proposed in this invention comprises the fermentation products of *Polyspora pinkis* HLY-1 and *Streptomyces tailanhei* NYC 1-5, with no inhibitory effect between the two. This invention provides the application of the compound microbial agent of *Polyspora pinkis* HLY-1 and *Streptomyces tailanhei* NYC 1-5 in the control of Fusarium root rot in crops. The core feature of this compound microbial agent containing these fungi and actinomycetes is that there is no inhibitory effect between the fungi and actinomycetes, and their combined application produces a synergistic effect.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Outstanding disease control effect: The greenhouse control efficacy of *Alternaria pinki* HLY-1 and *Streptomyces tailanhei* NYC 1-5 against Fusarium root rot of tomatoes and peppers reaches 65-73%. When the two are used in combination in a certain proportion, the control efficacy against the two root rot diseases reaches more than 78%, which is significantly higher than that of single strains, showing a significant synergistic effect.

[0021] 2. Dual Growth-Promoting Functions: The aforementioned *Alternaria pinki* and *Streptomyces tailanensis* not only effectively control fungal diseases but also significantly promote plant growth. Experiments show that application of the inoculants significantly improves tomato seed germination vigor, promotes seedling root development and plant growth, increases leaf chlorophyll content, and increases yield, achieving the dual effects of "disease control" and "growth promotion" (see Examples 5 and 11).

[0022] 3. Inducing Plant Disease Resistance: The compound microbial agent provided by this invention can systematically activate the plant's own defense system. As shown in Example 12, the biocontrol agent treatment significantly increased the activity of key antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in tomato roots, while reducing the content of malondialdehyde (MDA), a membrane lipid peroxidation product. This reduces oxidative damage, enhances the plant's systemic resistance, and provides a physiological basis for sustained disease control.

[0023] In summary, the *Alternaria pinki* and *Streptomyces tailanhei* provided by this invention can effectively control diseases through multiple mechanisms of action, while promoting crop growth and increasing yield. Their application provides a safe, efficient, and long-lasting solution for the green control of Fusarium root rot in fruits and vegetables. Attached Figure Description

[0024] 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 are briefly described below.

[0025] Figure 1 The images show the colony morphology and microstructure of HNF-P1, the pathogen causing root rot in tomatoes. A shows the colony morphology on PDA medium, and B shows the conidial morphology under a microscope.

[0026] Figure 2 Phylogenetic tree of the tomato root rot pathogen HNF-P1 strain constructed using the NJ method based on ITS sequences.

[0027] Figure 3 The parasitic effects of *Alternaria pinki* from different sources on *Tomato root rot* pathogens.

[0028] Figure 4 To determine the control efficacy of different biocontrol strains against tomato root rot in hydroponic culture of test-tube seedlings. From left to right: pathogen control, water control, and treatments with biocontrol strains HLY-1, SYP 1-1, NYC 1-5, and NYC 1-3.

[0029] Figure 5 The images show the morphological characteristics and microstructure of actinomycete strain NYC 1-5 on Gao's No. 1 medium. A shows the front of the colony; B shows the back of the colony; C shows the microstructure of hyphae and spores.

[0030] Figure 6 To construct a phylogenetic tree of NYC 1-5 strains based on housekeeping gene sequences.

[0031] Figure 7 The compatibility of *Polyspora pinkis* HLY-1 with *Streptomyces tailanhei* NYC 1-5 was determined.

[0032] Figure 8The study investigated the efficacy of biocontrol agents against root rot in greenhouse potted tomatoes. CK was the pathogen control; water was the blank control without pathogen or biocontrol agents; T1 was treated with *Polyspora pinkis* HLY-1; T2 was treated with *Streptomyces tailanhei* NYC 1-5; and T3 was treated with a compound microbial agent.

[0033] Figure 9 The effects of compound microbial inoculant treatment on the activity of antioxidant enzymes and the accumulation of peroxides in tomato roots. A: Superoxide dismutase (SOD); B: Peroxidase (POD); C: Catalase (CAT); D: Hydrogen peroxide (H2O2); E: Malondialdehyde (MDA). Detailed Implementation

[0034] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0035] Unless otherwise specified, all test materials and instruments used in this instruction manual are commercially available.

[0036] Unless otherwise specified, all parts in the examples are by weight, and all percentages are by weight percentages.

[0037] Example 1: Isolation and Identification of the Pathogen of Tomato Root Rot The pathogen was isolated from tomato root rot-infected plants collected from a vegetable field in Danzhou, Hainan. Tissue blocks were excised from the boundary between diseased and healthy tissue, disinfected with 3% sodium hypochlorite, rinsed with sterile water, and then placed on PDA plates for incubation at 25°C. After colony growth, single colonies were purified. Hyphae were picked and their morphology observed under an optical microscope. DNA was extracted from fresh mycelia, and the ITS sequence was amplified by PCR using universal primers ITS1 and ITS4 for the fungal ribosomal ITS region. The reaction volume was 25 μL, and the amplification program was: 94°C pre-denaturation for 5 min; 94°C denaturation for 45 s, 55°C annealing for 45 s, 72°C extension for 105 s, for a total of 30 cycles; and a final extension at 72°C for 10 min. After agarose gel electrophoresis, the amplified product was excised, purified, and sequenced. The obtained sequences were BLASTed for homology in the GenBank database, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA 7.0 software to conduct multiple sequence alignment and evolutionary relationship analysis.

[0038] After culturing on PDA medium at 28°C for 5 days, the colony diameter was 4.8 cm. The mycelium was initially white, with aerial hyphae appearing fluffy, later turning pink to reddish-pink and producing red pigment. Microscopic observation revealed numerous small conidia, oval or kidney-shaped; large conidia were crescent-shaped or sickle-shaped, slightly curved or bent, gradually tapering at both ends. Based on morphological observation, it was preliminarily identified as *Fusarium oxysporum*. Fusarium oxysporum . Figure 1The colony morphology of the pathogen on PDA medium (A) and the morphology of conidia under a microscope (B) are shown. Further sequencing results were compared with the Blast gene sequence in NCBI, showing a 99% similarity to *Fusarium oxysporum*, yielding the accession number TWD26. Phylogenetic analysis confirmed that the pathogen is *Fusarium oxysporum*, named HNF-P1. Figure 2 Phylogenetic tree of HNF-P1 strain of tomato root rot fungus based on ITS sequence.

[0039] Example 2: Pathogenicity determination of Fusarium oxysporum HNF-P1 in tomato and pepper seedlings Fusarium oxysporum HNF-P1 was inoculated onto PDA plates. After sporulation, the sporulation was washed away with sterile water and the concentration was adjusted to 1×10⁻⁶. 6 Spores / mL were prepared for use. Plump and uniform tomato and pepper seeds were selected and soaked in sterile water at 55℃ for 2 hours, then transferred to a bacterial suspension and soaked for 20 minutes. The treated seeds were placed in petri dishes lined with sterile, moistened gauze and incubated at 28℃ for germination. Ten seeds were used for each treatment, with sterile water soaking serving as a control. Germination rate, radicle and plumule growth status, and disease incidence were recorded daily. The treatment was repeated three times.

[0040] The results showed that the HNF-P1 strain was highly pathogenic to tomato and pepper seedlings. Seven days after inoculation, browning symptoms began to appear on the roots and stem base of the seedlings; by day 14, most of the roots and stem base had turned brown and rotted, with a disease incidence rate of 100% and disease indices of 85.0 and 78.3, respectively (Table 1).

[0041] Table 1. Pathogenicity of Fusarium oxysporum HNF-P1 strain to tomato and pepper

[0042] Example 3: Plate antagonistic activity of *Polyspora pinki* against *Fusarium oxysporum* HNF-P1 The antagonistic activity of *Polyspora pinki* against the pathogen HNF-P1 was determined using the plate confrontation method. Pathogen blocks with a diameter of 5 mm were inoculated 2 cm from the edge of a PDA plate, and the tested *Polyspora pinki* was inoculated symmetrically on the opposite side of the plate. A single pathogen-inoculated control was used, and the plates were incubated at 28°C for 10–15 days. The colony radius of the pathogen was measured using the cross-cross method, and the inhibition rate was calculated. Each treatment was repeated three times.

[0043] The results showed that most strains of *Polyspora pinkis* were able to parasitize the pathogen, exhibiting covering and attachment, ultimately leading to its degradation. Among them, strain HLY-1 showed high biocontrol efficacy. Strains HLD-1 and YJS-2-14 had relatively high inhibition rates and large spread distances; strains 54-1 and SYP-1-1 also performed well in terms of spread distance and inhibition rate, indicating that they possess both strong antagonistic and parasitic abilities. Figure 3 This figure illustrates the parasitic activity of several *Polyspora pinkis* strains on *Fusarium oxysporum*. The arrows indicate the spread distance of each strain on the pathogen HNF-P1, from left to right: HLY-1, M5, 54-1, and SYP 1-1. Strain HLY-1 was isolated from Liuyang City, Hunan Province, with the strain accession number CGMCC No. 42332, and is classified as *Polyspora pinkis*. Clonostachys rosea The deposit date is December 1, 2025, and the deposit location is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0044] Table 2. Inhibitory effects of *Alternaria pinki* from different sources on tomato root rot pathogens.

[0045] Example 4: Plate antagonistic activity of actinomycetes from different sources against Fusarium oxysporum HNF-P1 Actinomycetes with biocontrol effects, previously preserved in the laboratory, were selected, and their inhibitory effects on pathogens were determined using the cross-confrontation culture method. Actinomycetes were inoculated at the four corners of PDA plates (2 cm from the edge), with plates without actinomycetes serving as controls. Two days after inoculation, a 5 mm diameter Fusarium oxysporum mycelium disc was placed in the center of the plate, and the plates were incubated at 25°C for another 7 days. The diameter of the pathogen colonies was measured, and the inhibition rate was calculated. Each treatment was repeated three times.

[0046] The actinomycete strain NYC 1-5 in the table was isolated from vegetable field soil in Yinchuan City, Ningxia by our research team and is labeled as strain NYC1-5.

[0047] Table 3. Inhibitory effects of actinomycetes on Fusarium oxysporum HNF-P1

[0048] The results in Table 3 show that the inhibition rate of actinomycete NYC 1-5 against Fusarium oxysporum reached 66.4%, while that of NYC 1-3 was 56.3%, which is significantly better than that of other actinomycetes.

[0049] Example 5: Hydroponic Experiment to Determine the Control Efficacy and Growth-Promoting Ability of Biocontrol Strains Against Tomato Root Rot The 14 *Polyspora pinkis* and *Actinomyces* strains obtained from the initial screening were inoculated into PDB and Gao's No. 1 liquid medium (20 g soluble starch, 1 g KNO3, 0.5 g NaCl, 0.5 g K2HPO4·3H2O, 0.5 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, with water added to 1000 mL), respectively. The medium was cultured at 28°C with shaking at 180 r / min for 4 days, and the fermentation broth concentration was adjusted to 1×10⁻⁶. 7 Spores / mL for later use.

[0050] Tomato and pepper seeds were disinfected with 1% sodium hypochlorite, rinsed, soaked in warm water, and germinated in the dark with damp gauze for 24 hours. They were then sown in sterilized substrate soil and cultivated for 10 days. Select seedlings with uniform growth and true leaves, and transplant them into test tubes containing 15 mL of 1 / 8 MS medium (containing 1.9 g potassium nitrate, 1.65 g ammonium nitrate, 0.17 g potassium dihydrogen phosphate, 0.37 g magnesium sulfate, 0.44 g calcium chloride, 0.83 mg potassium iodide, 6.2 mg boric acid, 22.3 mg manganese sulfate, 8.6 mg zinc sulfate, 0.25 mg sodium molybdate, 0.025 mg copper sulfate, 0.5 mg cobalt chloride, 0.5 mg nicotinic acid, 37.3 mg disodium EDTA, 27.8 mg ferrous sulfate, 100 mg inositol, 2 mg glycine, 0.1 mg thiamine hydrochloride, and 0.5 mg pyridoxine hydrochloride). Inoculate with the respective biocontrol bacterial solutions, and after 0.5 h, inoculate with 1×10⁻⁶ cells / mL. 6 A pathogen suspension of [number] cells / mL was cultured at 25°C under a 16-h / 8-h light-dark cycle. Seed germination and root development were observed, and the seed vigor index was calculated as: Seed vigor index = germination rate × (root length + shoot length). After 10 days, plant height, root length, fresh weight, and dry weight were measured. Simultaneously, the disease incidence was investigated according to the tomato root rot grading standard: Grade 1: No root symptoms; Grade 3: Slight root discoloration, discolored area ≤ 25%; Grade 5: 25% < discolored area ≤ 50%; Grade 7: 50% < discolored area ≤ 75%; Grade 9: Discolored area > 75%. The disease index and control efficacy were calculated. An equal volume of sterile water was used as a control instead of the fermentation broth. Each treatment consisted of 15 seedlings, with three replicates.

[0051] Hydroponic experiments showed that the addition of biocontrol bacteria solution resulted in robust growth of tomato and pepper seedlings in vitro, with well-developed root systems and a significant reduction in root browning. Specifically, the *Aspergillus oryzae* HLY-1 strain achieved 88.9% control efficacy against tomato seedling root rot, while *Actinomyces NYC 1-5* achieved 74.6% efficacy. Against pepper root rot, the control effects of HLY-1 and NYC 1-5 strains reached 86.5% and 71%, respectively (Tables 4 and 5). After application of the inoculant, the germination rate of tomato and pepper seeds was 100%, and the seed vigor index was significantly improved. *Aspergillus oryzae* HLY-1, *Actinomyces NYC 1-5*, and other strains all exhibited significant growth-promoting effects, with significant increases in multiple growth indicators such as plant height, root length, and fresh and dry weight (Table 6). Figure 4 The study demonstrates the control efficacy of two strains of *Alternaria pinki* and two strains of actinomycetes against root rot in hydroponically grown tomato seedlings. From left to right, the treatments are: pathogen control, water control, and treatments with biocontrol agents HLY-1, SYP 1-1, NYC 1-5, and NYC 1-3.

[0052] Table 4. Effects of candidate *Polyspora pinki* and *Actinomyces* strains on root rot in hydroponic tomato and pepper seedlings.

[0053] Table 5. Effects of candidate *Polyspora pinkis* and *Actinomyces* strains on tomato seed germination and growth.

[0054] Example 6 Morphological and molecular biological identification of Actinomycete NYC 1-5 strains The actinomycete strain NYC 1-5 was inoculated onto Gao's No. 1 medium using the streak plating method and incubated at 28°C for 7 days. Observations showed that the NYC 1-5 colonies were initially white, turning milky white after 3 days. Aerial mycelium growth was vigorous, and the colonies exhibited a spreading growth pattern. The colonies were dense, dry, and powdery. Under a microscope, NYC 1-5 showed typical spiral spore chains with smooth spore surfaces. Figure 5 The colony morphology and microstructure of strain NYC-1-5 are shown. A is the front view of the colony; B is the back view of the colony; C is the microstructure of hyphae and spores.

[0055] Simultaneously, strain NYC-1-5 was inoculated into Gao's liquid medium and cultured at 28℃ with shaking at 180 r / min for 3 days. Bacterial cells were collected, and DNA was extracted using a bacterial genomic DNA extraction kit. The 16S rRNA gene sequence was amplified by PCR using universal primers 27F / 1492R. The amplified product was purified and sent to Sangon Biotech for sequencing. The obtained sequences were BLAST aligned in the NCBI database, and a phylogenetic tree was constructed based on the housekeeping gene. The results showed that NYC 1-5 is related to *Streptomyces tailanhei*. Streptomyces tailanensis (GCF-008386495.1) clustered into one branch in the phylogenetic tree, with a bootstrap value of 100. Based on morphological and molecular identification results, NYC 1-5 was identified as *Streptomyces tailanhei*. Figure 6 Phylogenetic tree of actinomycete NYC 1-5 strains constructed based on 16S rRNA gene sequence.

[0056] The NYC 1-5 strains were classified and named *Streptomyces tailanhei* (…). Streptomyces tailanensis The strain has accession number CGMCC No. 36965, was deposited on December 9, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0057] Example 7 Liquid fermentation of *Polyspora pinkis* strain HLY-1 The pink spiral polyspora HLY-1 strain was inoculated onto PDA and cultured for 7 days. After washing away the spores with 5 mL of sterile water, the culture was inoculated into PDB medium and cultured in the dark at 27°C on a shaker for 2 days to prepare a seed culture.

[0058] Fermentation medium was prepared containing 30 g sucrose, 6 g yeast extract, 6 g soybean meal powder, 1 g K₂HPO₄, 0.5 g NaCl, 0.5 g MgSO₄•7H₂O, 0.05 g FeSO₄•7H₂O, and 0.05 g ZnSO₄•7H₂O. Water was added to a final volume of 1000 mL, and the pH was adjusted to 6.0 with 10% HCl solution. The fermentation medium was dispensed into Erlenmeyer flasks and sterilized at 121℃ for 30 min. After cooling to room temperature, the seed culture was inoculated into the fermentation medium at a 2% inoculum. The culture was incubated at 27℃ with shaking at 180 r / min for 4 days. The concentration of the fermentation broth was determined to be 2.0 × 10⁻⁶ using a hemocytometer. 8 Spores / mL.

[0059] Example 8: Screening of *Streptomyces tailanhei* NYC 1-5 strains on culture medium To obtain a suitable microbial agent for production and field application, single-factor experiments were conducted to screen and optimize the fermentation medium for Streptomyces tyc. 1-5 strain from Tailan River.

[0060] The NYC 1-5 strain was streaked onto Gao's No. 1 solid medium plates and cultured at 28°C for 10 days to form mature colonies. 5×5 mm bacterial blocks were cut from the plates and inoculated into Gao's No. 1 liquid medium. The culture was then incubated at 28°C and 180 r / min for 60 h to prepare a seed culture.

[0061] Carbon source screening: Liquid culture media containing different carbon sources were prepared to screen suitable carbon sources for sporulation of *Streptomyces tailanhei*. Six treatments were set up: (1) glucose, (2) sucrose, (3) malt extract, (4) molasses, (5) soluble starch, and (6) glucose / soluble starch = 1:5 (mass ratio). The carbon source content in the culture medium was 30 g, and the other components were: 10 g yeast extract, 1 g K₂HPO₄, 1 g NaCl, 0.5 g MgSO₄•7H₂O, and 1 g CaCO₃. Water was added to 1000 mL, and the pH was adjusted to 7.4. 100 mL of liquid culture medium was added to a 500 mL Erlenmeyer flask, sterilized by high-temperature moist heat, and after cooling to room temperature, the seed culture was inoculated into the culture media with different carbon sources at a 1% inoculum rate. The culture was incubated at 26℃ and 180 r / min for 72 h to obtain the fermentation broth. The spore content in the different fermentation broths was determined by counting with a hemocytometer, with three replicates for each treatment. The results are shown in Table 6. When a combined carbon source of glucose and soluble starch was used, *Streptomyces tailanhei* NYC 1-5 produced the highest sporulation, reaching 1.26 × 10⁻⁶. 8 The number / mL was significantly different from other treatments. P <0.05).

[0062] Table 6. Effects of different carbon sources on sporulation of *Streptomyces tailanhei* NYC 1-5 deal with carbon source Spore count (x 10 8 cells / mL) 1 glucose 0.63±0.07 b 2 sucrose 0.27±0.01 a 3 Malt extract 0.75±0.11 bc 2 Molasses 0.87±0.09 c 4 Soluble starch 0.79±0.10 bc 6 Glucose / Soluble Starch 1.26±0.15 d Nitrogen source screening: Liquid culture media containing different nitrogen sources were prepared, and suitable nitrogen sources for sporulation of *Streptomyces tailanhei* NYC 1-5 were screened by shaking flask culture. A total of eight treatments were set up: (1) soybean meal, (2) yeast extract, (3) peptone, (4) urea, (5) (NH4)2SO4, (6) soybean meal / peptone / (NH4)2SO4 (nitrogen mass ratio 6:2:1), and (7) yeast extract / (NH4)2SO4 (nitrogen mass ratio 5:1). Different types of nitrogen sources were used in the experiment, including organic nitrogen, inorganic nitrogen, and organic-inorganic nitrogen combinations, to achieve higher fermentation levels and minimize costs. For safety reasons, urea and (NH4)2SO4 were used to replace KNO3 in Gao's No. 1 medium for inorganic salt screening.

[0063] The other ingredients were formulated as follows: 30 g soluble starch, 1 g K₂HPO₄, 1 g NaCl, 0.5 g MgSO₄•7H₂O, and 1 g CaCO₃, diluted with water to 1000 mL, and the pH was adjusted to 7.4. 100 mL of liquid culture medium was added to a 500 mL Erlenmeyer flask, and the mixture was sterilized by high-temperature moist heat. After cooling to room temperature, the seed culture was inoculated into culture media with different nitrogen sources at a 1% inoculum rate. The cultures were incubated at 28°C with shaking at 180 r / min for 72 h to obtain the fermentation broth. Spore content in different fermentation broths was determined using a hemocytometer, with three replicates for each treatment. The results are shown in Table 8. Significant differences were found among the different nitrogen source treatments. P <0.05). When organic nitrogen soybean flour, yeast extract, peptone, or inorganic nitrogen urea or (NH4)2SO4 are used as nitrogen sources, the fermentation level of NYC 1-5 strain is (0.65~1.60)×10⁻⁶. 8 Spores / mL. Inorganic nitrogen can quickly provide nitrogen, accelerate growth, and reduce raw material costs, but the overall spore production is relatively low; organic nitrogen is beneficial for microbial growth and metabolism. Combining organic and inorganic nitrogen, with (NH4)2SO4 in the culture medium as a readily available nitrogen source, allows biocontrol bacteria to germinate and grow rapidly, while soybean flour, peptone, or yeast extract as a sustained nitrogen source provides long-term benefits for cell growth and spore production. When soybean flour / peptone / (NH4)2SO4 is used as a composite nitrogen source, the fermentation level reaches 2.11 × 10⁻⁶. 8 The spores / mL were 31.9% and 52.9% higher than those from soybean meal and peptone alone, respectively.

[0064] Table 7. Effects of different nitrogen sources on sporulation of *Streptomyces tailanhei* NYC 1-5 deal with nitrogen source Spore count (x 10 8 cells / mL) 1 Soy flour 1.60±0.07 bc 2 Yeast extract 1.35±0.21 b 3 peptone 1.38±0.19 b 4 urea 0.65±0.25 a 5 (NH4)2SO4 0.81±0.17 a 6 Soy flour / peptone / (NH4)2SO4 2.11±0.20 d 7 <![CDATA[Yeast extract powder / (NH4)2SO4]]> 1.75±0.12 c Based on the above experimental results, the present invention selects the following formulation for the cultivation of Streptomyces tyrannus NYC 1-5 strain: Add 20-30 g of soluble starch, 5-10 g of glucose, 10-20 g of soybean flour, 1-5 g of peptone, 0.5-2 g of (NH4)2SO4, 0.5-2 g of K2HPO4, 0.5-2 g of NaCl, 0.1-0.5 g of MgSO4•7H2O, and 0.5-2 g of CaCO3 to water to 1000 mL and adjust the pH to 7.0-7.5.

[0065] Example 9: Liquid fermentation of Streptomyces tyrannus NYC 1-5 strains The NYC 1-5 strain was streaked onto Gao's No. 1 solid medium plates and cultured at 28°C for 10 days to form mature colonies. 5×5 mm bacterial blocks were cut from the plates and inoculated into Gao's No. 1 liquid medium. The culture was then incubated at 28°C and 180 r / min for 60 h to prepare a seed culture.

[0066] To prepare the fermentation medium, weigh out 25 g of soluble starch, 5 g of glucose, 12 g of soybean flour, 2 g of peptone, 0.5 g of (NH4)2SO4, 1 g of K2HPO4, 1 g of NaCl, 0.5 g of MgSO4•7H2O, and 1 g of CaCO3. Add water to 1000 mL and adjust the pH to 7.4 with 10% NaOH solution. Sterilize at 121℃ for 30 min. After cooling, inoculate the seed culture into the fermentation medium at a rate of 5%. Incubate at 28℃ with shaking at 200 r / min for 4 days. The concentration of the fermentation broth was determined to be 1.8 × 10⁻⁶ using a hemocytometer. 8 Spores / mL.

[0067] Example 10 Biocompatibility determination of *Polyspora pinkis* HLY-1 and *Streptomyces tailanhei* NYC 1-5 To investigate the interaction between *Streptomyces tailanhei* and *Alternaria pinki*, the plate confrontation method was used to assess whether the two biocontrol strains exhibited inhibitory effects. HLY-1 and NYC 1-5 strains were inoculated onto PDA and Gao's No. 1 media, respectively, and cultured at 28°C for 7 days. Mycelial discs with a diameter of 5 mm were punched using a sterile punch. Four actinomycete mycelial discs were placed at equal intervals around the perimeter of the plate and pre-cultured at 28°C for 2 days. Then, *Alternaria pinki* mycelial discs were inoculated into the center of the PDA plate and cultured further. The growth status, contact zone morphology, and inhibition zone formation of the two strains were observed. The results showed that after 7 days of co-culture, both *Alternaria pinki* HLY-1 and actinomycete NYC 1-5 colonies grew normally, and no obvious inhibition zone was formed in the contact area. Figure 7 The results showed that under the experimental conditions, the growth of the biocontrol bacteria *Polyspora pinkis* HLY-1 and *Streptomyces tailanhei* NYC 1-5 was not inhibited by each other, indicating that the two have high biocompatibility.

[0068] Example 11 Evaluation of the synergistic control and growth-promoting effects of compound microbial inoculants on tomato root rot under greenhouse conditions. The HLY-1 fermentation broth obtained in Example 7 and the NYC 1-5 fermentation broth obtained in Example 9, along with a compound microbial agent prepared by mixing them (spore ratio 1:1) (the spore content in T1 to T3 was adjusted to be the same, and in T3, the content of each was halved, while the total amount (volume) remained unchanged), were used to determine the effect on greenhouse potted plants against tomato root rot and on plant growth. The experiment included five treatments: T1: HLY-1 fermentation broth; T2: NYC 1-5 fermentation broth; T3: compound microbial agent; T4: pathogen control (CK); and T5: water blank control.

[0069] Take garden soil and sterilize it. Fill 15 cm × 12 cm plastic pots, 200 g per pot. Sow tomatoes in seedling trays. When the seedlings have 4-5 true leaves, select seedlings with uniform growth, dip their roots in a biocontrol agent (4 mL per plant), and then transplant them into plastic pots. Cultivate in a greenhouse at a constant temperature of 25 ℃ with a light / dark cycle of 16 h / 8 h. 24 h after transplanting, inoculate the rhizosphere with 2 mL of Fusarium oxysporum spore suspension (2 × 10⁻⁶). 7 (Spores / mL). Disease incidence was investigated 30 days later, the disease index was calculated, and the control effect was determined. Plant height, stem diameter, root length, plant fresh and dry weight, and chlorophyll content were also measured. Four pots were used per treatment, with three plants per pot. Each treatment was replicated three times.

[0070] The results showed that *Alternaria pinki* HLY-1 and *Actinomyces NYC1-5* had good control effects on tomato root rot, with a significant reduction in the disease index after treatment, and control efficiencies of 74.1% and 65.2%, respectively. The combined application of the two biocontrol agents showed a synergistic effect, achieving a control efficacy of 82.1%. Simultaneously, the biocontrol agent treatment significantly increased tomato plant height and above-ground and underground fresh weight, with chlorophyll and nitrogen content approaching the levels of healthy plants not treated with the pathogen, indicating that the biocontrol agents helped maintain normal plant physiological functions while mitigating disease. The combined agent treatment showed the most significant growth-promoting effect, increasing plant height by 36.8% and more than doubling above-ground and underground biomass compared to the control (CK). Figure 8 To investigate the efficacy of biocontrol agents in controlling tomato root rot in greenhouse potted plants, T1, T2, and T3 were compared with the incidence and growth of tomato root rot after treatment with *Alternaria pinki* HLY-1, *Streptomyces tailanhei* NYC 1-5, and a compound microbial agent, respectively.

[0071] Table 8. Control efficacy of biocontrol agents against tomato root rot under greenhouse pot cultivation conditions. deal with Disease index Preventive efficacy (%) CK 75.8±6.3 a - HLY-1 19.6±4.9 bc 74.1±3.9 b NYC 1-5 26.4±7.7 b 65.2±7.5 b Compound microbial agent 13.6±2.8 cd 82.1±2.6 a Table 9. Effects of biocontrol agents on tomato growth under greenhouse pot cultivation conditions. deal with Plant height (cm) Stem diameter (mm) Chlorophyll (SPAD) Nitrogen content (mg / g) Fresh weight on the ground (g) Dry weight on the ground (g) Fresh weight underground (g) Dry weight of underground soil (g) CK 25.08±0.51 d 3.24±0.08 a 35.87±0.94 b 11.29±0.90 b 10.29±1.35 b 2.23±1.09 a 3.96±1.30 d 0.84±0.77 a Clear water 37.24±1.10 a 3.25±0.14 a 42.42±2.03 a 13.26±0.44 a 12.74±1.54 b 2.91±0.82 a 7.30±0.72 b 1.17±0.36 a HLY-1 32.00±0.83 bc 3.45±0.15 a 41.82±0.87 a 13.08±0.29 a 15.38±3.34 ab 3.33±0.72 a 7.01±1.8a 1.26±0.58 a NYC 1-5 31.25±0.95 bc 3.25±0.09 a 41.19±3.70 a 12.20±1.18 a 14.33±0.31 ab 4.79±1.97 a 6.56±0.73 bc 1.11±0.46 a Compound microbial agent 34.30±1.25 a 3.29±0.12 a 42.34±0.54 a 13.15±0.16 ab 18.65±1.42 a 5.28±1.84 a 7.57±2.52 a 1.73±1.10 a Example 12 Effects of biocontrol agents on antioxidant enzyme activity and peroxide content in tomato plants Tomatoes were planted according to the method in Example 11, with the addition of HLY-1 fermentation broth obtained in Example 7 and NYC1-5 fermentation broth obtained in Example 9, along with a mixed compound microbial agent. Sixty days after inoculation, tomato roots from each treatment were collected, repeatedly rinsed with tap water, and dried. 0.1 g of root sample was weighed, and the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA), were determined using a kit. Each treatment was replicated three times.

[0072] Figure 9 This study demonstrates the effects of compound microbial inoculant treatment on the activity of antioxidant enzymes and the accumulation of peroxides in tomato roots. The upper part of the figure shows the activities of three antioxidant enzymes: superoxide dismutase, peroxidase, and catalase; the lower part shows the contents of peroxides—hydrogen peroxide and malondialdehyde. The results indicate that treatment with *Polyspora pinki* HLY-1 and *Actinomyces NYC 1-5* significantly increased the activity of antioxidant enzymes in tomato roots. P < 0.05), among which the activities of SOD and POD increased by 41.0% and 59.8% respectively after treatment with compound microbial agents. At the same time, the content of peroxidation product MDA decreased by 56.6% compared with the control, indicating that biocontrol agents can improve the activity of antioxidant enzymes, enhance the ability of plant cells to scavenge reactive oxygen species, reduce oxidative damage, and induce plant resistance.

[0073] Although the present invention has been described above through embodiments, those skilled in the art should understand that any improvements and modifications made to the present invention without departing from its spirit and essence should fall within the protection scope of the present invention.

Claims

1. A compound inoculant of fungi and actinomycetes for the prevention and control of root rot, characterized in that, The compound microbial agent contains *Polyspora pinki* with accession number CGMCC No. 42332 and *Streptomyces tailanhei* with accession number CGMCC No. 36965.

2. The fungal and actinomycete compound inoculant according to claim 1, characterized in that, The compound microbial agent contains the fermentation products of *Polyspora pinkis* and *Streptomyces tailanhei*, with the spore ratio of *Polyspora pinkis* to *Streptomyces tailanhei* being 1:0.5~2.

3. The fungal and actinomycete compound inoculant according to claim 1, characterized in that, The compound microbial agent is available in liquid, granule, or powder form. In the liquid form, the bacterial content of *Polyspora pinkis* and *Streptomyces tailanhei* is (5~50) × 10⁻⁶. 7 Spores / mL.

4. The fungal and actinomycete compound inoculant according to any one of claims 1 to 3, characterized in that, It is prepared through the following steps: 1) Plate culture: The *Polyspora pinkis* and *Streptomyces tailanhei* were inoculated onto potato dextrose agar and Gao's No. 1 medium, respectively, and cultured at 26-28℃ for 7-14 days until sporulation. 2) Seed culture: Elute the spores to prepare a spore suspension, or cut the mycelium blocks from the cultured plate and inoculate them into potato dextrose liquid medium and Gao's No. 1 liquid medium, respectively, and culture them in a shaker at 25~28℃ for 48~84 h to obtain the seed culture. 3) Fermentation culture: Inoculate the seed liquid into the liquid fermentation medium and culture for 60-120 h to obtain the fermentation broth; 4) Formulation: Mix the fermentation broth of *Polyspora pinkis* and *Streptomyces tailanhei* obtained in step 3) to obtain a liquid formulation of the fungal and actinomycete compound agent, or add fillers and adjuvants to prepare granules or powders.

5. The fungal and actinomycete compound inoculant according to claim 4, characterized in that, In step 3), the liquid fermentation medium for the fermentation culture of *Polyspora pinkis* is formulated as follows: 10-40 g sucrose, 5-15 g yeast extract, 5-15 g soybean meal powder, 0.5-2 g K₂HPO₄, 0.1-1 g NaCl, 0.1-1 g MgSO₄•7H₂O, 0.05-0.1 g FeSO₄•7H₂O, 0.01-0.1 g ZnSO₄•7H₂O, with water added to 1000 mL, and the pH adjusted to 5.5-6.

5.

6. The fungal and actinomycete compound inoculant according to claim 4, characterized in that, In step 3), the liquid fermentation medium for the fermentation culture of Streptomyces tailanhe is formulated as follows: 20-30 g soluble starch, 5-10 g glucose, 10-20 g soybean flour, 1-5 g peptone, 0.5-2 g (NH4)2SO4, 0.5-2 g K2HPO4, 0.5-2 g NaCl, 0.1-0.5 g MgSO4•7H2O, 0.5-2 g CaCO3, with water added to 1000 mL, and the pH adjusted to 7.0-7.

5.

7. The fungal and actinomycete compound inoculant according to claim 4, characterized in that, In step 3), the fermentation culture conditions of *Polyspora pinkis* and *Streptomyces tailanhei* are independent of each other: seed liquid inoculation amount 1~6%, rotation speed 150~220 r / min, culture temperature 25~30℃, and culture time 72~108 h.

8. The fungal and actinomycete compound inoculant according to claim 4, characterized in that, In step 4), the fermentation broth or fermentation broth mixture is subjected to operation A or B: A: add filler, mix and granulate to obtain granules; B: add filler and additives, dry and pulverize to obtain powder. The filler is one or more of diatomaceous earth, kaolin, bentonite, talc, starch, dextrin, and peat moss; the auxiliary agent is one or more of sodium carboxymethyl cellulose, chitosan, polyethylene glycol, sodium lignosulfonate, sodium dodecylbenzenesulfonate, amino oligosaccharide, and sodium alginate; the filler is added at 80% to 300% of the fermentation broth mass, and the auxiliary agent is added at 0.1% to 10% of the fermentation broth mass.

9. The application of the fungal and actinomycete compound agent according to any one of claims 1 to 8 in the prevention and control of root rot of tomatoes, peppers, strawberries, and watermelons caused by Fusarium, and in promoting plant growth.

10. The application according to claim 9, characterized in that, The application method is as follows: apply the compound fungi and actinomycetes at one or more of the following periods: seedling stage, transplanting stage, and growth stage. The application methods include seed soaking, seed mixing, root dipping, root irrigation, drip irrigation, or soil irrigation.

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