A strain of Streptomyces SK-2 and its application in the control of white cone rot.

Abstract

This invention discloses a strain of Streptomyces SK-2 and its application in the control of white cone rot. The Streptomyces strain of this invention (… Streptomyces SK-2 (sp.) was isolated from the rhizosphere soil of Chinese fir soil. This biocontrol bacterium can significantly inhibit the occurrence area of ​​white cone rot disease under indoor conditions, with a prevention effect of more than 68%, and has potential development and application value.

Description

Technical Field

[0001] This invention belongs to the field of biological technology, specifically relating to a strain of Streptomyces SK-2 and its application in the prevention and control of white cone rot. Background Technology

[0002] White cone ( Castanopsis faberi *Pinus chinensis*, belonging to the Fagaceae family, is an evergreen, deciduous tree and an excellent native broadleaf timber species in South China. This species possesses the dual function of improving soil structure and controlling soil erosion, making it a core species for constructing ecological public welfare forest systems. *Pinus chinensis* canker is a serious disease affecting its production and timber use, primarily damaging the branches, trunks, and roots, weakening the tree's growth. Previous research by the Forestry Biological Insecticide Team of the Guangdong Academy of Forestry Sciences has identified *Dispora trichophyton* as the pathogen causing *Pinus chinensis* canker. Lasiodiplodia sp.

[0003] Currently, there are few reports on research regarding *Dioscorea bulbifera* rot disease both domestically and internationally. *Dioscorea bulbifera* has a wide host range and is highly pathogenic. Traditional control methods, such as chemical and physical control, have limitations, including environmental pollution and pathogen resistance. Biocontrol bacteria, on the other hand, have advantages such as a broad antimicrobial spectrum and diverse mechanisms of action, showing great potential in controlling plant diseases and playing a crucial role in developing sustainable control strategies for *Dioscorea bulbifera* rot. Therefore, finding an environmentally friendly and efficient control method is urgently needed.

[0004] Actinomycetes possess significant advantages such as being non-toxic, residue-free, having a broad antibacterial spectrum, diverse mechanisms of action, and long-lasting efficacy, effectively overcoming many shortcomings of chemical pesticides. Their core mechanism of action lies in the fact that actinomycetes can secrete various secondary metabolites, which exhibit antibacterial, growth-promoting, and resistance-inducing properties in plant disease control. Furthermore, actinomycetes can transform soil substances into easily absorbed and utilized forms through nitrogen fixation, potassium solubilization, and phosphorus dissolution, effectively improving soil nutrients and promoting plant growth. This is of great significance for developing sustainable biological control strategies. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a Streptomyces SK-2 strain (which was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on April 27, 2025, with accession number GDMCC No. 66221) and its application in the prevention and control of white cone rot disease.

[0006] The first objective of this invention is to provide a Streptomyces SK-2, with accession number GDMCC No. 66221.

[0007] A second objective of this invention is to provide a biological agent containing a culture of the aforementioned Streptomyces SK-2 as an active ingredient.

[0008] Preferably, the concentration of the Streptomyces SK-2 culture in the formulation is OD. 600 =0.3 or above.

[0009] Preferably, the concentration of the Streptomyces SK-2 culture in the formulation is OD. 600 =0.3-0.6.

[0010] Preferably, the culture of Streptomyces SK-2 is obtained by inoculating Streptomyces SK-2 onto ISP1, ISP2, ISP3, ISP4, ISP5, ISP6, ISP7, NA, LB, KMB, PDA, Chapman's or Gao's No. 1 medium and culturing it.

[0011] A third object of the present invention is to provide the use of the Streptomyces SK-2 or the preparation thereof in at least one of the following (1)-(2):

[0012] (1) Promotes plant growth;

[0013] (2) Prevention and control of plant rot disease.

[0014] Preferably, the promotion of plant growth is achieved by secreting extracellular enzymes, fixing nitrogen, solubilizing potassium, producing iron, and / or dissolving organic phosphorus.

[0015] Preferably, the extracellular enzyme is a protease, cellulase, amylase, and / or lipase.

[0016] Preferably, the plant is Conophytum comosum.

[0017] A fourth objective of this invention is to provide a method for preventing and treating white cone rot, comprising the step of applying the Streptomyces SK-2 or the preparation to white cones.

[0018] The beneficial effects of this invention are:

[0019] 1. The Streptomyces SK-2 bacterial solution of the present invention is effective against Diplosporum tricolor, the pathogen of Conyza rot. Lasiodiplodia sp. has a good antagonistic effect and can be used to control white cone rot in forestry production. At the same time, it provides a new strain resource for the development of biological agents with broad-spectrum antibacterial activity, and has good research and application value.

[0020] 2. The Streptomyces SK-2 strain of the present invention is a biological control strain with potential application value in the prevention and control of white cone rot. It can be developed into a microbial fungicide, providing technical support for the green prevention and control of white cone rot, and has good application prospects.

[0021] 3. This invention supplements the morphological characteristics, physiological and biochemical properties, and detection of extracellular enzymes and life-promoting substances of Streptomyces SK-2. Among them, strain SK-2 has the ability to dissolve organophosphorus compounds, fix nitrogen, solubilize potassium and produce siderophores. It can also produce proteases, cellulases, amylases and lipases, which provides a certain theoretical basis for subsequent research on this strain.

[0022] Preservation Instructions

[0023] The present invention Streptomyces sp. SK-2 (Streptomyces SK-2) was deposited on April 27, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No. 66221, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0024] Figure 1 The images show the colony morphology of Streptomyces SK-2 on ISP2 medium on day 3; where A is the front view of a Streptomyces SK-2 colony and B is the back view of a Streptomyces SK-2 colony.

[0025] Figure 2 Image of the hyphae of Streptomyces SK-2 (Gram staining).

[0026] Figure 3 Phenotypic results of Streptomyces SK-2 in various differential culture media.

[0027] Figure 4 A phylogenetic tree constructed based on the 16S rDNA sequence of Streptomyces SK-2.

[0028] Figure 5 Phenotypes of Streptomyces SK-2 on lipase (A), protease (B), amylase (C), and cellulase (D) media.

[0029] Figure 6 Phenotypic results of Streptomyces SK-2 cultured on modified Ashby nitrogen-fixing medium (A), NBRIP inorganic phosphorus medium (E), Monkina organic phosphorus medium (B), potassium-solubilizing bacteria solid medium (C), and modified CAS solid medium (D).

[0030] Figure 7 The efficacy of Streptomyces SK-2 against white cone rot in potted plants was evaluated; where A was the control group and B was the treatment group I. 600 =0.3, C is the OD of treatment group II 600 =0.6. Detailed Implementation

[0031] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0032] Example 1: Isolation and Identification of Streptomyces SK-2

[0033] strain SK-2 ( Streptomyces The strain (species) was isolated from the rhizosphere soil of Chinese fir soil. Using *Dioscorea brasiliensis* BZ-FL-1 as the target bacterium, strain SK-2 was obtained by plate confrontation method, and the inhibition rate of *Dioscorea brasiliensis* BZ-FL-1 was 68.02%.

[0034] 2. Identification of strains

[0035] 2.1 Morphological identification

[0036] Strain strain SK-2 was cultured on a PDA (PDA prepared from fresh potato tubers, hereinafter referred to as PDA (tubers)) plate, and the morphology of the colonies is as follows. Figure 1 As shown, Figure 1 In this text, A represents the front view of a colony of strain SK-2. Figure 1 B in the text represents the reverse side of a colony of strain SK-2.

[0037] from Figure 1 As can be seen, on the PDA (block) plate, the colonies of strain SK-2 are round, pale yellow on the back, wrinkled on the surface, smooth at the edges, milky white in color, with fine white hairs on the central raised part, and the colony diameter is mostly 3-6 mm, and the colonies are opaque.

[0038] Under a microscope, the bacteria showed Gram-positive staining and were filamentous. Figure 2 It has branches and breaks, no septa, and no spores.

[0039] The phenotypic differences of strain SK-2 were significant in various differential culture media. For example... Figure 3As shown, the mycelium grew most vigorously on ISP1-ISP3 and KMB media, and least abundantly on Czapek's medium. The aerial mycelium was predominantly white and yellow, except for a light brown color on ISP6 and PDA (PDA prepared using potato dextrose solution from Guangzhou Huankai Biotechnology (catalog number: 021053), hereinafter referred to as PDA (powder)). The substrate mycelium was varying shades of yellow on ISP2, ISP4, ISP5, and ISP7, and off-white on ISP6; the color was consistent with the aerial mycelium on other media. Strain SK-2 produced soluble pigments on ISP2, ISP4-7, NA, KMB, and PDA (blocks); light brown on ISP2, ISP7, NA, and KMB media; dark brown on ISP6 and PDA (powder) media; and off-white on ISP4 and ISP5 media. In addition, strain SK-2 forms a transparent halo around its colonies on ISP3 medium and a white, blurry halo around its colonies on modified Gao's No. 1 medium.

[0040] 2.2 Physiological and Biochemical Characteristics

[0041] Physiological and biochemical characteristics of the target strain SK-2 were identified, including arginine reduction properties, xylose, lactose, sucrose, maltose, mannitol, nitrate reduction, hydrogen sulfide, melanin, VP, methyl red test MR-VP, glucose OF, DNase, acetamase, citrate, milk coagulation, milk peptone formation, and gelatin liquefaction.

[0042] Table 1 Physiological and biochemical characteristics of strain SK-2

[0043]

[0044] Note: "+" indicates a positive result; "-" indicates a negative result.

[0045] The physiological and biochemical results are shown in Table 1. The results of sucrose, hydrogen sulfide, melanin and gelatin liquefaction of strain SK-2 were positive, while the results of the other results were negative. This indicates that strain SK-2 can decompose sucrose, produce melanin and hydrogen sulfide, and secrete gelatinase.

[0046] 2.3 Molecular biological identification

[0047] The SK-2 strain was transferred to ISP2 medium and cultured for 3 days. The bacterial cells were then scraped off.

[0048] The genome of the strain was extracted by thermal lysis. A single colony was inoculated into a 1.5 mL centrifuge tube containing 50 μL of sterile water, lysed at 100℃ for 10 min, centrifuged at 10000 rpm for 5 min, and the supernatant was used as a template.

[0049] The 16S rDNA genome was amplified using universal primers 27F / 1492R (5'-AGAGTTGATCCTGGCTCAG-3', SEQ ID NO.1; 5'-TGGTTACCTTGTTACGACTT-3', SEQ ID NO.2).

[0050] The 25 μL PCR amplification reaction system consisted of 12.5 μL of 2×Taq PCR Mix, 1.0 μL each of forward and reverse primers, 1.0 μL of DNA template, and ddH2O to a final volume of 25 μL. The PCR amplification conditions were: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 0.5 min, 55℃ annealing for 0.5 min, 72℃ extension for 1.5 min, for 34 cycles, followed by a final extension at 72℃ for 10 min. The PCR products were sequenced using bidirectional sequencing performed by Shanghai Sangon Biotech Co., Ltd. The 16S rDNA sequencing results of SK-2 (its nucleotide sequence is shown in SEQ ID NO.3) were submitted to NCBI for sequence alignment. Homologous sequences with high similarity were screened, and sequence alignment was performed using ClustalW in MEGA 7.0 software. A phylogenetic tree of strain SK-2 was constructed using the neigbor-joining method. A bootstrap test was performed during phylogenetic tree construction, with 1000 repeated samplings. The phylogenetic tree is shown below. Figure 4 .

[0051] from Figure 4 It can be seen that strain SK-2 is similar to Streptomyces strains. Streptomyces sp . Based on the clustering of species in the same branch and the combined results of morphological identification and 16S rDNA sequence analysis, Streptomyces SK-2 was preliminarily identified as Streptomyces. Streptomyces sp.)SK-2.

[0052] Example 2: Detection of extracellular enzymes and life-promoting substances produced by Streptomyces SK-2

[0053] 1. Detection of the extracellular enzyme production capacity of Streptomyces SK-2

[0054] Streptomyces SK-2 was inoculated into protease-producing, cellulase-producing, amylase-producing, and lipase-producing mediums, with 3 replicates, and cultured in the dark at 30°C for 3 days.

[0055] from Figure 5 It can be seen that a turbid halo appeared around the Streptomyces SK-2 bacterial block on the lipase detection medium. Figure 5 In the A section, transparent hydrolysis zones appeared around Streptomyces SK-2 bacterial blocks on protease, amylase, and cellulase detection media. Figure 5The results (B, C, D) indicate that Streptomyces SK-2 possesses the ability to synthesize lipase, protease, amylase, and cellulase.

[0056] Streptomyces SK-2 was inoculated on modified Ashby nitrogen-fixing medium, NBRIP inorganic phosphorus medium, Monkina organic phosphorus medium, potassium-solubilizing bacteria solid medium and modified CAS solid medium, with 3 replicates for each.

[0057] from Figure 6 It can be seen that Streptomyces SK-2 can still grow normally after multiple subcultures on modified Ashby nitrogen-fixing medium. Figure 6 The A in the figure indicates that Streptomyces SK-2 has nitrogen-fixing ability; transparent hydrolysis zones were formed in the test media for organophosphates, potassium solubilizers, and iron-producing carriers. Figure 6 (B, C, D in the text), but no transparent hydrolysis zone appeared on the inorganic phosphorus medium ( Figure 6 The E in the figure indicates that Streptomyces SK-2 has the ability to dissolve organophosphates, solubilize potassium and produce iron carriers, but lacks the ability to dissolve inorganic phosphorus. In particular, the hydrolysis zone of Streptomyces SK-2 in organophosphate medium has a small diameter and low transparency, which preliminarily indicates that the organophosphate dissolving ability of Streptomyces SK-2 is relatively weak.

[0058] Example 3: Determination of the potted plant control efficacy of Streptomyces SK-2 against Conophytum rot

[0059] 1. Preparation of biocontrol fermentation broth

[0060] Activated Streptomyces SK-2 was transferred to a 250 mL Erlenmeyer flask (containing 200 mL of ISP2 liquid medium) and cultured at 28℃ and 180 r / min. After 32 h of culture, the culture solution was diluted and the OD was adjusted. 600 The values ​​are approximately 0.3 and 0.6, for future reference.

[0061] 2. Determination of the potted plant control efficacy of Streptomyces SK-2 against Conophytum rot

[0062] Control group (CK): Healthy white cone branches with uniform growth were surface-disinfected with 0.3% sodium hypochlorite solution for 1 min, repeated twice, and then rinsed 2-3 times with sterile water. A hole was punched in the treated branch using a sterile punch with a 6 mm aperture as an inoculation point. 1 mL of blank ISP2 liquid culture medium was spread at the hole, and the area was kept moist with sterile gauze. After 24 h, the gauze was removed, and a 6 mm diameter mycelial cake of *Dioscorea opposita* BZ-FL-1 was punched using a sterile punch and inoculated into the hole in the branch. The area was then wrapped with medical gauze, and finally, filter paper soaked in sterile water was wrapped around the gauze and sealed to retain moisture.

[0063] Treatment group I: The branches were disinfected and holes were punched using the same method as the control group, and 1 mL of OD was applied. 600 Apply 0.3% Streptomyces SK-2 biocontrol solution to the perforated area, keep it moist with sterile gauze, remove the gauze after 24 hours, use a sterile punch to extract a 6mm diameter Diplosporum mollissima BZ-FL-1 mycelial cake, inoculate it into the perforated area of ​​the branch, wrap it with medical gauze, then wrap filter paper soaked in sterile water around the gauze, and finally seal it to keep it moist.

[0064] Treatment group II: The branches were disinfected and holes were punched using the same method as the control group, and 1 mL of OD was applied. 600 =0.6% Streptomyces SK-2 biocontrol solution was applied to the perforated area, and the area was kept moist with sterile gauze. After 24 hours of incubation, the covering gauze was removed, and 6 mm diameter Mycobacterium tumefaciens BZ-FL-1 mycelial cakes were prepared using a sterile punch and inoculated into the perforated area of ​​the branch. The area was then wrapped with medical gauze, and filter paper soaked in sterile water was wrapped around the gauze. Finally, the area was sealed to keep it moist.

[0065] Three inoculation points were used for each potted plant, with five replicates. Seven days after inoculation, the disease incidence and lesion size of the white cone branches were measured. Diseased branches were wrapped with transparent plastic wrap, and the lesion area was circled with a marker. The area was then measured and calculated on a 1 mm × 1 mm grid paper. The calculation formulas are as follows: Disease incidence = (Number of diseased branches in the treatment group / Number of branches in the treatment group) × 100%; Control effect = [(Area of ​​lesions in the positive control - Area of ​​lesions in the treatment group) / Area of ​​lesions in the positive control group] × 100%

[0066] Table 2. Control efficacy of strain SK-2 against white cone rot.

[0067]

[0068] The preventive effect of Streptomyces SK-2 on Conifer rot is shown in Table 2. Table 2 shows that the incidence rate in the control group (CK) was 100%, while the incidence rate in the treatment groups was 66%. However, the Conifer branches treated with Streptomyces SK-2 significantly reduced the lesion area of ​​Conifer rot. The data in Table 2 indicate that Streptomyces SK-2 achieved a preventive effect of over 68% against the pathogen of Conifer rot, and the OD of the treatment groups... 600 The preventive effect of 0.3 was 68.87%, and the OD of the treatment group was [missing information]. 600 The preventive effect of 0.6 was 74.56%, indicating that the OD in the treatment group was significantly higher than that in the control group. 600 =0.6 has the best preventive effect against white cone rot in potted plants.

[0069] from Figure 7 It can be seen that the white cone branches treated with Streptomyces SK-2 bacterial solution can significantly reduce the lesion area of ​​white cone rot.

[0070] In conclusion, treatment of Conifer stalks with Streptomyces SK-2 bacterial solution significantly reduced the lesion area of ​​Conifer rot, indicating that Streptomyces SK-2 is a potentially valuable biocontrol strain for the prevention and control of Conifer rot.

Claims

1. A method of controlling white tip rot caused by Pestalotiopsis gisevii, characterized by, Including Streptomyces ( Streptomyces The steps involve applying a culture of Streptomyces SK-2 or Streptomyces SK-2 to Conophytum comosum; the Streptomyces SK-2 has the preservation number GDMCC No. 66221; the Streptomyces SK-2 culture is obtained by inoculating Streptomyces SK-2 onto ISP2 medium and culturing it.

2. The method of claim 1, wherein, The concentration of the culture of Streptomyces SK-2 is OD 600 = 0.3 or more.

3. The method of claim 2, wherein, The concentration of the culture of Streptomyces SK-2 is OD 600 = 0.3-0.

6.

4. Streptomyces ( Streptomyces Application of sp.) SK-2 or a culture of Streptomyces SK-2 in the prevention and control of white cone rot caused by Trichoderma spp.; the preservation number of Streptomyces SK-2 is GDMCC No. 66221; the culture of Streptomyces SK-2 is obtained by inoculating Streptomyces SK-2 onto ISP2 medium.

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