Streptomyces Roche BJSC2-5 and application thereof

Abstract

The invention provides streptomyces Roche BJSC2-5 and application thereof, and belongs to the technical field of biological control. The streptomyces Roche BJSC2-5 is classified and named as Streptomyces Roche, the preservation unit is China General Microbiological Culture Collection Center (CGMCC), the preservation date is July 9, 2025, and the preservation number is CGMCC NO.35153. The streptomyces Roche belongs to the bacteria field, actinomycetes, actinomycetes, streptomycetes, streptomycetes, streptomycetes and streptomycetes. The strain disclosed by the invention has a good effect of preventing and treating anise anthracnose and has no influence on the growth of anise.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, specifically to a Streptomyces roximatebestrol BJSC2-5 and its applications. Background Technology

[0002] star anise( Illicium verum Illicium lanceolatum (also known as star anise) is a plant belonging to the genus Illicium in the family Illicaceae. It is native to subtropical regions of China, primarily concentrated in Guangxi (accounting for over 80% of global production), Yunnan, and Guangdong. It thrives in warm, humid climates (average annual temperature 18-23℃, annual rainfall 1200-2000 mm). As an important economic crop in my country, it is not only an indispensable spice in traditional cooking but also a crucial raw material in the pharmaceutical and chemical industries. Its fruit is rich in anethole oil and shikimic acid, widely used in food seasoning, traditional Chinese medicine (such as the extraction of raw materials for the antiviral drug Tamiflu), and the production of daily chemical products, possessing extremely high economic value. However, star anise cultivation is threatened by various diseases, often caused by fungi of the genus *Anthracis* (*Illicium*). Colletotrichum spp.), Fusarium ( ) Fusarium Caused by fungi (spp.), the typical symptoms include brown spots on leaves and fruit rot and drop. Mild cases can lead to a 20%-30% reduction in yield, while severe cases can result in total crop failure, posing a huge challenge to farmers' income and the stability of the industrial chain.

[0003] Anthracnose and Fusarium are the core diseases threatening the star anise industry. While traditional chemical control can suppress pathogens in the short term, their long-term harm cannot be ignored: First, over-reliance on chemical pesticides leads to significant pesticide resistance in anthracnose and Fusarium; second, pesticide residues easily accumulate in star anise fruits, threatening the safety of food and medicines made from star anise; third, broad-spectrum fungicides indiscriminately kill soil microorganisms and pollinating insects, disrupting the understory ecological balance and further weakening the system's disease resistance. Currently, promoting a green control system of "microbial agents + disease-resistant varieties + ecological regulation" has become the only way to break through the predicament of chemical control and ensure the high-quality development of the star anise industry. Summary of the Invention

[0004] The purpose of this invention is to propose a Streptomyces roximately BJSC2-5 and its application, which is effective in preventing and controlling diseases caused by anthracnose and Fusarium moniliforme, and has no effect on the growth of star anise.

[0005] The technical solution of this invention is implemented as follows: This invention provides a strain of *Streptomyces roximatei* BJSC2-5, wherein *Streptomyces roximatei* BJSC2-5 is classified and named as follows: Streptomyces roietensis, the depositing unit is China General Microbiological Culture Collection Center, the address is No. 1, Xibei Road, Beichen, Beijing, China, the postal code is 100080, the depositing date is July 9, 2025, the depositing number is CGMCC NO. 35153, the Streptomyces rocheus belongs to Bacteria, Actinobacteria, Streptomycetales, Streptomycetaceae and Streptomyces.

[0006] As a further improvement of the application, the 16S rRNA sequence of the Streptomyces rocheus BJSC2-5 is shown as SEQ ID No. 1.

[0007] As a further improvement of the application, the strain has good inhibitory effect on multiple pathogens of Illicium verum, can inhibit the growth of the pathogenic bacteria, and does not affect the normal growth of Illicium verum.

[0008] The strain can observe that the multiple pathogens of Illicium verum are inhibited on the 4th day of confrontation culture, and the inhibition phenomenon is more obvious on the 7th day of confrontation culture, the inhibition rates of the four strains of pathogenic bacteria are 43.44±2.95%, 40.26±1.31%, 36.79±2.77% and 27.27±2.49% respectively, p<0.05 reaches significant inhibition, and the inhibition zones are 9, 9, 8 and 6 mm respectively.

[0009] The strain is obtained by the following specific steps: A, specimen collection: healthy Illicium verum root system soil samples collected from Funing County, Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province.

[0010] B, isolation and culture of the strain: dilution coating plate method is used to separate endophytic actinomycetes in healthy Illicium verum root system soil samples: 100 ml of sterile water, 10 g of soil sample, glass beads, 25℃, 180 rpm / min, overnight for 24 h. Centrifugation to obtain supernatant, dilute 10 -1 , 10 -2 , 10 -3 , 10 -4 , add 50 μL of diluent to each plate and coat on TWYE medium plate, cultivate at 28℃ for 7-10 d. After the formation of colonies, typical single colonies are picked out with a sterile inoculation loop, and the strain is purified by streaking method on PDA medium.

[0011] C, strain preservation: the pure colonies of the biocontrol preparation strain obtained in the experiment are inoculated on test tube slant, cultured at 28℃ for 10 d, and then stored in a refrigerator at 4℃ after the colonies are full. The preservation medium is PDA medium.

[0012] TWYE medium: Yeast extract 0.25 g, Potassium dihydrogen phosphate 0.5 g, Agar 20 g, distilled water 1 L, pH=7.0.

[0013] Potato dextrose agar (PDA) medium ingredients in g / L: potato 200 g, glucose 20 g, agar 20 g, distilled water 1 L, pH natural.

[0014] The sample collection site described in step A is Funing County, Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province.

[0015] The sample described in step A is collected from the soil around the healthy roots of star anise in Funing County, Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province.

[0016] In the sterile operation table described in step B, the operation is performed under an alcohol lamp, and the inoculation needle, inoculation ring, sterile water, tweezers, and empty medium are operated under the alcohol lamp flame.

[0017] The sterilization described in step B is to place the inoculation needle, inoculation ring, sterile water, tweezers, and empty medium required for the test in a high-pressure steam sterilizer and sterilize at 121℃ for 30 min.

[0018] The preferred isolation culture conditions described in step B are 28℃ in the dark for 7-10 d.

[0019] The preferred conditions for the purification culture and preservation described in step C are 28℃ in the dark for 10 d, and then stored at 4℃.

[0020] The application further protects the use of the above-mentioned Streptomyces rocheus BJSC2-5 in the biological control of various diseases of star anise.

[0021] As a further improvement of the application, the Streptomyces rocheus BJSC2-5 has an inhibitory effect on the pathogenic bacteria of star anise diseases.

[0022] As a further improvement of the application, the pathogenic bacteria of star anise anthracnose include BJYB 3-20 Colletotrichum gloeosporioides ( Colletotrichum fructicola ), BJYB 3-22 Haraea anthracnose ( Colletotrichum horii ), BJYB 3-29 Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), BJYB 3-6 Fusarium oxysporum ( Fusariumoxysporum ).

[0023] A method for the biological control of various diseases of star anise using the Streptomyces rocheus ( Streptomyces roietensis ) BJSC2-5, which includes the screening of biocontrol strains and the determination of biocontrol effects, specifically including the following steps: A, screening of biocontrol strains: the cross method is used for preliminary screening of antagonistic bacteria. The specific operation is as follows: eight kinds of pathogenic fungi BJYB3-20 Guosheng Colletotrichum ( Colletotrichum fructicola ), BJYB3-22 Ha Xie Colletotrichum ( Colletotrichum horii ), BJYB3-29 Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), BJYB3-6 Fusarium oxysporum ( Fusariumoxysporum ) are used as indicator bacteria, 6mm agar block of pathogenic bacteria is inoculated in the center of PDA medium plate, then the single colony of bacteria obtained by separation culture is inoculated at equal intervals at a distance of 2.5 cm from the pathogenic bacteria, 3 repeats are set, and after constant temperature culture at 28℃ for 4-7d, the size of inhibition zone is observed, and the strain with the best antagonistic effect is selected.

[0024] B, determination of biocontrol effect: in order to screen out strains with strong bacteriostatic activity, the plate confrontation growth method is used for rescreening of the strains obtained by preliminary screening: eight kinds of pathogenic fungi BJYB3-20 Guosheng Colletotrichum ( Colletotrichum fructicola ), BJYB3-22 Ha Xie Colletotrichum ( Colletotrichum horii ), BJYB3-29 Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), BJYB3-6 Fusarium oxysporum ( Fusariumoxysporum ) are used as indicator bacteria, first, the activated pathogenic bacteria agar block is punched with a 6mm puncher and placed on the plate, then the antagonistic bacteria are inoculated by point inoculation, the distance between the two bacteria is 2.5 cm, the control group is only inoculated with pathogenic bacteria agar block, 3 repeats are set, and after culture in a 28℃ incubator for 4-7d, the growth of pathogenic bacteria is observed, the inhibition zone is measured, the inhibition rate is calculated, and the best antagonistic bacteria are finally screened out. The inhibition rate calculation formula is: inhibition rate (%) = (control pathogenic bacteria colony diameter-treatment pathogenic bacteria colony diameter) / control pathogenic bacteria colony diameter x 100% TWYE medium: yeast extract 0.25 g, potassium dihydrogen phosphate 0.5 g, agar 20 g, constant volume to 1 L with distilled water, pH=7.0.

[0025] Potato glucose agar medium (PDA) composition is as follows in g / L: potato 200 g, glucose 20 g, agar 20 g, distilled water 1 L, pH is natural.

[0026] The second object of the application is achieved in that the application of Streptomyces rocheus ( Streptomyces roietensis ) BJSC2-5 in the prevention and control of multiple diseases of star anise.

[0027] The application has the following beneficial effects: 1. The strain has good effect on preventing and controlling multiple diseases of star anise, and has no influence on the normal growth of star anise.

[0028] 2. The strain described in this invention showed inhibition of multiple pathogens in star anise on the 4th day of confrontation culture, and the inhibition was more obvious on the 7th day of confrontation culture.

[0029] 3. The strains described in this invention are easy to obtain and have a short cultivation time, which is conducive to large-scale production and widespread application. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The Streptomyces roximatee in Example 1 ( Streptomyces roietensis Image A: Front view of the culture medium; Image B: Back view of the culture medium. B: Reverse view of the culture medium.

[0032] Figure 2 For Streptomyces roximatei strains based on plate confrontation method ( Streptomyces roietensis BJSC2-5 is a strain of *Anthracis chinensis* (also known as BJSC2-5). Colletotrichum fructicola Antagonistic effect of BJYB3-20; A: BJYB3-20 (pure culture, front and back); B: BJYB3-20 control group; C: BJSC2-5 vs. B3-20 confrontation training group.

[0033] Figure 3 Streptomyces roximatei strains based on plate confrontation method ( Streptomyces roietensis BJSC2-5 is a strain of *Anthracis octophyllum*. Colletotrichum horii Antagonistic effect of BJYB3-22; A: BJYB3-22 (pure culture, front and back); B: BJYB3-22 control group; C: BJSC2-5 vs B3-22 confrontation culture group.

[0034] Figure 4 Streptomyces roximate based on plate confrontation method Streptomyces roietensis BJSC2-5 is effective against *Colletotrichum octophyllum* strains ( Colletotrichum gloeosporioidesAntagonistic effect of BJYB3-29; A: BJYB3-29 control group (pure culture, front and back sides); B: BJYB3-29 control group; C: BJSC2-5 vs. B3-29 confrontation training group. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] This invention provides a strain of *Streptomyces roximatei* BJSC2-5, wherein *Streptomyces roximatei* BJSC2-5 is classified and named as follows: Streptomyces roietensis The depositary institution 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, 100080, China. The deposit date is July 9, 2025, and the accession number is CGMCC NO.35153. The described *Streptomyces roximatei* belongs to the kingdom Bacteria, phylum Actinobacteria, class Actinobacteria, order Streptomycetes, family Streptomycetes, and genus *Streptomyces*.

[0037] Example 1: Streptomyces roximatei ( Streptomyces roietensis Acquisition, identification and preservation of BJSC2-5 (1) Streptomyces roximatei Streptomyces roietensis Obtaining and identifying BJSC2-5.

[0038] The Streptomyces roximatee described in this invention ( Streptomyces roietensis BJSC2-5 was isolated from the root soil of healthy star anise in Funing County, Yunnan Province. The sample was collected from Funing County, Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province, and stored at 4℃ for later use. Endophytic actinomycetes were isolated from the healthy star anise root soil sample using the dilution-spreading method: 10 g of soil sample was added to 100 ml of sterile water and incubated overnight (24 h) at 25℃ and 180 rpm / min using a glass bead shaker. The supernatant was collected by centrifugation and diluted 10⁻⁶ times. -1 10 -2 10 -3 10 -4 Add 50 μL of diluent to each plate and spread it on TWYE agar plates. Incubate at 28°C for 7-10 days. After colony formation, pick out typical single colonies with a sterile inoculation loop and purify the strain by streak plating on PDA medium.

[0039] TWYE medium: 0.25 g yeast extract, 0.5 g potassium dihydrogen phosphate, 20 g agar, diluted to 1 L with distilled water, pH=7.0.

[0040] Potato glucose agar (PDA) medium consists of the following components in g / L: 200 g potato, 20 g glucose, 20 g agar, 1 L distilled water, and natural pH.

[0041] The morphological and molecular biological identification results of the isolated strain BJSC2-5 are recorded below: A. Morphological characteristics: The colonies of strain BJSC2-5 are green in the early stage and dark brown in the later stage. They are nearly round to irregular in shape, with raised and wrinkled surfaces and radially pubescent edges. In the later stage, they become powdery, dry and opaque, with radiating lines around the colonies.

[0042] B. 16S rDNA sequence analysis: The 16S rDNA sequence of the strain was determined, yielding *Streptomyces roximatei* (…). Streptomyces roietensis The 16S rDNA sequence of BJSC2-5 is shown in SEQ ID NO.1. Using total DNA from strain CPAT-W06 as a template, the 16S rDNA fragment of this strain was amplified by PCR using the universal 16S rDNA primers for actinomycetes 8-27F (5'-AGAGTTTGATCCTGGCTCAG-3') / 1523-1504R (5'-TTAAGGTGATCCAGCCGCA-3'). The PCR reaction system was 2×Power... Taq PCR MasterMix: 12.5 µL, Template DNA: 1 µL, Forward primer (10 μmol / L): 1 µL, Reverse primer (10 μmol / L): 1 µL, ddH2O: 9.5 µL, Total volume: 25 µL. PCR reaction program: After pre-denaturation at 95℃ for 5 min, the following cycles were performed: denaturation at 95℃ for 1 min, annealing at 55℃ for 1 min, extension at 72℃ for 2 min, for 35 cycles. After the final extension at 72℃ for 10 min, the PCR instrument was stored at 4℃.

[0043] The PCR product was sent to Kunming Qingke Biotechnology Co., Ltd. for purification and sequencing. After sequence assembly, it was submitted to EzBioCloud (https: / / www.ezbiocloud.net / ) for comparison. The comparison results showed that strain BJSC2-5 was similar to Streptomyces roximatei (…). Streptomyces roietensis ) The (WES2) 16S rDNA sequence similarity reached 99.13%. Based on sequence alignment and morphological characteristics, strain BJSC2-5 was identified as *Streptomyces roximatei*. Streptomyces roietensisThe sequence of strain BJSC2-5 described in this invention is shown in the sequence listing.

[0044] (2) Streptomyces roximatei Streptomyces roietensis Preservation of BJSC2-5.

[0045] Based on the above identification results, the strain was confirmed to be *Streptomyces roximatei*. Streptomyces roietensis The specimen, designated BJSC2-5, was deposited on July 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100080, China). Its accession number is 35153.

[0046] Example 2 Streptomyces roximatei ( Streptomyces roietensis Screening of BJSC2-5 strains.

[0047] (1) Screening of strains.

[0048] The cross-linking method was used for initial screening of antagonistic bacteria. The specific procedure is as follows: Multiple pathogens of star anise (BJYB3-20, *Anthracnose citrinum*) were screened. Colletotrichum fructicola ), BJYB3-22 Hari anthrax bacteria ( Colletotrichum horii ), BJYB3-29 Colloidal anthrax bacteria ( Colletotrichum gloeosporioides ), BJYB3-6 Fusarium oxysporum ( Fusariumoxysporum Using the pathogen as an indicator bacterium, a 6 mm agar block of the pathogen was inoculated in the center of a PDA medium plate. Then, single colonies of the isolated bacteria were inoculated at equal intervals 2.5 cm away from the pathogen. Three replicates were set up. After incubation at 28℃ for 4-7 days, the size of the inhibition zone was observed, and the strain with the best antagonistic effect was selected.

[0049] (2) Observation of results.

[0050] The isolated strains were screened using the cross-linking method, and the results showed that 8 strains had antagonistic effects against anthracnose and Fusarium.

[0051] Example 3 Streptomyces roximatei ( Streptomyces roietensis Determination of the biocontrol effect of strain BJSC2-5 (1) Determination of biocontrol effect.

[0052] Confrontation culture method: using multiple pathogens of star anise, BJYB3-20, fruit anthracnose fungus ( Colletotrichum fructicola ), BJYB3-22 Hari anthrax bacteria ( Colletotrichum horii), BJYB3-29 Colletotrichum gloeosprioides (Penz.) Sacc. Colletotrichum gloeosporioides ), BJYB3-6 Fusarium oxysporum (Schlecht) f. sp. cubense (E.F. Smith) Snyder et H. aynes Fusariumoxysporum ) as an indicator, first use 6 mm puncher to punch activated pathogenic bacteria agar block, place on the plate, then use point inoculation method to inoculate antagonistic bacteria, the distance between two bacteria is 2.5 cm, the control group is only inoculated with pathogenic bacteria agar block, set 3 repeats, cultivate in 28℃ incubator for 4-7 days, observe pathogenic bacteria growth, measure inhibition zone, calculate inhibition rate, finally screen out the best antagonistic bacteria. (2) Results observation.

[0053] The plate confrontation culture method is used for determining the biocontrol effect of the screened strains, 8 kinds of antagonistic bacteria have antagonistic activity on multiple pathogenic bacteria of star anise, and the inhibition effect of different antagonistic bacteria on the mycelium of the pathogenic bacteria has certain difference. The antagonistic activity of BJSC2-5 strain is strong, and the inhibition zone boundary is clear, the inhibition rates of BJSC2-5 on four pathogenic bacteria are 43.44±2.95%, 40.26±1.31%, 36.79±2.77% and 27.27±2.49% respectively, p<0.05 reaches significant inhibition, and the inhibition zones are 9, 9, 8 and 6 mm respectively, reaching the significant level. Although the culture time is prolonged, the inhibition zone always remains transparent, indicating that the antagonistic performance of the antagonistic bacteria is stable.

[0054] The above only describes the preferred embodiments of the present application and should not be used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A strain of Streptomyces rochei BJSC2-5, characterized in that, The classification name of the Streptomyces rochei BJSC2-5 is Streptomyces roietensis , the deposit unit is China General Microbiological Culture Collection Center, the deposit date is July 9, 2025, the deposit number is CGMCC NO.35153, and the Streptomyces rochei belongs to the bacterial kingdom, the actinobacteria phylum, the actinobacteria class, the Streptomyces order, the Streptomyces family and the Streptomyces genus.

2. The S. rochei BJSC2-5 of claim 1, wherein, The 16S rRNA sequence of the Streptomyces roietensis BJSC2-5 is shown as SEQ ID No.

1.

3. The use of the Streptomyces roietensis BJSC2-5 of claim 1 or 2 in the biological control of various diseases of star anise.

4. Use according to claim 3, characterized in that, The Streptomyces roietensis BJSC2-5 has inhibitory effect on the pathogenic fungi of star anise diseases.

5. Use according to claim 4, characterized in that, The anthracnose pathogen mentioned includes BJYB 3-20 fruit anthracnose fungus ( Colletotrichum fructicola ), BJYB3-22 Hari anthrax bacteria ( Colletotrichum horii ), BJYB3-29 Colloidal anthrax bacteria ( Colletotrichum gloeosporioides ), BJYB3-6 Fusarium oxysporum ( Fusarium oxysporum ).

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