Streptomyces amylase HTR-4 and application thereof

Abstract

The invention discloses a strain of streptomyces diastatus HTR-4 and an application of the strain of streptomyces diastatus HTR-4. The strain HTR-4 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 9, 2024, and the preservation number is CGMCC NO.30538. The strain HTR-4 is named as HTR-4. The strain has a good antagonistic effect on fusarium graminearum, and a sterile fermentation broth of the strain has an inhibiting effect on mycelial growth and spore germination of the fusarium graminearum and also has good stability. The strain HTR-4 provided by the invention can provide a strain resource for biological prevention and control of wheat scab. In addition, the strain HTR-4 also has an antagonistic ability to alternaria alternata, botryosphaeria dothidea, corn northern leaf blight, colletotrichum gloeosporioides, pyrethrum lasiocarpum and fusarium oxysporum.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of amylase Streptomyces HTR-4 and its applications. Background Technology

[0002] Wheat is an important food crop in my country and plays a vital role in the national economy. Fusarium head blight, caused primarily by the fungus *Fusarium graminearum*, is a significant disease affecting wheat production in my country. It leads to reduced grain quantity and the production of shriveled grains. Besides yield loss, *Fusarium graminearum* infection can also secrete various fungal toxins, such as deoxyfucoidanol and zearalenone, which can negatively impact human and animal health. Therefore, *Fusarium graminearum* poses a serious threat to safe wheat production under the current circumstances.

[0003] Due to the lack of effective resistant varieties, chemical control remains the primary method for combating wheat scab. However, the long-term, excessive, and irrational use of chemical agents not only affects environmental safety but also leads to the proliferation of drug-resistant strains in the field, ultimately reducing the effectiveness of chemical control. Using biological control methods to manage wheat scab plays a crucial role in ensuring the safe and sustainable production of wheat. Summary of the Invention

[0004] The purpose of this invention is to provide an amylase-producing Streptomyces HTR-4 and its applications.

[0005] A strain of *Streptomyces diastaticus*, with accession number CGMCC No. 30538, was named HTR-4.

[0006] The application of Streptomyces diastaticus in the control of wheat scab.

[0007] The aforementioned *Streptomyces diastaticus* exhibits antagonistic activity against *Fusarium graminearum*, which causes wheat scab.

[0008] The application of Streptomyces diastaticus in the prevention and control of plant pathogens.

[0009] The plant pathogens mentioned are Alternaria alternata, Botryosphaeria dothidea, Exserohilum turcicum, Colletorichum gloeosporioides, Diaporthe eres, or Fusarium oxysporum.

[0010] A microbial pesticide for controlling wheat scab, comprising the aforementioned Streptomyces diastaticus.

[0011] A fermentation broth of Streptomyces diastaticus, obtained by fermentation of the Streptomyces diastaticus.

[0012] A microbial fungicide for controlling wheat scab, said fungicide containing the aforementioned Streptomyces diastaticus and / or its fermentation products.

[0013] The *Streptomyces diastaticus* strain HTR-4 of this invention is classified and named as *Streptomyces diastaticus*. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 9, 2024, and has proof of viability. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 30538.

[0014] The beneficial effects of this invention are as follows: *Streptomyces amylase* HTR-4 exhibits good antagonistic activity against *Fusarium graminearum*. After fermentation culture of strain HTR-4 on KMB medium, its sterile fermentation broth significantly inhibited the colony growth of *Fusarium graminearum*, with an inhibition rate of 82.3%. Furthermore, the sterile fermentation broth of strain HTR-4 also demonstrates good stability. In terms of antibacterial spectrum, strain HTR-4 also exhibits antagonistic activity against *Alternaria alternata*, *Staphylococcus aureus*, *Corynebacterium zebrina*, *Colletotrichum gloeosporioides*, *Ceratophyllum demersum*, and *Fusarium oxysporum*. These results indicate that strain HTR-4 can provide strain resources and a theoretical basis for developing highly efficient biocontrol microbial agents against *Fusarium graminearum*. Attached Figure Description

[0015] Figure 1 The image shows the antagonistic effect of strain HTR-4 on Fusarium graminearum; A represents Fusarium graminearum, and B represents the confrontation culture between strain HTR-4 and Fusarium graminearum.

[0016] Figure 2 The colony morphology (A, B) and sporulation characteristics (C) of strain HTR-4 on Gao's No. 1 medium.

[0017] Figure 3 A neighbor-joining phylogenetic tree for strain HTR-4 constructed based on the 16S rRNA gene sequence.

[0018] Figure 4 The effect of sterile fermentation broth of strain HTR-4 on the colony growth of Fusarium graminearum; A is the control of Fusarium graminearum; B is the effect of sterile fermentation broth of strain HTR-4 on Fusarium graminearum.

[0019] Figure 5 The effect of aseptic fermentation of strain HTR-4 on the germination of Fusarium graminearum spores.

[0020] Figure 6 A represents the stability of the sterile fermentation broth of strain HTR-4; B represents thermal stability; C represents acid-base stability; D represents ultraviolet stability; and D represents protease stability.

[0021] Figure 7 A represents the disease control effect of strain HTR-4; B represents the effect on Fusarium graminearum infection of wheat ears; C represents the effect on Fusarium graminearum infection of coleoptiles.

[0022] Figure 8 The study investigated the antagonistic effects of strain HTR-4 against *Alternaria alternata*, *Staphylococcus aureus*, *Gnaphalium affine*, *Colletotrichum gloeosporioides*, *Ceratophyllum demersum*, and *Fusarium oxysporum*. A represents the *Alternaria alternata* control; B represents the confrontation culture of strain HTR-4 with *Alternaria alternata*; C represents the *Staphylococcus aureus* control; D represents the confrontation culture of strain HTR-4 with *Staphylococcus aureus*; E represents the *Staphylococcus aureus* control; F represents the confrontation culture of strain HTR-4 with *Staphylococcus aureus*; G represents the *Colletotrichum gloeosporioides* control; H represents the confrontation culture of strain HTR-4 with *Colletotrichum gloeosporioides*; I represents the *Ceratophyllum demersum* control; J represents the confrontation culture of strain HTR-4 with *Ceratophyllum demersum*; K represents the *Fusarium oxysporum* control; and L represents the confrontation culture of strain HTR-4 with *Fusarium oxysporum*. Detailed Implementation

[0023] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0024] Example 1: Isolation and screening of strain HTR-4.

[0025] Soil samples were collected from wheat fields in Hongqi District, Xinxiang City, Henan Province in May 2023. 10g of soil sample was placed in 90mL of sterile water and shaken for 30min at 28℃ and 180r / min. The suspension was then serially diluted with sterile water to a concentration of 10. -1 10 -2 and 10 -3 100 μL of each concentration was plated onto Gao's No. 1 agar plates containing 50 μg / mL potassium dichromate and incubated at 28°C for 6 days. Single bacteria were then picked for purification.

[0026] Streptomyces cells were symmetrically inoculated onto PDA plates 2.5 cm from the center and incubated at 28°C for 2 days. Then, Fusarium graminearum mycelium (0.5 cm) was inoculated into the center of the plate. A PDA plate inoculated only with Fusarium graminearum served as a control. The antagonistic effect was observed after 3 more days of incubation. The resulting antagonistic strain of Fusarium graminearum, designated HTR-4, showed an inhibition rate of 67.7 ± 1.3%. Figure 1 ).

[0027] Example 2: Classification and identification of strain HTR-4.

[0028] (1) Morphological characteristics of strain HTR-4

[0029] Strawberry strain HTR-4 was streaked onto Gao's No. 1 medium and incubated at 28°C for 7 days. The shape, color, and texture of the colonies were observed. Sterile coverslips were inserted near the HTR-4 colonies, and after 10 days of incubation, the morphological characteristics of the hyphae and spores on the coverslips were observed. Figure 2 As shown, the aerial hyphae of strain HTR-4 are grayish-white, while the substrate hyphae are pale yellow. No soluble pigments are produced, and the spore filaments are spiral-shaped.

[0030] (2) Physiological and biochemical characteristics of strain HTR-4

[0031] Physiological and biochemical tests were performed on strain HTR-4 according to "Rapid Identification and Systematic Classification of Actinomycetes". The specific results are shown in Table 1.

[0032] Table 1 Physiological and biochemical characteristics of strain HTR-4

[0033]

[0034]

[0035] (3) 16S rRNA gene sequence analysis of strain HTR-4

[0036] Genomic DNA was extracted from strain HTR-4 using universal primer 27F(5 ′ -AGAGTTTGATCCTGGCTCAG-3 ′;SEQ ID NO: 2) and 1492R (5 ′ -GGTTACCTTGTTACGACTT-3 ′ The 16S rRNA gene sequence (SEQ ID NO: 3) was amplified, and the amplification product was purified and sequenced by Sangon Biotech (Shanghai) Co., Ltd. (gene sequence shown in SEQ ID NO: 1). The sequencing results were compared for homology in the NCBI database, and a phylogenetic tree was constructed using the neighbor-joining method in MEGA 7.0 software. Figure 3 As shown, strain HTR-4 clustered with *Streptomyces amylase* DSD1912, FJAT-31547, and SCSIO GJ056. Therefore, based on morphological and physiological-biochemical characteristics, strain HTR-4 was ultimately identified as *Streptomyces amylase*.

[0037] Example 3: Inhibitory effect of sterile fermentation broth of strain HTR-4 on Fusarium graminearum.

[0038] (1) Preparation of sterile fermentation broth

[0039] Freshly cultured HTR-4 bacterial blocks (2cm) were placed in 50mL KMB medium and cultured at 28℃ and 180r / min for 5 days. The bacterial solution was collected and centrifuged at 12000r / min for 15min. The supernatant was then filtered through a 0.22μm microporous membrane to remove the bacterial cells, thus obtaining the sterile fermentation broth of the strain.

[0040] KMB medium formula: 20.0g peptone, 10.0g glycerol, 1.5g K2HPO4, 1.5g MgSO4·7H2O, 1L distilled water, pH 7.2.

[0041] (2) Effect of sterile fermentation broth on the growth of Fusarium graminearum colonies

[0042] The aseptic fermentation broth of strain HTR-4 was added to the melted PDA medium at a final concentration of 10%. After the medium solidified, Fusarium graminearum mycelium (0.5 cm) was inoculated into the center of the plate. A PDA plate with an equal volume of sterile water was used as a control. After incubation at 28°C for 3 days, the colony diameter was measured and the inhibition rate was calculated. Figure 4 As shown, the aseptic fermentation broth of strain HTR-4 can inhibit the colony growth of Fusarium graminearum, with an inhibition rate of 84.7±1.2%.

[0043] (3) Effect of sterile fermentation broth on spore germination of Fusarium graminearum

[0044] 1 cm pieces of Fusarium graminearum were placed in CMC medium and cultured at 28°C and 150 rpm for 5 days. After collecting the spores, a 1×10⁻⁶ concentration of Fusarium graminearum was prepared using YEPD medium.6 A spore suspension of 10 spores per mL was prepared. Sterile fermentation broth of strain HTR-4 was added to the spore suspension at final concentrations of 10% and 50%, respectively, with an equal volume of sterile water as a control. After incubation at 28℃ and 150 rpm for 2 h and 6 h, respectively, spore germination was observed and statistically analyzed. Figure 5 As shown, at 2 h, the spore germination rate of the *Fusarium graminearum* control group was 90.17 ± 0.89%. The 10% concentration of sterile fermentation broth for strain HTR-4 had no significant effect on spore germination, with a germination rate of 91.15 ± 0.74%. However, when the concentration of the sterile fermentation broth increased to 50%, the spore germination rate decreased to 77.52 ± 0.94%, and the germ tube length became significantly shorter. At 6 h, the spore germination rate of the control group increased to 98.92 ± 0.94%. Both 10% and 50% concentrations of sterile fermentation broth had no significant effect on the spore germination rate, with germination rates of 98.73 ± 1.10% and 97.09 ± 0.33%, respectively. However, the 50% concentration of sterile fermentation broth still affected the elongation of the germ tube after spore germination.

[0045] Example 4: Stability analysis of sterile fermentation broth of strain HTR-4.

[0046] (1) Thermal stability

[0047] The aseptic fermentation broth of strain HTR-4 was placed in a water bath at 40℃, 60℃, 80℃, and 100℃ for 30 min, respectively. After cooling to room temperature, it was mixed with PDA medium at a final concentration of 10%, poured into plates, and then inoculated with Fusarium graminearum mycelium (0.5 cm). Untreated aseptic fermentation broth served as a control, and PDA medium without aseptic fermentation broth served as a blank control. After incubation at 28℃ for 3 days, the relative inhibition rates of the aseptic fermentation broths after different treatments were calculated. Figure 6 As shown in Figure A, the antibacterial effect of the sterile fermentation broth was almost unaffected after treatment at 40℃; however, the relative inhibition rate decreased with increasing temperature. After treatment at 60℃, the relative inhibition rate dropped to 77.0±1.4%; after treatment at 100℃, the relative inhibition rate was only 46.1±3.2%. This indicates that the sterile fermentation broth of strain HTR-4 has a certain degree of thermal stability.

[0048] (2) Acid-base stability

[0049] The pH of the sterile fermentation broth of strain HTR-4 was adjusted to 3, 5, 7, 9, and 11 respectively using 1 mol / L HCl and 1 mol / L NaOH. After standing at room temperature for 4 hours, the pH was adjusted back to 7. Untreated sterile fermentation broth was used as a control, and PDA medium without sterile fermentation broth was used as a blank control. After incubation at 28℃ for 3 days, the relative inhibition rates of the sterile fermentation broths after different treatments were calculated. Figure 6As shown in Figure B, the inhibition rate of the sterile fermentation broth remained unaffected at pH 7 and 9. Under acidic conditions, the relative inhibition rate decreased, reaching 84.0 ± 3.0% at pH 3. At pH 11, the inhibition rate significantly decreased, with a relative inhibition rate of only 8.6 ± 2.3%. This indicates that the sterile fermentation broth of strain HTR-4 is acid-resistant but not alkali-resistant.

[0050] (3) UV stability

[0051] The sterile fermentation broth of strain HTR-4 was irradiated under a 40W UV lamp (30cm distance) for 0.5h, 1h, 2h, and 4h. Untreated sterile fermentation broth served as a control, and PDA medium without sterile fermentation broth served as a blank control. After incubation at 28℃ for 3 days, the relative inhibition rates of the sterile fermentation broths after different treatments were calculated. Figure 6 As shown in Figure C, the antibacterial effect of the sterile fermentation broth was not affected after 0.5 hours of ultraviolet irradiation; the inhibition rate remained relatively stable with increasing irradiation time, and after 4 hours of irradiation, the relative inhibition rate was still 98.0 ± 3.6%. This indicates that the sterile fermentation broth of strain HTR-4 is stable to ultraviolet irradiation.

[0052] (4) Protease stability

[0053] Pepsin, trypsin, and proteinase K were added to the sterile fermentation broth of strain HTR-4 at a final concentration of 1 mg / mL, and the mixture was incubated at 37°C for 2 hours. Untreated sterile fermentation broth was used as a control, and PDA medium without sterile fermentation broth was used as a blank control. After incubation at 28°C for 3 days, the relative inhibition rates of the sterile fermentation broths after different treatments were calculated. Figure 6 As shown in Figure D, the sterile fermentation broth was not sensitive to the treatment with the three proteases, with relative inhibition rates ranging from 95.8% to 98.9%. This indicates that the sterile fermentation broth of strain HTR-4 is resistant to the tested proteases.

[0054] Example 5: Analysis of the disease control effect of strain HTR-4

[0055] (1) Inhibitory effect on Fusarium graminearum infection of wheat ears

[0056] Fermentation broth of strain HTR-4 and spore suspension of Fusarium graminearum were prepared according to the method in Example 3. The fermentation broth of strain HTR-4 and the spore suspension of Fusarium graminearum were mixed in equal proportions, and the final concentration of the spore suspension of Fusarium graminearum was 5 × 10⁻⁶. 5 / mL. During the wheat flowering stage, 10 μL of Fusarium graminearum spore liquid and a mixture of fermentation broth of strain HTR-4 and Fusarium graminearum spore liquid were injected into one of the lower spikelets, respectively, and the mixture was bagged and kept moist for 2 days. The disease incidence in the wheat spikelet was observed 14 days after inoculation. Figure 7 As shown in Figure A, after inoculation with Fusarium graminearum alone, the entire wheat ear shrivels and dies. However, after inoculation with a mixture of fermentation broth from strain HTR-4 and Fusarium graminearum spore liquid, only the inoculated grains on the wheat ear showed disease. This indicates that strain HTR-4 can effectively reduce the occurrence of wheat scab.

[0057] (2) Inhibitory effect on Fusarium graminearum infection of wheat coleoptiles

[0058] After selecting plump wheat seeds and cleaning them thoroughly, they were placed in petri dishes lined with filter paper and cultured at 25°C under alternating light and dark conditions (12h:12h) for 3 days. The tips of the wheat coleoptiles were then removed using sterile scissors. Sterile cotton balls soaked in a mixture of Fusarium graminearum spores and fermentation broth of strain HTR-4 with Fusarium graminearum spores were then applied to the cut surface of the coleoptile. The mixture was then kept moist for another 7 days, during which the disease development of the coleoptiles was observed. Figure 7 As shown in Figure B, wheat coleoptiles developed obvious brown lesions after inoculation with *Fusarium graminearum* alone. However, after inoculation with a mixture of fermentation broth from strain HTR-4 and *Fusarium graminearum* spore liquid, the wheat coleoptiles only turned brown at the cut surfaces. This indicates that strain HTR-4 has a good inhibitory effect on *Fusarium graminearum* infection of wheat coleoptiles.

[0059] Example 6: Antibacterial spectrum analysis of strain HTR-4.

[0060] Using an in-plate confrontation method, strain HTR-4 was first symmetrically inoculated 2.5 cm from the center of a PDA plate and incubated at 28°C for 2 days. Then, six plant pathogenic fungi, including Alternaria, were inoculated into the center of the plate, and the antagonistic effect was observed after another 4 days of incubation. Figure 8 As shown, strain HTR-4 exhibited the strongest antagonistic activity against *Staphylococcus aureus*, with an inhibition rate of 83.0 ± 3.0%; followed by *Alternaria alternata*, with an inhibition rate of 81.3 ± 1.3%; the inhibition rates against *Macrophyllum oleraceum* and *Colletotrichum gloeosporioides* were both around 70%; the inhibition rate against *Metaphyllum sacchariformis* was 63.5 ± 2.6%; and the inhibition rate against *Fusarium oxysporum* was relatively low, at 50.0 ± 1.1%. Therefore, strain HTR-4 possesses a good antibacterial spectrum.

[0061] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A Streptomyces diastaticus strain, with preservation number CGMCCNo.30538, named HTR-4.

2. The application of the amylase-producing Streptomyces diastaticus as described in claim 1 in the prevention and control of wheat scab.

3. The application according to claim 2, characterized in that, The aforementioned *Streptomyces diastaticus* exhibits antagonistic activity against *Fusarium graminearum*, which causes wheat scab.

4. The application of the amylase-producing Streptomyces diastaticus as described in claim 1 in the prevention and control of plant pathogens.

5. The application according to claim 4, characterized in that, The plant pathogens mentioned are Alternaria alternata, Botryosphaeria dothidea, Exserohilum turcicum, Colletorichum gloeosporioides, Diaportheeres, or Fusarium oxysporum.

6. A microbial pesticide for controlling wheat scab, characterized in that, It includes the Streptomyces diastaticus as described in claim 1.

7. A fermentation broth of *Streptomyces diastaticus*, characterized in that, It is obtained by fermentation of the amylase Streptomyces diastaticus as described in claim 1.

8. A microbial fungicide for controlling wheat scab, characterized in that, The bactericide contains the Streptomyces diastaticus as described in claim 1 and / or its fermentation products.

Images