A live attenuated strain of Staphylococcus aureus SdBd-6 and its application

Abstract

This invention belongs to the field of biological control of plant diseases, specifically relating to a virulent strain of *Botrytis cinerea* SdBd-6 and its applications. The virulent strain of *Botrytis cinerea* SdBd-6 is classified and named... Botryosphaeria dothidea The attenuated strain SdBd-6 of Staphylococcus aureus was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 14, 2026, with accession number GDMCC: 67656. Walsa money The growth of the fungus has a certain inhibitory function. At the same time, after 10 generations of continuous inoculation, the virus contained in the attenuated strain SdBd-6 of *Botrytis cinerea* was not lost, and its growth characteristics and pathogenicity remained attenuated strain characteristics. This provides materials and lays the foundation for the green control of plant fungal diseases caused by the pathogenic fungus *Botrytis cinerea*.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a virulent strain of Staphylococcus aureus SdBd-6 and its applications. Background Technology

[0002] Staphylococcus aureus ( Botryosphaeria dothidea *Pestalotiopsis theae chrysovirus* is a common and important pathogenic fungus found globally, infecting apple, pear, poplar, and walnut trees, causing symptoms such as branch and trunk canker, gumming, and fruit rot. In severe cases, it can kill the entire tree, seriously impacting the healthy and sustainable development of my country's forestry and fruit industry. In production, diseases caused by *Pestalotiopsis theae chrysovirus* mainly rely on traditional chemical control. However, the long-term use of chemical agents has led to increasingly significant problems with drug resistance, safety, and product quality. Therefore, there is an urgent need to find a scientific and effective green control technology. Utilizing fungal viruses to mediate the weak pathogenicity of fungi to control fungal diseases can greatly reduce the use of chemical agents, lower production costs, and maintain ecological balance, representing a green and efficient new strategy for fungal disease control. There are many successful cases of using fungal viruses to control fungal diseases in production. For example, the fungal virus *Cryphonectria hypovirus 1* (CHV1) has been successfully used to control European chestnut blight; and the fungal virus *Pestalotiopsis theae chrysovirus 1* (PtCV1) has been used to control diseases caused by... Pestalotiopsis theae Diseases caused by Sclerotinia sclerotiorum; control of fungal diseases caused by Sclerotinia sclerotiorum using Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1).

[0003] Therefore, identifying fungal viruses that attenuate pathogenic fungi and clarifying their attenuating effect on pathogenic fungi can provide important candidate materials for green and efficient control of plant fungal diseases. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a virulent strain of Staphylococcus aureus, SdBd-6, and its applications.

[0005] The technical solution of this invention is as follows:

[0006] In a first aspect, the present invention provides a potent strain of *Botrytis cinerea* SdBd-6, wherein the potent strain of *Botrytis cinerea* SdBd-6 is classified as... Botryosphaeria dothidea It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 14, 2026, with accession number GDMCC:67656.

[0007] Based on high-throughput sequencing and PCR detection, SdBd-6 was found to contain six fungal viruses: Botryosphaeria dothidea chrysovirus 1 (BdCV1), Botryosphaeria dothidea partitivirus 1 (BdPV1), Botryosphaeria dothidea partitivirus 3 (BdPV3), Botryosphaeria dothidea partitivirus 4 (BdPV4), Botryosphaeria dothidea Vivivirus 1 (BdVivivirus1), and Botryosphaeria dothidea fusarivirus 2 (BdFV2).

[0008] The nucleotide sequences of BdCV1-RNA1, BdCV1-RNA2, BdCV1-RNA3, and BdCV1-RNA4 are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; the nucleotide sequences of BdPV1 RNA1 and BdPV1 RNA2 are shown in SEQ ID NO:6; the nucleotide sequences of BdPV3 RNA1 and BdPV3 RNA2 are shown in SEQ ID NO:8; the nucleotide sequences of BdPV4 RNA1 and BdPV4 RNA2 are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively; the nucleotide sequences of Bd Vivivirus 1 RNA1 and Bd Vivivirus 1 RNA2 are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively; and the nucleotide sequence of BdFV2 is shown in SEQ ID NO:13.

[0009] In a second aspect, the present invention provides a method for culturing a staphylococcal attenuated strain SdBd-6, comprising inoculating the staphylococcal attenuated strain SdBd-6 into a PDA medium and culturing it under light at 20-26°C for 4-6 days.

[0010] A third aspect of the present invention provides the application of the attenuated strain of *Botrytis cinerea* SdBd-6 in the suppression of plant rot.

[0011] Furthermore, the plant rot disease is caused by... Valsa mali The resulting putrefaction disease.

[0012] Furthermore, the plant in question is an apple tree.

[0013] A fourth aspect of the present invention provides the use of the attenuated strain of *Botrytis cinerea* SdBd-6 in the preparation of a preparation that inhibits plant pathogens or diseases caused by plant pathogens.

[0014] Furthermore, the plant pathogen is *Botrytis cinerea*. 。

[0015] A fifth aspect of the present invention provides a method for preventing and controlling plant rot, comprising applying a virulent strain of *Botrytis cinerea* SdBd-6 to the plant; wherein the plant rot is caused by... Valsa mali The plant causing the rot disease is an apple tree, pear tree, poplar tree, or walnut tree.

[0016] A sixth aspect of the present invention provides a method for preventing and controlling plant diseases, comprising applying a virulent strain of *Botrytis cinerea* SdBd-6 to a plant, wherein the plant disease is a plant disease caused by *Botrytis cinerea*; and wherein the plant is an apple tree, pear tree, poplar tree, or walnut tree.

[0017] The beneficial technical effects of one or more of the above technical solutions are as follows: This invention discovered a weakened strain, SdBd-6, from *Staphylococcus aureus*, the pathogen of apple ring rot. Compared to the highly virulent strain LS-11, strain SdBd-6 exhibits significantly reduced growth rate and pathogenicity. Testing revealed that strain SdBd-6 contains six fungal viruses. This strain is similar to the pathogen of apple canker, a major disease of apple trees. Valsa mali Controlled experiments showed that SdBd-6 has a significant effect on... Valsa mali The growth of SdBd-6 has a certain inhibitory function, indicating that the metabolites of SdBd-6 have an inhibitory effect on its growth. Valsa mali It has control potential. After 10 consecutive generations of inoculation with SdBd-6, no virus was lost from the strain, and its growth characteristics and pathogenicity remained those of a weakly virulent strain. This strain can be used as a biocontrol resource for controlling forest and fruit diseases caused by *Botrytis cinerea*. Attached Figure Description

[0018] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0019] Figure 1 The colony morphology characteristics of strain SdBd-6 in the embodiments of the present invention; Figure 2 This is a pathogenicity determination of strain SdBd-6 in the embodiments of the present invention; Figure 3 This diagram illustrates the RT-PCR detection of the virus contained in strain SdBd-6 in this embodiment of the invention. In the figure, M: DL2000 Marker; 1: Detection of BdCV1-RNA1; 2: Detection of BdCV1-RNA2; 3: Detection of BdCV1-RNA3; 4: Detection of BdCV1-RNA4; 5: Detection of BdPV1 RNA1; 6: Detection of BdPV1 RNA2; 7: Detection of BdPV3 RNA1; 8: Detection of BdPV3 RNA2; 9: Detection of BdPV4 RNA1; 10: Detection of BdPV4 RNA2; 11: Detection of Bd Vivivirus 1 RNA1; 12: Detection of Bd Vivivirus 1 RNA2; 13: Detection of BdFV2. Figure 4 This is an electrophoresis image of the RT-PCR detection results of the protoplast-transformed regenerated strain SdBd-6-10 containing the virus in this embodiment of the invention; where M: DL2000 Marker; A: Detection of BdCV1 virus, 1: Detection of BdCV1-RNA1, 2: Detection of BdCV1-RNA2, 3: Detection of BdCV1-RNA3, 4: Detection of BdCV1-RNA4; B: Detection of BdPV1 virus, 1: Detection of BdPV1-RNA1, 2: Detection of BdPV1-RNA2; C: Detection of BdPV3 virus, 1: Detection of BdPV3-RNA1, 2: Detection of BdPV3-RNA2; D: Detection of BdPV4 virus, 1: Detection of BdPV4-RNA1, 2: Detection of BdPV4-RNA2; E: Detection of BdFV2 virus; F: Detection of Bd Vivivirus virus, 1: Detection of Bd Vivivirus1-RNA1, 2: Detection of Bd... Detection of Vivivirus-RNA2; Figure 5 This is an analysis of differences in colony morphology in this embodiment of the invention; where A represents differences in colony morphology; and B represents a significant analysis of differences in colony growth rate. This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. This indicates that P < 0.0001; Figure 6This is an example of determining the pathogenicity of the strains on apple fruit in this invention embodiment; in the figure, A is the pathogenicity test; B is the analysis of significant differences in pathogenicity. This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. This indicates that P < 0.0001; Figure 7 In this embodiment of the invention, strain SdBd-6 is used to... Valsa mali Biological defense potential test. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise stated, the experimental methods disclosed in this invention employ conventional techniques in the art, and the reagents and raw materials used in the examples are commercially available.

[0021] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, or combinations thereof.

[0022] This invention has identified a weakened strain, SdBd-6, from *Staphylococcus aureus*, the pathogen of apple ring rot. This strain has been deposited at the Guangdong Provincial Microbial Culture Collection Center (strain number: GDMCC 67656). Compared to the highly virulent strain LS-11, strain SdBd-6 exhibits significantly reduced growth rate and pathogenicity. Testing revealed that strain SdBd-6 contains six fungal viruses. This strain is similar to the pathogen of apple tree rot, a major disease of apple trees. Valsa mali Controlled experiments showed that SdBd-6 has a significant effect on... Valsa mali The growth of SdBd-6 has a certain inhibitory function, suggesting that the metabolites of SdBd-6 have an inhibitory effect on its growth. Valsa mali It has control potential. After 10 consecutive generations of inoculation with SdBd-6, no virus was lost from the strain, and its growth characteristics and pathogenicity remained those of a weakly virulent strain. This strain can be used as a biocontrol resource for controlling forest and fruit diseases caused by *Botrytis cinerea*.

[0023] Example 1: Identification and characteristics of strain SdBd-6 A virulent strain of *Gnaphalium affine*, SdBd-6, was isolated from apple branch samples infected with apple ring rot in Feicheng City, Shandong Province. The strain was identified by comparing the ITS and EF1-α sequences using NCBI; the primers used for identification are shown in Table 1. After culturing on PDA medium at 25°C for 5 days, compared with LS-11, SdBd-6 showed deformed colonies, a dark brown color, and slow mycelial growth. Figure 1 ). Pathogenicity testing was performed on healthy apples inoculated with SdBd-6 via the puncture method. After 7 days of incubation at 25°C, the lesions were significantly smaller than those in apples inoculated with LS-11. Figure 2 ).

[0024] The strain was deposited at the Guangdong Provincial Microbial Culture Collection Center and classified and named. Botryosphaeria dothidea, The accession number is GDMCC 67656, and the accession address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0025] Example 2 Identification of fungal viruses contained in strain SdBd-6 Extraction of total RNA from strain SdBd-6: 5 g of mycelium cultured on PDA medium lined with cellophane for 7 days was taken, and total RNA was extracted using the Trizol method and the RNA quality was measured. After extraction, electrophoresis was performed and the total RNA quality was determined using Nanodrop. If the RNA quality was qualified, high-throughput sequencing was performed.

[0026] High-throughput sequencing identified fungal viruses in SdBd-6: Total RNA extracted was sequenced using Illumina, and raw data were processed using ACGT101-miR (LCSciences, Houston, Texas, USA). Sequences were assembled using Velvet software to obtain longer contigs, and viral information was obtained by annotating the NT and NR databases. Finally, it was determined that strain SdBd-6 contained a total of 6 viruses, namely BdCV1, BdPV1, BdPV3, BdPV4, Bd Vivivirus1, and BdFV2.

[0027] RT-PCR identification of fungal viruses contained in SdBd-6: Based on high-throughput sequencing results, virus detection primers were designed using the obtained viral contig data. Total RNA from SdBd-6 was reverse transcribed using the HiScript® II 1st Strand cDNA Synthesis Kit (Vazyme) to obtain cDNA. Using the reverse-transcribed cDNA as a template, PCR amplification was performed using the designed primers for the detection of six viruses. Figure 3). Sequencing of the amplified products followed by BLAST analysis using the NCBI website revealed that strain SdBd-6 contained six viruses: BdCV1, BdPV1, BdPV3, BdPV4, Bd Vivivirus1, and BdFV2. The nucleotide sequences of BdCV1-RNA1, BdCV1-RNA2, BdCV1-RNA3, and BdCV1-RNA4 are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; BdPV1-RNA1 and BdPV1-RNA2 are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively; BdPV3-RNA1 and BdPV3-RNA2 are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively; BdPV4-RNA1 and BdPV4-RNA2 are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively; and Bd Vivivirus1... The nucleotide sequence of RNA1 is shown in SEQ ID NO:11; the nucleotide sequence of BdVivivirus 1 RNA2 is shown in SEQ ID NO:12; and the nucleotide sequence of BdFV2 is shown in SEQ ID NO:13. These results are consistent with high-throughput sequencing results. Primer information used is shown in Table 1.

[0028] Table 1 Primer Information

[0029] Example 3: Stability test of strain SdBd-6 Fungal viruses can be lost after multiple generations of host fungi. To verify the stability of the viruses contained in strain SdBd-6 and the stability of the pathogenicity characteristics of the strain, apples were continuously inoculated with strain SdBd-6 and Koch assay was performed. The 10th generation strain SdBd-6-10 was used to detect the types of viruses contained in it and to test the pathogenicity of the strain.

[0030] Detection of viruses in strain SdBd-6-10: Total RNA was extracted from SdBd-6-10 using the Trizol method, and the presence of six candidate viruses was detected by RT-PCR. Six viruses were detected simultaneously in strain SdBd-6-10; the electrophoresis results are shown below. Figure 4 The primers used are shown in Table 1.

[0031] Culture characteristics of strain SdBd-6-10: Mycelial cakes were collected from the edges of strains SdBd-6, SdBd-6-10, and LS-11 after 5 days of culture using a 5 mm punch. These cakes were then inoculated into the center of PDA medium and cultured at 25°C under light. Colony morphology was observed after 5 days, and the diameter of each group was measured using the cross-cross method. Data were recorded and photographed. Each treatment was repeated 5 times, and SPSS was used for statistical significance analysis. Figure 5 After 5 days of cultivation, strain LS-11 covered the plate completely, with vigorous aerial hyphae, white colonies with neat edges, and regular circular shape, with a colony diameter of 8.45±0.21 cm. Strains SdBd-6 and SdBd-6-10, on the other hand, had irregular colony edges, diverse colony shapes, and slower colony growth rates. The average colony diameter of SdBd-6-10 was 3.83±0.46 cm, and the average colony diameter of SdBd-6 was 2.98±0.98 cm.

[0032] Pathogenicity analysis of strain SdBd-6-10: Healthy Fuji apples were selected and inoculated with mycelial cakes of strains SdBd-6, SdBd-6-10, and LS-11 (5 mm in diameter, cultured for 5 days) using the puncture inoculation method. The control group was inoculated with PDA medium. Each treatment was performed in 5 replicates. Five days after inoculation, the disease incidence and lesion size of the apples were measured and photographed.

[0033] Five days later, apples inoculated with LS-11 developed the disease, with lesion diameters of 3.81±0.49 cm; apples inoculated with SdBd-6-10 developed the disease, with lesion diameters of 2.19±0.54 cm; apples inoculated with SdBd-6 developed the disease, with lesion diameters of 1.81±0.49 cm; the control group showed no disease. Figure 6 ).

[0034] Example 4: Strain SdBd-6 against Valsa mali Biosecurity potential test Using a 5 mm diameter punch, mycelial cakes were collected from the edges of strain SdBd-6 and the rot pathogen Valsa mali, both cultured for 5 days. These were then inoculated opposite each other in the center of the same PDA medium and cultured at 25°C under light. Colony morphology was observed and photographed after 5 days, with a total of 5 replicates. After 5 days of culture, a distinct inhibition zone was formed around strain SdBd-6, inhibiting the growth of the rot pathogen. The average diameter of the inhibition zone was 9 ± 2.21 mm. Figure 7 This result suggests that the metabolites of strain SdBd-6 have the ability to control the spread of the virus. Valsa mali The application and development prospects of apple tree rot caused by this disease.

[0035] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A potent strain of Staphylococcus aureus, SdBd-6, characterized in that, The attenuated strain of Staphylococcus aureus SdBd-6 is classified as follows: Botryosphaeria dothidea It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 14, 2026, with accession number GDMCC: 67656.

2. The attenuated Staphylococcus aureus strain SdBd-6 as described in claim 1, characterized in that, It contains 6 fungal viruses: BdCV1, BdPV1, BdPV3, BdPV4, Bd Vivivirus1, and BdFV2.

3. The method for culturing the attenuated strain SdBd-6 of Staphylococcus aureus according to claims 1-2, characterized in that, This includes inoculating the attenuated strain of Staphylococcus aureus SdBd-6 into PDA medium and culturing it under light at 20-26°C for 4-6 days.

4. The application of the attenuated strain SdBd-6 of *Botrytis cinerea* as described in claims 1-2 in inhibiting plant rot.

5. The application as described in claim 3, characterized in that, The plant rot disease is caused by Valsa mali The resulting putrefaction disease.

6. The application as described in claims 4-5, characterized in that, The plant in question is an apple tree.

7. The use of the attenuated strain SdBd-6 of *Botrytis cinerea* as described in claims 1-2 in the preparation of a preparation that inhibits plant pathogens or diseases caused by plant pathogens.

8. The application as described in claim 6, characterized in that, The plant pathogen is B. dothidea .

9. A method for preventing and controlling plant rot, characterized in that, This includes applying the attenuated strain of *Botrytis cinerea* SdBd-6 as described in claim 1 to the plant; the plant is an apple tree.

10. A method for preventing and controlling plant diseases, characterized in that, This includes applying the attenuated strain SdBd-6 of *Botrytis cinerea* as described in claim 1 to the plant plant, wherein the plant disease is... B. dothidea Plant diseases caused by this.

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