Epimedium endophytic bacteria with multiple disease resistance and growth promotion functions, combinations and applications thereof

Abstract

This invention discloses an endophytic bacterium for Epimedium with multiple disease-resistant and growth-promoting functions, its combination, and its application, belonging to the field of biocontrol technology. The endophytic bacterium for Epimedium described in this invention is *Bacillus belye* B-Y-2. The combination of endophytic bacteria for Epimedium in this invention includes *Bacillus belye* B-Y-2, *Bacillus halophilus* A-G-7, and *Bacillus subtilis* D-1. The endophytic bacteria and combination of this invention exhibit good inhibitory activity against the main pathogens causing Epimedium diseases, and possess nitrogen-fixing, phosphorus-solubilizing, and potassium-solubilizing effects. Icariin and IAA can be detected in the fermentation broth, which can increase soil nutrient content, promote plant growth, and improve Epimedium quality. When combined, they synergistically enhance their effects. As an effective component of foliar fertilizer for controlling Epimedium diseases, they have a long-lasting effect, good control efficacy, and are environmentally safe. When applied to the soil as a microbial fertilizer, they can increase the content of available nitrogen, phosphorus, and potassium in the soil, improve soil fertility, promote plant growth, and reduce the use of chemical fertilizers.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, and in particular relates to an endophytic bacterium of Epimedium with multiple disease-resistant and growth-promoting functions, as well as its combination and application. Background Technology

[0002] Epimedium ( Epimedium brevicomu It belongs to the genus Epimedium in the family Berberidaceae. Epimedium The plant *Epimedium spp.* is the main source of the traditional Chinese medicine *Epimedium sagittatum*, with its dried leaves used medicinally. According to the 2020 edition of the *Chinese Pharmacopoeia*, it belongs to the same genus as *Epimedium sagittatum*. E. sagittatum Epimedium with soft hairs E. pubescens and North Korean Epimedium E. Korean Epimedium is also used in traditional Chinese medicine. With the discovery of more and more of its pharmacological effects, Epimedium has become an important raw material for many traditional Chinese medicines and health products. Epimedium mainly grows in the wild, but with increasing market demand, the area of ​​large-scale artificial cultivation has gradually increased, and diseases have also occurred and worsened year by year. The leaves are the main medicinal part of Epimedium, and the content of active ingredients such as flavonoids in the leaves directly affects the quality and efficacy stability of the medicinal material. Therefore, if leaf diseases such as sclerotinia rot, gray mold, leaf spot, and anthracnose occur during field production, they will not only weaken photosynthesis, cause premature leaf drop, and reduce yield, but also significantly affect the medicinal quality by damaging leaf tissue and altering the accumulation of secondary metabolites.

[0003] Currently, chemical pesticides are the main means of controlling these diseases. However, traditional chemical pesticides easily lead to pesticide resistance and environmental pollution, and residues reduce the safety of medicinal herbs, contradicting the production concept of green medicinal herbs. Meanwhile, nitrogen, phosphorus, and potassium are essential macronutrients for plant growth in the soil, but the content of available nitrogen, phosphorus, and potassium that can be directly absorbed and utilized by plants is often low. While traditional fertilizer application can supplement soil nutrients to some extent, excessive application can cause soil compaction, environmental pollution, and resource waste. Therefore, finding a safe, efficient, and environmentally friendly method for controlling plant diseases and soil improvement measures is of significant practical importance. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an endophytic bacterium of Epimedium that has significant resistance to sclerotinia rot, gray mold, anthracnose and leaf spot, and also has the functions of producing indoleacetic acid, fixing nitrogen, dissolving phosphorus and potassium. It can be used for the prevention and control of fungal diseases of Epimedium leaves and soil improvement, reducing the use of chemical pesticides and fertilizers.

[0005] Another object of the present invention is to provide an endophytic bacterial composition of Epimedium.

[0006] Another object of the present invention is to provide a culture.

[0007] Another objective of this invention is to provide an epimedium-based product for disease resistance and growth promotion.

[0008] Another object of the present invention is to provide the application of the Epimedium endophytic bacteria, the Epimedium endophytic bacteria combination, the culture, or the product in improving the quality and yield of Epimedium or preventing Epimedium fungal diseases.

[0009] Another objective of this invention is to provide a method for improving the quality and yield of Epimedium or for preventing Epimedium fungal diseases.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an endophytic bacterium from Epimedium, wherein the endophytic bacterium is Bacillus belye (…). Bacillus velezensis BY-2, accession number CCTCC NO: M 20252587.

[0011] This invention provides an endophytic bacterium from Epimedium, wherein the endophytic bacterium is a halophilic Bacillus (Bacillus). Bacillus halotolerans )AG-7, with accession number CCTCC NO: M 20252586.

[0012] This invention provides an endophytic bacterium from Epimedium, wherein the endophytic bacterium is Bacillus subtilis (Bacillus subtilis). Bacillus subtilis D-1, with accession number CCTCC NO: M 20252588.

[0013] This invention also provides an endophytic bacterial composition of Epimedium, wherein the endophytic bacterial composition of Epimedium includes Bacillus belye ( Bacillus velezensis BY-2, Halophilic Bacillus ( Bacillus halotolerans AG-7 and Bacillus subtilis ( Bacillus subtilis The Bacillus berberis BY-2 has the accession number CCTCC NO: M 20252587, the Bacillus halophilus AG-7 has the accession number CCTCC NO: M 20252586, and the Bacillus subtilis D-1 has the accession number CCTCC NO: M 20252588.

[0014] Preferably, the 16S rRNA gene sequence of Bacillus belyss BY-2 is shown in SEQ ID NO.7, and the gyrB gene sequence is shown in SEQ ID NO.8; the 16S rRNA gene sequence of Bacillus halophilus AG-7 is shown in SEQ ID NO.5, and the gyrB gene sequence is shown in SEQ ID NO.6; the 16S rRNA gene sequence of Bacillus subtilis D-1 is shown in SEQ ID NO.9, and the gyrB gene sequence is shown in SEQ ID NO.10.

[0015] Preferably, the viable cell concentration ratio of Bacillus belyi BY-2, Bacillus halophilus AG-7 and Bacillus subtilis D-1 is (1~5):(1~5):(1~5).

[0016] The present invention also provides a culture, which is a culture of the epimedium endophytic bacteria or a combination of the epimedium endophytic bacteria.

[0017] Preferably, the culture comprises the fermentation product of the Epimedium endophytic bacteria or a combination of the Epimedium endophytic bacteria.

[0018] Preferably, the fermentation product includes fermentation broth or sterile fermentation broth.

[0019] The present invention also provides a method for preparing the culture, comprising inoculating the Epimedium endophytic bacteria or a combination of Epimedium endophytic bacteria into a culture medium for fermentation culture to obtain a fermentation broth; Alternatively, the fermentation broth can be further centrifuged and filtered sequentially through 0.45μm and 0.22μm filter membranes to obtain sterile fermentation broth.

[0020] Preferably, the culture medium comprises LB liquid culture medium.

[0021] Preferably, the fermentation culture temperature is 25~30℃.

[0022] Preferably, the oscillation speed of the fermentation culture is 150~200 r / min.

[0023] Preferably, the fermentation culture time is 2 to 7 days.

[0024] The present invention also provides an epimedium disease-resistant and growth-promoting product, the product comprising epimedium endophytic bacteria or a combination of epimedium endophytic bacteria or the culture.

[0025] Preferably, the product includes seed treatment agents, fungicides, growth promoters, fertilizers, or soil conditioners.

[0026] The present invention also provides the application of the epimedium endophytic bacteria, the epimedium endophytic bacteria combination, the culture, or the product in improving the quality and yield of epimedium or preventing epimedium fungal diseases.

[0027] Preferably, the Epimedium fungal diseases include any one or more of sclerotinia rot, gray mold, leaf spot, and anthracnose.

[0028] The present invention also provides a method for improving the quality and yield of Epimedium or for preventing Epimedium fungal diseases. The method for improving the quality and yield of Epimedium includes: spraying the Epimedium endophytic bacteria or a combination of the Epimedium endophytic bacteria or the culture or the product onto the above-ground parts of the Epimedium plant or applying it into the soil. The method for preventing and controlling Epimedium fungal diseases includes: spraying the Epimedium endophytic bacteria, the Epimedium endophytic bacteria combination, the culture, or the product onto the above-ground parts of the Epimedium plant.

[0029] The beneficial effects of this invention are: This invention is the first to discover the endophytic antagonistic strain of Epimedium, Bacillus belye ( Bacillus velezensis BY-2, Halophilic Bacillus ( Bacillus halotolerans AG-7 and Bacillus subtilis ( Bacillus subtle D-1. The *Bacillus vesiculosus* BY-2, *Bacillus halophilus* AG-7, and *Bacillus subtilis* D-1 of this invention exhibit good inhibitory activity against four major pathogens causing diseases of *Epimedium*, and can be used for the prevention and control of *Epimedium* sclerotinia rot, gray mold, leaf spot, and anthracnose. They also possess nitrogen-fixing, organophosphate-dissolving, and potassium-solubilizing effects. Icariin and IAA can be detected in the fermentation broth, which can increase soil nutrient content, promote plant growth, and improve the quality of *Epimedium*. When used in combination, they exhibit synergistic effects, achieving plate inhibition rates of over 80.0% against all four pathogens. They can be used as effective foliar fertilizer components to control *Epimedium* diseases, characterized by long-lasting effects, good control efficacy, and environmental safety. When applied to the soil as a microbial fertilizer, they can increase the content of available nitrogen, phosphorus, and potassium in the soil, improve soil fertility, promote plant growth, and reduce the use of chemical fertilizers. This is of great significance in the field of high-yield, high-efficiency, green, and safe traditional Chinese medicine.

[0030] Biological Preservation Information This invention is based on Bacillus belesiensis ( Bacillus velezensis BY-2, categorized and named Bacillus from Velez The specimen is deposited at the China Center for Type Culture Collection (CCTCC) on November 19, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252587.

[0031] This invention relates to halophilic Bacillus ( Bacillus halotolerans AG-7, classified and named Bacillus halotolerant The specimen is deposited at the China Center for Type Culture Collection (CCTCC) on November 19, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252586.

[0032] This invention relates to Bacillus subtilis ( Bacillus subtilis D-1, classified and named Bacillus subtilis The specimen is deposited at the China Center for Type Culture Collection (CCTCC) on November 19, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252588. Attached Figure Description

[0033] Figure 1 The images show the colony morphology and scanning electron microscope (SEM) images of the three endophytic bacteria strains in Example 1 on beef extract peptone medium. AC represents the colony morphology of Bacillus belyssus BY-2, Bacillus halophilus AG-7, and Bacillus subtilis D-1, respectively, while DF represents the SEM images of Bacillus belyssus BY-2, Bacillus halophilus AG-7, and Bacillus subtilis D-1, respectively. Figure 2 In Example 1, phylogenetic trees of the strains were constructed using 16S rRNA (A) and gyrB gene sequences (B), respectively. Figure 3 This refers to the interaction among the three endophytic bacteria strains in Example 1; Figure 4 This is a plate culture diagram showing the confrontation between endophytic bacteria and Sclerotinia sclerotiorum, Botrytis cinerea, Bacillus anthracis, and Alternaria alternata in Example 2. Figure 5 This is a plate contrast diagram of the composite strain in Example 2 with Sclerotinia sclerotiorum, Botrytis cinerea, Bacillus anthracis, and Alternaria alternata. Figure 6 The results of the IAA colorimetric reaction of endophytic bacteria in Example 3 are shown. Detailed Implementation

[0034] This invention provides an endophytic bacterium from Epimedium, wherein the endophytic bacterium is Bacillus belye (…). Bacillus velezensis BY-2, accession number CCTCC NO: M 20252587.

[0035] The *Bacillus belyssus* BY-2 strain described in this invention was isolated from the leaves of *Epimedium brevicornu* grown in an artificial cultivation base in Zhang County, Dingxi City, Gansu Province. It was deposited at the China Center for Type Culture Collection (CCTCC) on November 19, 2025, with accession number CCTCCNO: M 20252587. The 16S rRNA gene sequence of *Bacillus belyssus* BY-2 is shown in SEQ ID NO.7, and the gyrB gene sequence is shown in SEQ ID NO.8.

[0036] The Bacillus berberis described in this invention is positive for the methyl red test, VP test, starch hydrolysis test, gelatin liquefaction test and cellulose decomposition test, and negative for the 7% salt tolerance test, hydrogen sulfide gas production test and citrate test.

[0037] In plate tests, *Bacillus belye* BY-2 showed inhibition rates of 83.7%, 88.9%, 82.5%, and 87.3% against *Sclerotinia sclerotiorum*, *Botrytis cinerea*, *Alternaria alternata*, and *Anthracnose*, respectively. When the aseptic fermentation broth concentration was 20%, the inhibition rates against *Sclerotinia sclerotiorum*, *Botrytis cinerea*, *Alternaria alternata*, and *Anthracnose* all exceeded 90.6%. This indicates that *Bacillus belye* BY-2 and its metabolites exhibit good inhibitory activity against four major pathogens causing diseases of *Epimedium*, and can be used for the prevention and control of *Epimedium* sclerotinia rot, gray mold, leaf spot, and anthracnose.

[0038] The *Bacillus belyssus* BY-2 of this invention possesses nitrogen-fixing and organophosphate-dissolving properties. Furthermore, after culturing *Bacillus belyssus* BY-2 in LB liquid medium, the aseptic fermentation broth showed a picaridin content of 0.074 μg / mL, which improves the quality of *Epimedium*; the fermentation broth also showed an IAA content of 23.377 mg / L, which significantly promotes plant growth.

[0039] This invention provides an endophytic bacterium from Epimedium, wherein the endophytic bacterium is a halophilic Bacillus (Bacillus). Bacillus halotolerans )AG-7, with accession number CCTCC NO: M 20252586.

[0040] The *Bacillus halophilus* AG-7 described in this invention was isolated from the roots of *Epimedium* from an artificial cultivation base in Zhang County, Dingxi City, Gansu Province, and was deposited at the China Center for Type Culture Collection (CCTCC) on November 19, 2025, with accession number CCTCC NO:M 20252586. The 16S rRNA gene sequence of *Bacillus halophilus* AG-7 is shown in SEQ ID NO.5, and the gyrB gene sequence is shown in SEQ ID NO.6.

[0041] The *Bacillus halophilus* AG-7 of this invention is positive in the methyl red test, VP test, starch hydrolysis test, gelatin liquefaction test, 7% salt tolerance test, and cellulose decomposition test, and negative in the hydrogen sulfide gas production test and citrate test.

[0042] In plate tests, *Bacillus halophilus* AG-7 showed inhibition rates of 81.4%, 77.8%, 85.0%, and 77.2% against *Sclerotinia sclerotiorum*, *Botrytis cinerea*, *Alternaria alternata*, and *Anthracnose*, respectively. When the aseptic fermentation broth concentration was 50%, the inhibition rates against *Sclerotinia sclerotiorum*, *Botrytis cinerea*, *Alternaria alternata*, and *Anthracnose* reached 100%, 100%, 96.5%, and 80.5%, respectively. This indicates that *Bacillus halophilus* AG-7 of this invention has good inhibitory activity against four major pathogens causing diseases of *Epimedium*, and can be used for the prevention and control of *Epimedium* sclerotinia rot, gray mold, leaf spot, and anthracnose.

[0043] The present invention relates to a halophilic Bacillus AG-7 strain that exhibits nitrogen-fixing activity. Furthermore, after culturing halophilic Bacillus AG-7 in LB liquid medium, the aseptic fermentation broth showed a picaridin content of 0.052 μg / mL, which improves the quality of Epimedium; the fermentation broth also showed an IAA content of 13.387 mg / L, which significantly promotes plant growth.

[0044] This invention provides an endophytic bacterium from Epimedium, wherein the endophytic bacterium is Bacillus subtilis (Bacillus subtilis). Bacillus subtilis D-1, with accession number CCTCC NO: M 20252588.

[0045] The Bacillus subtilis D-1 strain described in this invention was collected from the roots of Epimedium brevicornu in an artificial cultivation base in Zhang County, Dingxi City, Gansu Province, and was deposited at the China Center for Type Culture Collection (CCTCC) on November 19, 2025, with accession number CCTCC NO: M20252588. The 16S rRNA gene sequence of Bacillus subtilis D-1 is shown in SEQ ID NO.9, and the gyrB gene sequence is shown in SEQ ID NO.10.

[0046] The Bacillus subtilis strain described in this invention is positive in the D-1 methyl red test, VP test, starch hydrolysis test, gelatin liquefaction test, 7% salt tolerance test, cellulose decomposition test, and citrate test, and negative in the hydrogen sulfide gas production test.

[0047] In plate tests, Bacillus subtilis D-1 showed inhibition rates of 72.1%, 86.1%, 75.0%, and 82.3% against Sclerotinia sclerotiorum, Botrytis cinerea, Alternaria alternata, and Bacillus anthracis, respectively. When the concentration of the aseptic fermentation broth was 50%, the inhibition rates against these four pathogens reached 100%, 100%, 100%, and 74.0%, respectively. This indicates that Bacillus subtilis D-1 of this invention has good inhibitory activity against the four major pathogens causing diseases of Epimedium and can be used for the prevention and control of Sclerotinia sclerotiorum rot, gray mold, leaf spot, and anthracnose in Epimedium.

[0048] The Bacillus subtilis D-1 of this invention has nitrogen-fixing and potassium-solubilizing effects. Furthermore, after culturing Bacillus subtilis D-1 in LB liquid medium, the IAA content in the fermentation broth was 10.157 mg / L, which significantly promoted plant growth.

[0049] This invention also provides an endophytic bacterial composition of Epimedium, wherein the endophytic bacterial composition of Epimedium includes Bacillus belye ( Bacillus velezensis BY-2, Halophilic Bacillus ( Bacillus halotolerans AG-7 and Bacillus subtilis ( Bacillus subtilis )D-1.

[0050] In this invention, the preferred ratio of viable bacterial concentrations of Bacillus belyi BY-2, Bacillus halophilus AG-7, and Bacillus subtilis D-1 is (1~5):(1~5):(1~5), further preferably (1~3):(1~3):(1~3), and even more preferably 1:1:1.

[0051] In plate experiments, the three endophytic bacteria, fermented in a 1:1:1 volume ratio, exhibited inhibition rates of 81.4%, 86.1%, 80.0%, and 82.3% against *Sclerotinia sclerotiorum*, *Botrytis cinerea*, *Alternaria alternata*, and *Anthracnose*, respectively. Furthermore, the streaking on the plates showed no breakage at the cross-sections of the three strains, indicating no antagonistic effect among them. The combination of *Bacillus belye* BY-2, *Bacillus halophilus* AG-7, and *Bacillus subtilis* D-1 in this invention demonstrates good inhibitory activity against four major pathogens causing diseases of *Epimedium*, and can be used for the control of *Epimedium* sclerotinia rot, gray mold, leaf spot, and anthracnose.

[0052] In this invention, Bacillus berleis BY-2, Bacillus halophilus AG-7, and Bacillus subtilis D-1 all have nitrogen-fixing activity, Bacillus berleis BY-2 has the ability to dissolve organic phosphorus, and Bacillus subtilis D-1 has the ability to solubilize potassium. Meanwhile, after culturing Bacillus vesicularis BY-2 and Bacillus halophilus AG-7 in LB liquid medium at 28℃ and 150 r / min for 7 days, the icariin content in the sterile fermentation broth was detected at 0.074 μg / mL and 0.052 μg / mL, respectively. Furthermore, the icariin content in the sterile fermentation broth after mixed fermentation of the three reached 0.090 μg / mL, which can improve the quality of Epimedium. The IAA content in the fermentation broth of Bacillus vesicularis BY-2, Bacillus halophilus AG-7, and Bacillus subtilis D-1 was 23.377, 13.387, and 10.157 mg / L, respectively, which can significantly promote plant growth.

[0053] The present invention also provides a culture, which is a culture of the epimedium endophytic bacteria or a combination of the epimedium endophytic bacteria.

[0054] In this invention, the culture preferably comprises the fermentation product of the Epimedium endophytic bacteria or a combination of the Epimedium endophytic bacteria. The fermentation product preferably comprises fermentation broth or sterile fermentation broth.

[0055] In this invention, the preferred method for preparing the culture includes inoculating the Epimedium endophytic bacteria or a combination of Epimedium endophytic bacteria into a culture medium for fermentation culture to obtain a fermentation broth; Alternatively, the fermentation broth can be further centrifuged and filtered sequentially through 0.45μm and 0.22μm filter membranes to obtain sterile fermentation broth.

[0056] In this invention, the culture medium preferably includes a liquid culture medium; the liquid culture medium preferably includes LB liquid culture medium.

[0057] In this invention, the fermentation culture temperature is preferably 25~30℃, for example 25, 26, 27, 28, 29 or 30℃; the fermentation culture time is preferably 2~7 days, for example 2, 3, 4, 5, 6 or 7 days; the fermentation culture is preferably cultured under shaking conditions, and the shaking speed is preferably 150~200 r / min, for example 150, 160, 170, 180, 190 or 200 r / min.

[0058] In some embodiments, it is preferable to prepare a seed liquid by combining the Epimedium endophytic bacteria or Epimedium endophytic bacteria, and then inoculate the seed liquid into a culture medium for fermentation to obtain a fermentation broth.

[0059] The preferred method for preparing the Epimedium endophytic bacteria seed culture includes inoculating the Epimedium endophytic bacteria into LB liquid medium and culturing with shaking to prepare a seed culture. The culture temperature is preferably 25-30℃, for example, 25, 26, 27, 28, 29, or 30℃; the culture time is preferably 24-48h, for example, 24, 28, 32, 36, 40, 44, or 48h; and the shaking speed is preferably 150-200 r / min, for example, 150, 160, 170, 180, 190, or 200 r / min.

[0060] The preferred method for preparing the Epimedium endophytic bacterial combination seed solution includes inoculating Bacillus belye BY-2, Bacillus halophilus AG-7, and Bacillus subtilis D-1 into LB liquid medium and culturing them with shaking to prepare corresponding seed solutions; after mixing the seed solutions, the Epimedium endophytic bacterial combination seed solution is obtained. The preferred mixing volume ratio of the seed solutions is (1~5):(1~5):(1~5), more preferably (1~3):(1~3):(1~3), and even more preferably 1:1:1. The preferred culture temperature is 25~30℃, for example 25, 26, 27, 28, 29, or 30℃; the preferred culture time is 24~48h, for example 24, 28, 32, 36, 40, 44, or 48h; the preferred shaking speed is 150~200r / min, for example 150, 160, 170, 180, 190, or 200r / min.

[0061] The preferred viable bacteria concentration in the seed liquid is 10. 5 ~10 7 cfu / mL, more preferably 10 6 cfu / mL. The inoculation amount of the seed culture is preferably 1% to 5% of the culture medium volume, for example, 1%, 2%, 3%, 4% or 5%.

[0062] In some embodiments of this invention, the method for preparing the sterile fermentation broth preferably includes centrifuging the fermentation broth and filtering it sequentially through 0.45 μm and 0.22 μm filter membranes to obtain the sterile fermentation broth. The centrifugation speed is preferably 5000~10000 r / min, more preferably 8000~10000 r / min; the centrifugation time is preferably 5~20 min.

[0063] In this invention, the sterile fermentation broth preferably contains indoleacetic acid and icariin.

[0064] The present invention also provides an epimedium disease-resistant and growth-promoting product, the product comprising epimedium endophytic bacteria or a combination of epimedium endophytic bacteria or the culture.

[0065] In this invention, the type of product is not specifically limited and can be selected according to actual needs; in some embodiments, the product preferably includes seed treatment agents, fungicides, growth promoters, fertilizers, or soil conditioners, etc. The product may also include product-acceptable excipients, and there is no specific limitation on the type of excipients, including but not limited to one or more of carriers, diluents, excipients, preservatives, surfactants, and antioxidants.

[0066] In some embodiments of the present invention, the endophytic bacteria of Epimedium or a combination of the endophytic bacteria of Epimedium with a carrier are used to prepare a stable formulation that can extend the field shelf life; the carrier preferably includes sodium alginate microcapsules or attapulgite microcapsules.

[0067] The present invention also provides the application of the epimedium endophytic bacteria, the epimedium endophytic bacteria combination, the culture, or the product in improving the quality and yield of epimedium or preventing epimedium fungal diseases.

[0068] In this invention, the Epimedium refers to the Epimedium genus of the Berberidaceae family. Epimedium Epimedium (plant) Epimedium brevicomu ).

[0069] In this invention, the fungal diseases affecting Epimedium preferably include one or more of sclerotinia rot, gray mold, leaf spot, and anthracnose. The pathogens of these fungal diseases preferably include *Sclerotinia sclerotiorum* (…). Sclerotinia sclerotia ), Botrytis cinerea ( Botrytis cinerea ), Alternaria alternifolia ( Alternaria alternata ) and anthrax bacteria ( Colletotrichum incanum Any one or more of the following.

[0070] The present invention also provides a method for improving the quality and yield of Epimedium or for controlling Epimedium fungal diseases. The method for improving the quality and yield of Epimedium preferably includes: spraying the Epimedium endophytic bacteria or a combination of Epimedium endophytic bacteria or the culture or the product onto the above-ground parts of the Epimedium plant or applying it into the soil.

[0071] There are no particular limitations on the spraying method; in some embodiments, an electric sprayer is used. The above-ground parts of the Epimedium plant preferably include stems or leaves.

[0072] In this invention, improving the quality and yield of Epimedium preferably includes increasing the dry weight, fresh weight, leaf area, and icariin content of Epimedium. Applying the Epimedium endophytic bacteria, a combination of Epimedium endophytic bacteria, the culture, or the product as a microbial fertilizer to the soil can increase the content of available nitrogen, phosphorus, and potassium in the soil, improve soil fertility, promote plant growth, and reduce the use of chemical fertilizers.

[0073] In this invention, the method for preventing and controlling Epimedium fungal diseases preferably includes: spraying the Epimedium endophytic bacteria, the Epimedium endophytic bacteria combination, the culture, or the product onto the above-ground parts of the Epimedium plant.

[0074] Using the endophytic bacteria of Epimedium, or a combination of the endophytic bacteria of Epimedium, or the culture or the product as a biological pesticide, applied to plants by spraying or other methods, can effectively prevent and control diseases such as sclerotinia rot, gray mold, anthracnose and leaf spot, and reduce the use of chemical pesticides.

[0075] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0076] Unless otherwise specified, the following embodiments are all conventional methods.

[0077] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0078] Example 1: Isolation and Identification of Endophytic Bacteria from Epimedium 1. Isolation of endophytic bacteria (1) Sample collection: Vigorous and disease-free Epimedium plants were collected in Zhang County, Dingxi City. 50g of roots, stems and leaves were selected as separation materials, put into sterile plastic bags and brought back to the laboratory.

[0079] (2) Surface disinfection: Take 1g each of plant roots, stems and leaves, first soak them in 75% ethanol for 1 minute, then rinse them twice with sterile water; then soak them in 1% sodium hypochlorite solution for 30 seconds, and finally rinse them three times with sterile water to ensure that the surface is free of bacteria.

[0080] (3) Isolation of endophytic bacteria: After disinfection, samples from different parts were placed in a sterile mortar, and 1.5 mL of sterile water was added for grinding. After grinding, the samples were placed in a laminar flow hood for 10 min. The resulting supernatant was the stock solution. The stock solution was serially diluted, and 100 μL was pipetted onto beef extract peptone agar plates using a sterile pipette. Each sample was spread onto 3 plates and incubated in a 28℃ incubator for 2-3 days. Once colonies grew on the plates, single colonies with different morphologies were picked and streaked multiple times for purification until uniform single colonies were obtained. The purified strains were then inoculated onto slant agar plates and stored at 4℃ for later use.

[0081] 2. Identification of endophytic bacteria (1) Morphological observation: A small number of purified bacterial colonies were streaked on LB solid medium and incubated at 28°C for 24 hours. The morphological characteristics of the colonies were observed, and the cell structure was observed using scanning electron microscopy. The results are as follows: Figure 1 As shown.

[0082] As can be seen, on LB plates, strains AG-7, BY-2, and D-1 are pale yellow, with opaque colonies and irregular edges; all three showed positive Gram staining. Scanning electron microscopy revealed that the three strains were rod-shaped, occurring singly or in long chains, with blunt ends, no flagella, and no spores. Strain AG-7 was 1.016–1.988 μm long and 0.554–0.985 μm in diameter, relatively elliptical, and had a smooth surface; strain BY-2 was 1.041–1.516 μm long and 0.318–0.864 μm in diameter, relatively slender, and had a rough surface; strain D-1 was 1.024–1.639 μm long and 0.453–0.961 μm in diameter, relatively slender, and had a rough surface (n=50). Preliminary identification indicates that all three endophytic bacteria belong to the genus *Bacillus*. Bacillus ).

[0083] (2) Physiological and biochemical characteristics determination: The physiological and biochemical characteristics of the three Epimedium endophytic bacteria isolated were determined according to the method in the "Manual of Systematic Identification of Common Bacteria", including oxidase test, catalase test, starch hydrolysis test, etc. The results are shown in Table 1.

[0084] Table 1 Results of physiological and biochemical assays

[0085] Note: + indicates positive; - indicates negative.

[0086] It can be seen that the three strains were positive for methyl red test, VP test, starch hydrolysis test, gelatin liquefaction test and cellulose decomposition test, and negative for hydrogen sulfide gas production test; D-1 was positive for 7% salt tolerance test and citrate test; AG-7 was positive for 7% salt tolerance test and negative for citrate test; BY-2 was negative for both 7% salt tolerance test and citrate test.

[0087] (3) Genomic DNA was extracted from endophytic bacteria and 16S rRNA gene sequence was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2); gyrB gene sequence was amplified by PCR using primers UP1 (5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3', SEQ ID NO.3) and UP2R (5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTAT-3', SEQ ID NO.4).

[0088] The PCR reaction system consisted of 25 μL, including 12.5 μL of 2×Master Mix, 1 μL each of primers (10 μM), 1 μL of template DNA, and 9.5 μL of ddH2O.

[0089] The PCR reaction conditions were as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ (16S rRNA) / 60℃ (gyrB) annealing for 30 seconds, 72℃ extension for 1 minute, for a total of 35 cycles; 72℃ extension for 10 minutes.

[0090] After agarose gel electrophoresis, the PCR products were sent to a sequencing company for sequencing. The sequencing results showed that the 16S rRNA gene sequence of AG-7 is shown in SEQ ID NO.5, and the gyrB gene sequence is shown in SEQ ID NO.6; the 16S rRNA gene sequence of BY-2 is shown in SEQ ID NO.7, and the gyrB gene sequence is shown in SEQ ID NO.8; the 16S rRNA gene sequence of D-1 is shown in SEQ ID NO.9, and the gyrB gene sequence is shown in SEQ ID NO.10. The obtained 16S rRNA and gyrB gene sequences were compared with known sequences in the GenBank database to construct a phylogenetic tree of the strains to determine the species of endophytic bacteria. The results are as follows: Figure 2 As shown.

[0091] It can be seen that BY-2 is Bacillus belye ( Bacillus velezensis ), AG-7 is a halophilic bacillus ( Bacillus halotolerans D-1 is Bacillus subtilis ( Bacillus subtilis The following bacteria were deposited at the China Center for Type Culture Collection on November 19, 2025: BY-2 was named Bacillus belyestris BY-2, with accession number CCTCC NO: M 20252587; AG-7 was named Bacillus halophilus AG-7, with accession number CCTCC NO: M 20252586; and D-1 was named Bacillus subtilis D-1, with accession number CCTCC NO: M 20252588.

[0092] 3. Interactions among endophytes After activating the three bacterial strains on LB agar plates, pairwise plate interaction experiments were conducted using the cross-plotting method to determine whether there were antagonistic or inhibitory effects among the strains. The results are as follows: Figure 3 As shown.

[0093] It can be seen that the three strains did not break at the points where they crossed each other, indicating that there was no antagonistic effect between the strains.

[0094] Example 2: Determination of antibacterial activity of endophytic bacteria 1. Preparation of pathogens: The four main pathogens of Epimedium (Sclerotinia sclerotiorum, Botrytis cinerea, Alternaria alternata, and Bacillus anthracis) that were previously isolated, identified and preserved were inoculated on PDA medium and incubated at 25°C for 5-7 days. After the mycelium covered the plate, pathogen mycelial cakes with a diameter of 5 mm were prepared using a punch.

[0095] Preparation of compound bacterial culture: *Bacillus belyeis* BY-2, *Bacillus halophilus* AG-7, and *Bacillus subtilis* D-1 were inoculated into 100 mL LB liquid medium and cultured at 28℃ with shaking at 160 rpm for 24 h to prepare seed culture; a viable bacterial concentration of 10... 6 Seed cultures of Bacillus berreatus BY-2, Bacillus halophilus AG-7, and Bacillus subtilis D-1, at a volume ratio of 1:1:1, were added to LB liquid medium for fermentation. After culturing for 5 days at 28°C and 180 rpm, a compound bacterial culture was obtained.

[0096] Plate confrontation culture method: ① Place the cultured fungal cake in the center of a prepared PDA plate. Mark four points on the back of the plate using a cross-marking method, approximately 2.5 cm away from the indicator bacteria. Inoculate the strain activated and cultured on NA medium for 48 h at each of these four points. Use the unmarked biocontrol as a control. Incubate at 25℃. When the control pathogen has fully colonized the plate, measure the width of the inhibition zone (i.e., the distance from the edge of the biocontrol colony to the edge of the tested pathogen colony) and calculate the inhibition rate. Repeat three times and take the average value. ② Place the pathogen fungal cake in the center of the PDA medium. Make holes on all four sides at 2.5 cm away from the cake. Add 10 µL of endophytic compound bacterial solution to each hole. Set up a blank control group (sterile water). After incubation at 25℃ for 7 days, measure the diameter of the inhibition zone and the width of the inhibition band with calipers to evaluate its antibacterial activity. Colony growth inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100. The results are shown in Table 2 and... Figure 4 , Figure 5 As shown.

[0097] Table 2. Antagonistic and inhibitory effects of endophytic bacteria on four major pathogenic fungi of Epimedium.

[0098] It can be seen that Bacillus berleis BY-2, Bacillus halophilus AG-7, and Bacillus subtilis D-1 all have good inhibitory activity against the four major pathogens causing diseases of Epimedium. Moreover, the inhibitory activity is significantly enhanced after the three strains are combined, and they can be used for the prevention and control of Epimedium sclerotinia rot, gray mold, leaf spot and anthracnose.

[0099] 2. Single colonies of the purified strain were inoculated into LB liquid medium and cultured at 28℃ and 200 rpm for 2 days to obtain the fermentation broth. The broth was centrifuged at 10000 rpm for 20 min, and the supernatant was collected. The supernatant was filtered once through a 0.45 μm disposable microporous membrane and then once through a 0.22 μm disposable microporous membrane to obtain sterile fermentation broth, which was stored at 4℃ for later use. The obtained antagonistic strain sterile fermentation broth was poured into 250 mL Erlenmeyer flasks containing 100 mL of PDA medium at 50℃ at volume fractions of 10%, 20%, 30%, 40%, and 50%, respectively. After thorough mixing, the flasks were poured into plates, and a 5 mm diameter bacterial pellet was inoculated into the center of each plate. PDA plates inoculated only with the pathogen without the antagonistic strain sterile fermentation broth served as a control. The plates were incubated at 25℃ for 4 days, and the colony diameter was measured to calculate the inhibition rate. Each treatment was repeated 5 times. Inhibition rate = (colon diameter of control - colony diameter of treatment) / colony diameter of control × 100%. The results are shown in Table 3.

[0100] Table 3. Inhibitory effects of aseptic fermentation broth of endophytic bacteria on four pathogens.

[0101] It can be seen that as the concentration of the sterile fermentation broth increases, the inhibition rate of the sterile fermentation broth of the three endophytic bacteria against Sclerotinia sclerotiorum, Botrytis cinerea, Alternaria alternata, and Bacillus anthracis increases. Among them, the sterile fermentation broth of the three bacteria can achieve a high inhibition rate against Sclerotinia sclerotiorum at a relatively low concentration. The sterile fermentation broth of the three bacteria has the highest inhibition rate against Sclerotinia sclerotiorum and Botrytis cinerea, reaching 100%. When the concentration of the sterile fermentation broth of Bacillus belyss BY-2 is 30%, the inhibition rate against all four pathogenic fungi reaches 100%, indicating that its metabolites have a strong antagonistic effect.

[0102] Example 3: Determination of the growth-promoting characteristics of endophytic bacteria 1. *Bacillus belyeis* BY-2, *Bacillus halophilus* AG-7, and *Bacillus subtilis* D-1 were inoculated into 100 mL LB broth and cultured at 28°C with shaking at 160 rpm for 24 h to prepare seed cultures. The seed cultures were then inoculated into NFM, Monkina organophosphate, and potassium feldspar agar media and incubated at 28°C for 3 days. Colony growth was observed. The three strains were qualitatively assessed on agar plates using nitrogen fixation, phosphorus solubilization, and potassium solubilization as indicators to evaluate their growth-promoting abilities.

[0103] 2. The seed cultures of the three purified strains were inoculated into LB medium containing L-tryptophan, and the colorimetric reactions were observed using the Salkowski colorimetric method. The indoleacetic acid (IAA) production of the strains was qualitatively determined, and the results are as follows: Figure 6 As shown, from left to right, the strains are AG-7, BY-2, and D-1.

[0104] 3. Inoculate 4% seed culture into 50 mL LB liquid medium, adding 500 μL of tryptophan to every 1% seed culture. Incubate at 28℃ with shaking at 160 rpm for 48 h to obtain the bacterial fermentation broth. Take 10 mL of the bacterial fermentation broth and centrifuge at 8000 rpm for 5 min. Take 5 mL of the supernatant and add an equal volume of Salkowski colorimetric reagent to make up to the final volume. After incubating at room temperature in the dark for 30 min, measure its OD using a UV spectrophotometer. 535 The nm value was used to calculate the IAA content per unit volume of fermentation broth by referring to the standard curve. The results are shown in Table 4.

[0105] Table 4. Determination of the growth-promoting ability of endophytic bacteria

[0106] It can be seen that *Bacillus belliesi* BY-2, *Bacillus halophilus* AG-7, and *Bacillus subtilis* D-1 all have nitrogen-fixing activity, *Bacillus belliesi* BY-2 has the ability to dissolve organic phosphorus, and *Bacillus subtilis* D-1 has the ability to solubilize potassium. Meanwhile, the IAA content in the fermentation broths of *Bacillus belliesi* BY-2, *Bacillus halophilus* AG-7, and *Bacillus subtilis* D-1 were 23.377, 13.387, and 10.157 mg / L, respectively, all of which significantly promoted plant growth.

[0107] Example 4: Determination of Secondary Metabolites of Endophytic Bacteria Purified single colonies of *Bacillus belyssioides* BY-2, *Bacillus halophilus* AG-7, and *Bacillus subtilis* D-1 were inoculated into LB liquid medium and cultured for 7 days at 28°C and 150 rpm to obtain the fermentation broth. Simultaneously, a compound bacterial culture was prepared using the same method as in Example 2. The fermentation broths of the three strains or the compound bacterial culture were centrifuged at 10000 rpm for 20 min, and the supernatant was collected. The supernatant was first filtered through a 0.45 μm disposable microporous membrane, and then through a 0.22 μm disposable microporous membrane to obtain the sterile fermentation broth. Secondary metabolites in the sterile fermentation broths of the three strains and the compound bacterial culture were analyzed by ultraviolet spectrophotometry combined with LC-MS. The results are shown in Table 5. It can be seen that the contents of icariin in the sterile fermentation broth of Bacillus vesiculosus BY-2 and Bacillus halophilus AG-7 were 0.074 μg / mL and 0.052 μg / mL, respectively; after the three strains were mixed and fermented, the contents of icariin in the sterile fermentation broth were 0.090 μg / mL.

[0108] Table 5. Determination of secondary metabolites of endophytic bacteria

[0109] Example 5 Field Trial The live bacteria concentration was 10. 6 Seed cultures of *Bacillus belyssioides* BY-2, *Bacillus halophilus* AG-7, and *Bacillus subtilis* D-1 (cfu / mL) were added to LB broth at a volume ratio of 1:1:1 for fermentation. After 5 days of culture at 28°C and 150 rpm, the cultures were stored at 4°C for later use. In spring, after *Epimedium* had developed two true leaves, the plants were sprayed with a mixture of the compound microbial agent and sterile water, once each, every 15 days for a total of three applications. Each treatment was applied to 100 plants, with three replicates. In mid-July, growth and disease incidence were assessed for each treatment.

[0110] The results showed that, compared with the group treated with sterile water, the application of compound microbial agents significantly increased the dry weight, fresh weight, leaf area and icariin content of Epimedium, and reduced the incidence of diseases such as Sclerotinia sclerotiorum, gray mold, anthracnose and leaf spot.

[0111] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An endophytic bacterium of Epimedium, characterized in that, The endophytic bacteria in Epimedium are Bacillus belysinus (… Bacillus velezensis BY-2, accession number CCTCC NO: M 20252587.

2. An endophytic bacterial assemblage of Epimedium, characterized in that, The endophytic bacterial assemblage of Epimedium includes Bacillus belye (… Bacillus velezensis BY-2, Halophilic Bacillus ( Bacillus halotolerans AG-7 and Bacillus subtilis ( Bacillus subtilis The Bacillus subtilis BY-2 has the accession number CCTCC NO: M20252587, the Bacillus halophilus AG-7 has the accession number CCTCC NO: M 20252586, and the Bacillus subtilis D-1 has the accession number CCTCC NO: M 20252588.

3. The epimedium endophytic bacteria assemblage according to claim 2, characterized in that, The 16S rRNA gene sequence of *Bacillus belyssiensis* BY-2 is shown in SEQ ID NO.7, and the gyrB gene sequence is shown in SEQ ID NO.8; the 16S rRNA gene sequence of *Bacillus halophilus* AG-7 is shown in SEQ ID NO.5, and the gyrB gene sequence is shown in SEQ ID NO.6; the 16S rRNA gene sequence of *Bacillus subtilis* D-1 is shown in SEQ ID NO.9, and the gyrB gene sequence is shown in SEQ ID NO.

10. And / or the viable cell concentration ratio of the aforementioned Bacillus berleis BY-2, Bacillus halophilus AG-7 and Bacillus subtilis D-1 is (1~5):(1~5):(1~5).

4. A culture, characterized in that, The culture is a combination of Epimedium endophytic bacteria as described in claim 1 or any one of claims 2-3.

5. The culture according to claim 4, characterized in that, The culture comprises the fermentation product of the Epimedium endophytic bacteria as described in claim 1 or the combination of Epimedium endophytic bacteria as described in any one of claims 2 to 3; And / or the fermentation product includes fermentation broth or sterile fermentation broth.

6. The method for preparing the culture according to claim 5, characterized in that, This includes inoculating the Epimedium endophytic bacteria of claim 1 or the Epimedium endophytic bacteria combination of any one of claims 2 to 3 into a culture medium for fermentation culture to obtain a fermentation broth; Alternatively, the fermentation broth can be further centrifuged and filtered sequentially using 0.45μm and 0.22μm filter membranes to obtain sterile fermentation broth; And / or the culture medium includes LB liquid medium; And / or the fermentation temperature is 25~30℃; the oscillation speed of the fermentation is 150~200r / min; and the fermentation time is 2~7d.

7. A disease-resistant and growth-promoting product made from Epimedium, characterized in that, The product comprises the Epimedium endophytic bacteria as described in claim 1, or the Epimedium endophytic bacteria combination as described in any one of claims 2-3, or the culture as described in any one of claims 4-5.

8. The product according to claim 7, characterized in that, The products include seed treatment agents, fungicides, growth promoters, fertilizers, or soil conditioners.

9. The use of the Epimedium endophytic bacteria of claim 1, or the combination of Epimedium endophytic bacteria of any one of claims 2-3, or the culture of any one of claims 4-5, or the product of any one of claims 7-8, in improving the quality and yield of Epimedium or in preventing Epimedium fungal diseases.

10. A method for improving the quality and yield of Epimedium or for controlling Epimedium fungal diseases, characterized in that, The method for improving the quality and yield of Epimedium includes: spraying the Epimedium endophytic bacteria of claim 1, or the combination of Epimedium endophytic bacteria of any one of claims 2-3, or the culture of any one of claims 4-5, or the product of any one of claims 7-8 onto the above-ground parts of the Epimedium plant or applying it into the soil. The method for preventing and controlling Epimedium fungal diseases includes: spraying the Epimedium endophytic bacteria of claim 1, or the combination of Epimedium endophytic bacteria of any one of claims 2-3, or the culture of any one of claims 4-5, or the product of any one of claims 7-8 onto the above-ground parts of the Epimedium plant.

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