Streptomyces virginiae m54 and its use in the control of plant diseases

Abstract

The application discloses streptomyces virginiae M54 and application thereof in preventing and treating plant diseases. Streptomyces virginiae The application discloses streptomyces virginiae M54 and application thereof in preventing and treating plant diseases. The application discloses streptomyces virginiae M54 and application thereof in preventing and treating plant diseases. The application discloses streptomyces virginiae M54 and application thereof in preventing and treating plant diseases. The application discloses streptomyces virginiae M54 and application thereof in preventing and treating plant diseases. The application discloses streptomyces virginiae M54 and application thereof in preventing and treating plant diseases.

Description

Technical Field

[0001] This invention relates to the field of microbial and plant disease control technology, specifically to Streptomyces Virginiae M54 and its application in the control of plant diseases. Background Technology

[0002] blueberry( Blueberry L.) belongs to the genus Vaccinium (Ericaceae). Blueberry Blueberries are a type of small berry. Yunnan Province, with its high altitude, strong sunshine, and large diurnal temperature range, has become a major early-ripening and off-season blueberry producing area in my country. Currently, blueberries are grown in Kunming, Yuxi, Qujing, Honghe, Wenshan, and Dali, among other areas in Yunnan Province. However, with the expansion of cultivation, blueberries are suffering from various diseases to varying degrees, seriously affecting their quality and grade.

[0003] Common blueberry diseases currently include: gray mold, stem base rot, branch blight, fruit rot, leaf spot, stem brown spot, and root rot. Currently identified pathogens causing blueberry diseases include: Yan Qian et al. (2024) isolated a strain of *Botrytis cinerea* from diseased blueberry leaves in Majiang County, Guizhou Province. Botrytis cinerea Yang Xiumei et al. (2022) isolated, identified, and determined the pathogenicity of blueberry plants with typical symptoms of stem base rot in Jiangchuan District, Yuxi City, and identified the pathogen causing stem base rot in Yunnan Province as *Ilex chinensis* (Holly redbud). Calonectria ilicicola Zhang Xiaoyan et al. (2023) identified the pathogen of blueberry twig blight in Sanming, Fujian Province as a fungus of the genus *Metacarpa*. Australian Diaporthe ), and first reported that the fungus could infect blueberry branches; Wang Fei et al. (2021) isolated Botrytis cinerea and Alternaria from diseased blueberry fruit in California. Alternaria Nees These two pathogens can cause blueberry fruit rot; Bai Jianbo et al. (2024) isolated the pathogen of blueberry leaf spot disease in Mengzi City, Yunnan Province, and found that the pathogen causing blueberry leaf spot disease in Mengzi City, Yunnan Province is Alternaria alternifolia (Alternaria alternifolia). Alternaria alternata Yang Xiumei et al. (2022) identified the pathogen of blueberry leaf spot disease in Yuxi City, Yunnan Province as *Cyclocarya paliurus*. Calonectria colhounii Sun Can et al. (2023) identified the pathogen causing stem brown spot disease in blueberries in Changchun City, Jilin Province as Alternaria spp. (Alternaria spp.) Alternaria tenuissima The study screened the fungicides fluazinam and difenoconazole, finding them to have good inhibitory effects against Alternaria spp. Zhou Yanan et al. (2020) discovered a new blueberry root rot disease in Qingdao, Shandong, and identified the pathogen as Pythium terrestris (…). Pythium irregulare This pathogen was first reported in China to cause blueberry root rot. Currently, *Cladosporium spp.* has not been observed to cause this disease. Cladosporium tenuissimum There are reports that Cooke can cause blueberry diseases.

[0004] Currently, plant disease control mainly relies on chemical pesticides. However, the overuse of chemical pesticides has led to a series of drawbacks, including environmental pollution, pesticide residues, and the development of pesticide resistance in pathogens. In particular, the use of chemical pesticides makes it difficult to guarantee the safety of economic crops for consumption. Therefore, there is an urgent need to explore an economical, effective, and environmentally friendly biological control method. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned problems by providing a *Streptomyces virginiana* M54 and its application in the prevention and control of plant diseases.

[0006] To achieve its objective, the present invention employs the following technical solution: The first aspect of the present invention provides a strain of Streptomyces Virginiae ( Streptomyces virginiae M54 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20252043.

[0007] A second aspect of the present invention provides a microbial agent whose active ingredient comprises the above-mentioned Streptomyces Virginiae M54 or its fermentation broth or its spore liquid.

[0008] The fermentation broth is prepared by activating Streptomyces Virginiae M54 and inoculating it into a culture medium, then shaking and culturing it at 25-37°C until the OD600 is 0.6-1.5 (preferably 0.6-0.8), thus obtaining the fermentation broth.

[0009] The method for preparing the spore solution is as follows: after activating the Virginia Streptomyces M54 strain, inoculate it onto a culture medium plate and incubate it at 25-37°C. After it produces spores, scrape the spores and suspend them in sterile water. Filter to remove the mycelium to obtain the spore solution.

[0010] Preferably, the culture medium is Gao's No. 1 culture medium, and the culture temperature is 25~32℃.

[0011] More preferably, the culture temperature is 28~32℃ (preferably 28~30℃).

[0012] A third aspect of the invention provides the use of the above-described Streptomyces Virginiae M54 or fungal agent in any of the following: (1) Use in inhibiting plant pathogenic fungi or in preventing and controlling plant diseases caused by said plant pathogenic fungi; (2) Use in the preparation of products that inhibit plant pathogenic fungi or prevent plant diseases caused by said plant pathogenic fungi; (3) Applications in promoting plant growth.

[0013] Among them, the plant pathogenic fungi include Botrytis cinerea. Botrytis cinerea Anthrax pathogen Colletotrichum phyllanthi Cucumber wilt pathogen Fusarium oxysporum f. sp cucumber Fusarium wilt of tomatoes Fusarium oxysporum f. sp . lycopersiciSnyder et Hansen, Fusarium rotundifolium Fusarium solani Horsetail Fusarium horsetail Rice black spore fungus Nigrospora oryzae Alternaria microphylla Alternaria tenuissima Tall Mucor Chaetomium elatum Fusarium graminearum Fusarium grasses Clostridium difficile Neofusicoccus minor Phytophthora indicum Phytophthora nicotianae Phytophthora palmis Phytophthora palmivora Or Alternaria alternifolia Alternaria alternata . The fourth aspect of the present invention provides the use of the above-described Streptomyces Virginiae M54 or fungal agent in any of the following: (1) Inhibits Cladosporium Cladosporium tenuissimum Cooke or its application in the prevention and control of plant diseases caused by Cladosporium; (2) Application in the preparation of products that inhibit Cladosporium or prevent plant diseases caused by Cladosporium.

[0014] The plant mentioned includes blueberries, and the plant diseases caused by Cladosporium include blueberry bud blight / calyx purple shriveling disease.

[0015] The beneficial effects of this invention are: the *Streptomyces Virginiae* strain screened by this invention (… Streptomyces virginia M54 exhibits strong inhibitory effects against various filamentous fungal pathogens, showing significant control over leek leaf rot caused by *Alternaria alternifolia*. It is particularly effective in controlling a newly emerging blueberry disease caused by *Cladosporium falciparum*, preventing brown spot on blueberry buds and fruit deformities caused by *Cladosporium falciparum*. Growth-promoting function verification showed that M54 produces IAA, fixes nitrogen, and produces protease, promoting seed germination and plant growth. The *Streptomyces Virginiae* M54 screened in this invention possesses excellent biocontrol and growth-promoting properties, showing promising application prospects as a biocontrol agent for plant diseases, especially providing an effective biocontrol agent for diseases caused by the newly discovered blueberry pathogen *Cladosporium falciparum*. Attached Figure Description

[0016] Figure 1 The following are displayed: A and B: Field symptoms of blueberry bud blight; C and D: Field symptoms of purplish calyx wilt disease.

[0017] Figure 2The symptoms of disease 3 days after the isolated pathogen was inoculated into blueberry buds, leaves and fruits are shown: A, B, C and D are LM-1, LM-2, LM-3 and LMG-1 inoculated into blueberry buds, leaves and fruits, respectively.

[0018] Figure 3 Morphological characteristics of Cladosporium: A: Front view of colony; B: Back view of colony; C: Sporophorides and conidia; D and E: Conidia.

[0019] Figure 4 Phylogenetic tree of strains LM-1, LM-2, LM-3, and LMG-1.

[0020] Figure 5 This is a colony morphology diagram of M54.

[0021] Figure 6 This is a phylogenetic tree diagram of M54.

[0022] Figure 7 The image shows the results of the plate confrontation method for verifying antagonistic bacteria.

[0023] Figure 8 The results show the antagonistic effect of M54 on Cladosporium.

[0024] Figure 9 Results of an experiment involving the inoculation of detached chive leaves with Alternaria alternifolia.

[0025] Figure 10 Results of an experiment involving inoculating isolated blueberry apical buds with Cladosporium.

[0026] Figure 11 The results validate the nitrogen fixation function of the M54 fermentation broth.

[0027] Figure 12 To verify the protease production function of M54 bacterial culture. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0029] Example 1: Identification of the pathogen causing a new disease in blueberries In 2025, our research team discovered a blueberry bud blight and calyx purple rot disease in a blueberry-growing area of ​​Jianshui County, Yunnan Province. The pathogen initially causes the blueberry terminal buds to wither, then transfers to the blueberry fruit, leading to purple rot and shriveling of the calyx. To identify the pathogen, this study employed morphological observation, molecular biology, and pathogenicity assays to investigate the pathogens of blueberry bud blight and calyx purple rot diseases, discovering a new blueberry pathogen—*Cladosporium spp.* (…). Cladosporium tenuissimum Cooke).

[0030] 1. Materials and Methods 1.1 Materials 1.1.1 Blueberry Disease Sample Collection Blueberry disease samples were collected on February 21, 2025, from Jianshui County, Honghe Prefecture, Yunnan Province. The characteristics and symptoms of the disease were recorded at the time of collection. Plant protection personnel from the blueberry company were consulted to understand the occurrence and damage of the disease.

[0031] 1.1.2 Tested blueberry varieties Two-year-old F6 plants grown in substrate.

[0032] 1.1.3 Test culture medium and main reagents Rose Bengal agar medium; PDA medium; 2×Taq Plus Master Mix II; 0.05mol / L NaOH solution.

[0033] 1.2 Methods 1.2.1 Isolation and purification of pathogens Diseased buds and fruits were brought back to the laboratory and cut into 5 mm x 5 mm pieces using surgical scissors. They were placed in 9 cm sterile petri dishes, rinsed three times with sterile water, then disinfected with 75% alcohol for 3 minutes, rinsed three times with sterile water, disinfected with 2% sodium hypochlorite for 3 minutes, and rinsed three times with sterile water. After blotting dry with sterile filter paper, the pieces were transferred to Rosacea agar and PDA plates, sealed, and incubated at 28°C. Observations were made daily. Once hyphae appeared on the pathogenic tissue, they were picked from the edge with sterile forceps and transferred to a new PDA plate. This transfer process was repeated 2-3 times to complete purification.

[0034] 1.2.2 Pathogenicity determination of pathogens Take healthy F6 blueberry leaves, buds, and berries from Jianshui County. Wash the leaves, buds, and berries with 75% alcohol, then rinse twice with sterile water. Prepare cotton balls and filter paper moistened with sterile water beforehand, and place the filter paper at the bottom of a food storage container. Wrap the leaf petioles, the lower ends of the buds, and the berry stems with cotton balls moistened with sterile water, place them in the food storage container, and then lightly prick the leaves, buds, and berries with a dissecting needle.

[0035] Using a scalpel, the pathogens growing on the PDA were cut into 5 mm x 5 mm pieces. Three leaves were used for each treatment, with two pieces of pathogen inoculated on each leaf, repeated three times. Three buds were used for each treatment, with four pieces of pathogen inoculated on each bud, repeated three times. Ten fruits were used for each treatment, with one piece of pathogen inoculated at the calyx of each fruit, repeated three times. The container was covered, and the disease incidence was observed after three days. The pathogens were isolated from the diseased leaves, buds, and fruits, and Koch's postulates were used for verification.

[0036] 1.2.3 Morphological observation of pathogens Inoculate the center of a 6 cm culture dish with a circular mycelial cake or spore liquid containing the pathogen with a diameter of 6 mm and observe its growth. After sporulation, rinse the surface of the mycelium with sterile water and observe the spore liquid under a microscope.

[0037] 1.2.4 Molecular biological identification of pathogens DNA was rapidly extracted using an alkaline lysis method. The PCR system consisted of: 2×Taq Plus Master Mix II (12.5 μL), ITS1 (0.5 μL), ITS4 (0.5 μL), template DNA (1 μL), and ddH2O (10.5 μL). The PCR reaction conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 3 s, 72℃ extension for 90 s, for a total of 30 cycles, followed by a final extension at 72℃ for 10 min, and storage at 4℃. After passing 1% agarose gel electrophoresis, the amplified products were purified and sequenced by Sangon Biotech Co., Ltd. The assembled sequencing results were subjected to BLAST homology analysis in NCBI, and the sequences were submitted to GenBank. Sequences of strains with high similarity to the amplified products were downloaded from GenBank, and a phylogenetic tree was constructed using neighbor-joining (NJ) with MEGA11 software to identify the species of each pathogen.

[0038] 2 Results and Analysis 2.1 Field symptoms of blueberry bud blight and calyx purple shriveling disease The affected blueberry variety is F6. The pathogen causes the blueberry terminal buds to wither, and the symptoms of bud withering are shown in the figure. Figure 1 As shown in -AB); blueberry fruit disease mainly occurs during the fruit ripening stage, and the pathogen causes the fruit tip to turn purple and wither (as shown in -AB); Figure 1 -C), by using a dissecting needle to prick open the purplish and atrophied part, a brown lesion layer can be seen ( Figure 1 -D), the diseased fruit will not rot. Bud wilting began in 2024, mainly from October to December each year; the diseased fruit appeared from December 2024 to February 2025.

[0039] 2.2 Results of pathogen isolation and purification Three pathogens with different morphologies were isolated and purified from withered buds and named LM-1, LM-2, and LM-3, respectively. One pathogen was isolated and purified from diseased fruit and named LMG-1.

[0040] 2.3 Pathogenicity determination of pathogens When the pathogen was inoculated onto blueberry buds, leaves, and fruit, large areas of brown lesions caused by LM-1 pathogen were observed on blueberry buds and leaves after 3 days, and large areas of disease layer appeared on the fruit. Figure 2 -A). LM-2 pathogen causes brown spots on blueberry buds and leaves, and spots begin to appear at wounds on the fruit. Figure 2 -B). LM-3 and LMG-1 pathogens cause lesions on the terminal buds of blueberries, but do not affect older leaves. Instead, they cause disease at the calyx of the blueberry fruit, resulting in an inward depression at the calyx. Figure 2 -CD).

[0041] 2.4 Observation of Pathogen Morphological Characteristics Strain LM-1, cultured at 28℃ on a PDA for 4 days, completely filled a 6 cm culture dish. Colonies grew rapidly, with gray, velvety hyphae and radial growth. Colonies had irregular edges and a grayish-brown underside. With prolonged culture, the aerial fungi became dense and velvety, producing numerous conidia, but no black sclerotia were observed during growth. Conidiophores were erect, slender, branching at the apex in a dendritic manner, with swollen ends bearing numerous conidia. Conidia were spherical, oval, or sesame-shaped, with smooth surfaces, and approximately 5-8 x 12-16 μm in size.

[0042] The LM-2 strain, cultured at 28℃ on a PDA for 4 days, completely filled a 6 cm culture dish. The hyphae spread out like a carpet, with relatively dense aerial hyphae that were brown, long, and septate. A few conidia were darker in color and had two sporophytic chains. The conidia were obclavate, brown, and had varying degrees of brick-like septa, with dark brown septa at the septa. A few conidia had only transverse septa and no longitudinal septa.

[0043] LM-3 and LMG-1 have the same colony morphology and structure. After 7 days of growth on PDA medium, the colony diameter is 3 cm, with few hyphae, consisting mainly of white outer rings. Colony growth is slow, and during growth, spores are produced simultaneously. Figure 3 -A). The colony surface is dark green, the reverse side is black, and the outermost edge has a ring of white hyphae ( Figure 3 -B). Colonies bulge upwards and develop cracks; hyphae are dense and typically blanket-like. Figure 3 -A). Conidiophores are erect, with numerous conidia at the apex, resembling branches ( Figure 3-C). Conidia are relatively small, oval, oblong, or fusiform in shape, measuring 1-3 x 2-10 μm. Figure 3 -DE).

[0044] 2.5 Molecular biological identification results of pathogens The ITS sequence lengths of strains LM-1, LM-2, LM-3, and LMG-1 are shown in Table 1 below. A phylogenetic tree constructed based on the ITS sequences is shown in Figure 1. Figure 4 As shown in the image, the sequences of the four bacterial strains have been uploaded to NCBI and obtained their corresponding access numbers. BLAST alignment of the sequences revealed that LM-1 matches MN077161.1. Botrytis cinerea The similarity is 100%, and they are on the same branch of the phylogenetic tree. LM-2 and MN856385.1 Alternaria alternata The similarity reached 99.83%, and it is similar to OQ001035.1. Alternaria alternata The similarity reached 99.48%, and the three strains were located on the same branch of the phylogenetic tree. LM-3 and MG873070.1 Cladosporium tenuissimum Cook showed a similarity of 99.64% and LMG-1 showed a similarity of 99.82%, and they were both on the same branch of the phylogenetic tree.

[0045] Table 1 Sequencing results of the isolated strains

[0046] 3. Analysis and Summary Strains LM-1, LM-2, and LM-3 were isolated from diseased blueberry buds in Jianshui County, Yunnan Province, and strain LMG-1 was isolated from diseased fruit. Pathogenicity was determined using the needle prick method to verify Koch's postulates. Based on the morphological structure and molecular biological identification results, LM-1 and LM-2 were identified as *Botrytis cinerea* and *Alternaria*, respectively; LM-3 and LMG-1 were identified as *Cladosporium*. Cladosporium tenuissimum Cook. When inoculated onto healthy buds, fruits, and leaves, LM-1 caused buds and leaves to turn brown, resulting in large areas of lesions and severe fruit rot; LM-2 also caused buds and leaves to turn brown and fruits to rot; LM-3 and LMG-1 caused lesions at the very tips of buds but did not cause leaf disease, but led to purple and shriveled blueberry calyxes. The results indicate that blueberry bud blight is caused by a combination of *Botrytis cinerea*, *Alternaria*, and *Cladosporium*; calyx rot is caused by *Cladosporium*.

[0047] Numerous studies, both domestically and internationally, have focused on *Botrytis cinerea* and *Alternaria*, two common pathogens that cause leaf, flower, and fruit rot under high humidity conditions. However, reports on *Cladosporium* are scarce. The earliest domestic report on *Cladosporium* dates back to 1983, when He Pingxun et al. (1987) discovered that *Cladosporium* damages the terminal buds and shoots of larch trees in Jilin Province, causing bud blight. Lu Haiju et al. (2011) found that *Cladosporium* was the pathogen causing pomegranate mold in Mengzi, Yunnan. This pathogen damages older pomegranate leaves, forming dark brown lesions with an olive-green mold layer, leading to premature fruit drop. In November 2021, Xiao Y et al. first discovered *Cladosporium* causing Dekopon fruit brown spot disease in an orchard of the Citrus Research Institute in Jiangxi Province, China. In early October 2016, Xie XW et al. first observed that *Cladosporium wiltii* caused spotting on carnation leaves in carnations planted at the Chinese Academy of Agricultural Sciences and in parks in Xi'an. International reports on *Cladosporium wiltii* indicate that this pathogen infects cucumber fruits, causing numerous small, round swellings on the peel, making the fruit unsuitable for market (Batta YA et al., 2004). *Cladosporium wiltii* causes necrosis of flowers, pedicels, and small fruits on Mexican mango inflorescences, with the affected organs covered by gray, cottony mycelium and olive-green to gray spores (Guillén-Sánchez D et al., 2007).

[0048] This study marks the first discovery that *Cladosporium fragrans* can cause blueberry terminal bud wilting and calyx a purplish-red coloration and shrinkage. The pathogen affects blueberry terminal buds from October to December. If not properly controlled, it can transfer to the blueberry fruit from December to February of the following year, causing the calyx to turn purple and shrink. The disease primarily affects blueberry fruit during the ripening stage. Previously, *Cladosporium fragrans* was first reported to infect citrus and carnations in October or November, indicating a clear seasonality of the disease. Therefore, targeted fungicides can be selected before October each year to control the disease and prevent significant economic losses. Accurate identification of the pathogen provides biological material for further research on the disease and lays the foundation for blueberry disease control in the following year.

[0049] Example 2: Obtaining biocontrol bacterium M54 and its antibacterial and plant disease control experiments. I. Strains Isolation In November 2024, soil samples were uniformly collected from a leek field in Sankai, Mile City, Yunnan Province. 10 g of the collected soil sample was weighed and added to 90 mL of sterile water. The sample was then incubated at 180 rpm for 30 min at room temperature. The supernatant was then aspirated and used in a 10-fold serial dilution method on Gao's No. 1 medium for biocontrol bacterial isolation. 100 μL of the soil dilution was evenly spread on each plate and incubated in the dark at 28℃ for 3-5 days. After colonies grew, bacteria with different colony morphologies were selected, purified, propagated, and stored for later use.

[0050] II. Strain Identification Take 1 mL of liquid Gao's No. 1 culture base and transfer it to a 2 mL sterile centrifuge tube. Add a single colony of M54, obtained by dipping a sterile pipette tip, to the tube. Figure 5 Seal and incubate at 30℃ and 180 rpm for 12 h.

[0051] 16S rDNA was directly amplified by PCR from bacterial culture using universal primer pairs (primers 27F and 1492R). The PCR amplification reaction system consisted of: 1 μL bacterial culture sample, 12.5 μL 2 × Rapid Taq Master Mix (Vazyme, China), 1 μL each of forward and reverse primers (10 μM), and ddH2O to a final volume of 25 μL. The PCR amplification conditions were: 95℃ for 3 min; 95℃ for 15 sec, 55℃ for 15 sec, 72℃ for 15 sec, for 35 cycles; 72℃ for 5 min.

[0052] The amplified products were detected by 1% agarose gel electrophoresis and purified before being sent to a sequencing company for DNA sequencing. The assembled sequences were submitted to the NCBI database for sequence alignment. Homologous sequences were downloaded from the NCBI database, and a phylogenetic tree was constructed using MEGA 11.0 software. The 16S rDNA gene sequence of strain M54 was submitted to GeneBank, and homology alignment revealed that the 16S rDNA gene sequence of strain M54 was consistent with that of strain MW391646.1. Streptomyces virginiae The strains showed a similarity of 99.65%, and a phylogenetic tree was constructed based on the 16S rDNA gene sequence. Figure 6 M54 and MW391646.1 Streptomyces virginiae On one branch, with 100% support, strain M54 was identified as *Streptomyces virginiana*. Streptomyces virginiae ).

[0053] The 16S rDNA sequence (SEQ ID NO.1) of strain M54 is as follows:

[0054] The preservation information for strain M54 is as follows: Strain M54 was deposited at the China Center for Type Culture Collection (CCTCC) in September 2025, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province. The deposit date was September 15, 2025; accession number: CCTCC NO: M 20252043; classification and naming: Streptomyces virginiae M54.

[0055] III. Plate confrontation method for verifying antagonistic bacteria 1. Preparation of single colonies of Streptomyces Virginiae: The frozen M54 strain was streaked onto Gao's No. 1 medium plates and incubated at 28°C for 3 days. After colonies grew, single colonies were picked and cultured in liquid Gao's No. 1 medium at 30°C with shaking at 180 rpm until OD600≈1.5. This culture was then used for a confrontation inhibition test against pathogenic strains.

[0056] 2. Antagonistic bacterial plate confrontation Four points were marked on a PDA plate 2.5 cm from the center using the streak method. A 10 mm diameter blank antimicrobial susceptibility test strip was then placed at each of the four points. Fungal pellets were punched from the edge of the pathogenic fungal colony using a 5 mm punch and placed in the center of the streak. 100 μL of M54 bacterial suspension (OD600≈1.5) was added to each blank antimicrobial susceptibility test strip. After the bacterial suspension dried, the plates were inverted and incubated in the dark at 28℃ for 1–10 days (the incubation time varies depending on the pathogen; incubation continued until the size of the lesions no longer changed). A control group was set up, and incubation continued until the control pathogenic fungus hyphae completely covered the plate, allowing for the selection of pathogens exhibiting clear confrontation.

[0057] 3. Screening criteria for antagonistic bacteria Antagonistic screening was performed on pathogens from different plant species, and the colony diameter of the pathogens was measured to calculate the inhibition rate.

[0058]

[0059] The results are as follows Figure 7 As shown in Table 2, M54 exhibited antagonistic effects against all 14 tested pathogenic fungi. This indicates that M54 is a good broad-spectrum antagonistic microorganism that can effectively inhibit the growth of various pathogens.

[0060] Table 2. Statistics on the width of the inhibition zone of bacteria M54

[0061] 4. Verification of antagonism between Cladosporium and other microsporidiae Since Cladosporium produces relatively few hyphae, it is difficult to conduct antibacterial experiments using the plate confrontation method. Since Cladosporium reproduces by spores, antibacterial experiments are conducted using spore liquid.

[0062] Take 100 μL of *Cladosporium spores* cultured to an OD600 value of 0.8 and spread it evenly on a PDA plate. Use a 5 mm punch to create a mycelial cake from an M54 mycelial plate and place it in the center of the plate. Invert the plate and incubate in the dark at 28℃ for 1-6 days, with a control group included, until a clear inhibition zone appears. Figure 8 The results show the bacterial growth after 6 days of culture, with a clear inhibition zone in the center of the plate while other areas are covered with Cladosporium, demonstrating that M54 can inhibit the germination of Cladosporium spores.

[0063] Example 3: In vitro leaf inoculation experiment I. Leek Experimental Group After activating the M54 strain, culture it in liquid Gao's No. 1 medium at 180 rpm and 28℃ with shaking until OD600≈0.6. The unfiltered fermentation broth is then used.

[0064] Alternaria alternifolia spore suspension (OD600=0.6) and M54 (OD600=0.6) fermentation broth were mixed at a volume ratio of 1:1, resulting in a mixed broth OD=0.6. Uniformly growing chive leaves were selected and inoculated with the mixed broth as the experimental group. Two control groups were established: one group inoculated solely with Alternaria alternifolia spore suspension (+CK), and another group inoculated with water (-CK). The inoculation method was as follows: Chive leaves are tender and succulent; punctures or friction can cause wilting and rotting. Therefore, a direct inoculation method was used. 5 μL of the broth (or water) was inoculated at a distance of 5 cm from the tip of the chive leaf. After one day of incubation at 28℃ in the dark, the leaves were transferred to a 28℃ light (12 h dark: 12 h light) incubator for 3 days. Disease incidence was observed and recorded. Each experimental group, +CK group, and -CK group was tested in triplicate, with three chive leaves used for each replicate.

[0065] Depend on Figure 9 It can be seen that in the +CK group, which was inoculated only with Alternaria alternifolia spore liquid, the chive leaves turned yellow and soft overall, with browning and rotting at the tips; the chive leaves in the -CK group were firm and green, and grew well; the chive leaves in the mixed bacterial solution experimental group were firm and green, and grew well. This indicates that M54 has a good control effect on chive leaf rot caused by Alternaria alternifolia (+CK), proving that M54 has a significant effect on the control of chive leaf rot.

[0066] II. Blueberry Experimental Group: In Vitro Seed Inoculation Experiment Preparation of M54 spore solution: M54 strain was inoculated onto Gao's No. 1 culture medium plate and cultured at 28°C. After spore production, the spores were scraped off and suspended in sterile water. The mycelium was filtered through four layers of gauze to prepare a spore solution with OD600=0.8.

[0067] Preparation of compound spore solution for the experimental group: Using Cladosporium spore solution with OD600 value = 0.8 as the pathogen, it was mixed with M54 spore solution (OD600 = 0.8) at a volume ratio of 1:1 to prepare a compound spore solution for later use.

[0068] Uniformly growing blueberry terminal buds were cut and placed in sterile inoculation boxes. The experimental groups were inoculated with a compound spore solution. Groups inoculated only with *Cladosporium ferruginosa* spore solution were designated as the +CK group, and those inoculated with water were designated as the -CK group. After one day of incubation at 28°C in the dark, the buds were transferred to a 28°C light (12 h dark: 12 h light) incubator for 5 days. Disease incidence was observed and recorded. The inoculation method was the puncture method: a small hole was punctured on each side of the blueberry terminal bud's apical bud using a syringe needle, and then 10 μL of spore solution (or water) was inoculated into each bud. Each experimental group, +CK group, and -CK group was performed in triplicate, with three terminal buds from each replicate.

[0069] The experimental results showed that all blueberry buds in the +CK group turned brown and withered, while no symptoms were observed in the buds of the experimental group and the -CK group. Figure 10 The results show that M54 spore liquid has a good control effect on blueberry apical bud browning and necrosis caused by Cladosporium wilt (+CK), proving that M54 has a significant effect on the control of Cladosporium wilt.

[0070] Example 4: Verification of the fertility-promoting function Rhizosphere growth-promoting bacteria typically have functions such as IAA production, nitrogen fixation, and protease production. This example verifies the growth-promoting function of M54.

[0071] I. Verification of Nitrogen Fixation Function Nitrogen-fixing microorganisms can convert nitrogen in the soil into liquid ammonia that plants can absorb, increasing the nitrogen content in the soil and promoting plant growth and development. Nitrogen-fixing Ashby medium: 0.2 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 0.2 g sodium chloride, 0.1 g calcium sulfate heptahydrate, 5.0 g calcium carbonate, 10 ml 0.5% Congo red solution, 18 g agar powder, 1000 ml distilled water, 121℃, 15 min. Inoculate the center of the nitrogen-fixing medium with a susceptibility testing tablet containing 10 μL of M54 bacterial suspension (OD600=0.6), incubate at 30℃ for 3 days, and observe and record the results. Figure 11As shown: A clear clear zone appears around M54 on the nitrogen-fixing medium. The formation of the clear zone is due to the organic acid secreted by the nitrogen-fixing bacteria dissolving the calcium carbonate. The presence of the clear zone indicates that strain M54 has nitrogen-fixing activity.

[0072] II. Verification of Protease Production Function Microorganisms that produce proteases can break down proteins into amino acids, providing nitrogen sources for plants, increasing soil nitrogen content, and promoting leaf growth, chlorophyll synthesis, and plant immune responses. A protease assay medium was used: 15 g skim milk powder, 15 g agar, 1000 ml distilled water, sterilized at 121℃ for 15 min. A susceptibility testing tablet containing 10 μL of M54 bacterial suspension (OD600=0.6) was inoculated in the center of the protease monitoring medium, and the medium was incubated at 30℃ for 3 days. The results were observed and recorded. Figure 12 As shown: A clear zone appears around M54 on the culture medium, indicating that strain M54 has the function of producing protease. The clear zone is caused by protein hydrolysis due to protease activity.

[0073] Experiments of this invention show that strain M54 has a strong inhibitory effect on various filamentous fungal pathogens, especially on chive leaf rot caused by Alternaria alternifolia. It also shows significant control effects on newly emerging blueberry terminal bud brown spot and fruit malformation caused by Cladosporium fasciatus. After verification of its growth-promoting function, strain M54 has nitrogen-fixing and protease-producing functions, which can promote seed germination and plant growth and development. It has good biocontrol function and growth-promoting effect, and has excellent application prospects as a biocontrol fungus for the prevention and control of plant diseases.

Claims

1. A strain of Streptomyces Virginiae ( Streptomyces virginiae M54 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20252043.

2. A microbial agent, the active ingredient of which comprises Streptomyces Virginiae M54 as described in claim 1, or its fermentation broth or its spore liquid.

3. The microbial agent as described in claim 2, characterized in that: The fermentation broth is prepared by activating Streptomyces Virginiae M54 and inoculating it into a culture medium. The culture is then shaken at 25-37°C until the OD600 is 0.6-1.5, thus obtaining the fermentation broth.

4. The microbial agent as described in claim 2, characterized in that: The method for preparing the spore solution is as follows: after activating the Virginia Streptomyces M54 strain, inoculate it onto a culture medium plate and incubate it at 25-37°C. After it produces spores, scrape the spores and suspend them in sterile water. Filter to remove the mycelium to obtain the spore solution.

5. The microbial agent as described in claim 3 or 4, characterized in that: The culture medium is Gao's No. 1 medium, and the culture temperature is 25~32℃.

6. The microbial agent as described in claim 5, characterized in that: The culture temperature is 28~32℃.

7. The use of *Streptomyces Virginiae* M54 as described in claim 1 or the inoculum as described in claim 2 in any of the following: (1) Use in inhibiting plant pathogenic fungi or in preventing and controlling plant diseases caused by said plant pathogenic fungi; (2) Use in the preparation of products that inhibit plant pathogenic fungi or prevent plant diseases caused by said plant pathogenic fungi; (3) Applications in promoting plant growth.

8. The application according to claim 7, characterized in that: The plant pathogenic fungi include *Botrytis cinerea*. Botrytis cinerea Anthrax pathogen Colletotrichum phyllanthi Cucumber wilt pathogen Fusarium oxysporum f. sp cucumerinum Fusarium wilt of tomatoes Fusarium oxysporum f. sp . lycopersiciSnyder et Hansen, Fusarium rotundifolium Fusarium solani Horsetail Fusarium equiseti Rice black spore fungus Nigrospora oryzae Alternaria microphylla Alternaria tenuissima Tall Mucor Chaetomium elatum Fusarium graminearum Fusarium graminearum Clostridium difficile Neofusicoccum parvum Phytophthora indicum Phytophthora nicotianae Phytophthora palmis Phytophthora palmivora Or Alternaria alternifolia Alternaria alternata .

9. The use of *Streptomyces Virginiae* M54 as described in claim 1 or the inoculum as described in claim 2 in any of the following: (1) Inhibits Cladosporium Cladosporium tenuissimum Cooke or its application in the prevention and control of plant diseases caused by Cladosporium; (2) Application in the preparation of products that inhibit Cladosporium or prevent plant diseases caused by Cladosporium.

10. The application according to claim 9, characterized in that: The plant includes blueberries, and the plant diseases caused by Cladosporium include blueberry bud blight / calyx purple shriveling.

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