Purple protocyst and application of purple protocyst in biological prevention and control of wheat stem rot

Abstract

The invention discloses a strain of protocyst violaceum and application of the protocyst violaceum in biological prevention and treatment of wheat stem rot, and the strain WE12 has a preservation number of CGMCC (China General Microbiological Culture Collection Center) No.36395 and is classified and named as the protocyst violaceum. The protocyst purple WE12 disclosed by the invention has efficient predatory activity on various pathogenic bacteria causing wheat stem rot, and meanwhile, degerming fermentation filtrate and volatile metabolites of the strain also show remarkable bacteriostatic activity on the wheat stem rot, so that the protocyst purple WE12 has multiple antibacterial mechanisms. Indoor potting and field control effect tests show that the protocyst purpureum WE12 not only has a good control effect on wheat stem rot caused by single pathogenic bacteria, but also has a good control effect on compound infection of multiple pathogens, and also has remarkable growth promoting and yield increasing effects. The application potential in the aspect of biological prevention and control of the wheat stem rot is huge.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of *Protozoa purpureus* and its application in the biological control of wheat stem base rot. Background Technology

[0002] Wheat crown rot (FCR) is a worldwide soil-borne fungal disease caused by the combined infection of various Fusarium species, severely impacting wheat yield and quality. The main pathogens causing wheat crown rot include *Fusarium pseudograminearum*, *Fusarium graminearum*, *Fusarium culmorum*, *Fusarium equiseti*, *Fusarium fujikuroi*, *Fusarium proliferatum*, *Fusarium oxysporum*, and *Fusarium acuminatum*. Among these, *Fusarium pseudograminearum*, *Fusarium graminearum*, and *Fusarium culmorum* are the dominant pathogens causing wheat crown rot in wheat-growing areas of my country.

[0003] The control of wheat stem rot primarily relies on chemical control. Currently, the fungicides recommended by the National Agricultural Technology Extension Center mainly include cyazofamid, tebuconazole, and difenoconazole. However, the overuse of chemical agents can lead to a series of environmental and food safety problems. Biological control has received widespread attention due to its safety, environmental friendliness, and long-lasting effectiveness. Existing biological control systems mainly focus on two types of microbial resources: fungi, represented by genera such as *Trichoderma* and *Chaetomium*, and bacteria, primarily represented by genera such as *Bacillus*, *Pseudomonas*, and *Streptomyces*. Because existing biological control microorganisms are mostly concentrated on a few "star" strains, the diversity of the biocontrol microbial resource bank is insufficient. Furthermore, existing biocontrol strains and their formulations are mostly limited to screening and evaluating the efficacy of single pathogens (especially *Fusarium graminearum*). This single-target control model fails to fully consider the inherent characteristic of this disease—multiple pathogens infecting it—leading to potentially narrow control spectra in the resulting biocontrol strains and formulations. This limits their application potential in complex field pathogen environments, making it difficult to guarantee efficacy. Therefore, developing novel biocontrol formulations that can simultaneously target multiple dominant pathogens of wheat stem rot and achieve broad-spectrum, high-efficiency control has become an urgent need to address the shortcomings of existing technologies and promote the development of green control technologies for wheat stem rot.

[0004] Myxobacteria are a group of higher prokaryotes with social and predatory behaviors, and are considered "native" bacteria in the soil. Myxobacteria not only actively prey on other microorganisms through "wolf pack" hunting behavior and gliding motion, but they can also differentiate into highly resistant myxospores, exhibiting strong environmental adaptability. Furthermore, they can produce a rich variety of secondary metabolites and hydrolytic enzymes. Moreover, as myxobacteria occupy the apex of the soil microbial food web, their predation of soil-borne pathogens directly affects the soil microecological environment, playing a crucial role in maintaining soil microecological balance and plant health. These characteristics endow myxobacteria with unique biocontrol advantages, making them considered a novel type of biocontrol microorganism.

[0005] Archangium violaceum belongs to the phylum Myxobacteria, class Myxococci, order Myxococciales, family Archangycetes, and genus Archangium (including 7 species). Currently, there are relatively few Archangium resources protected by patents for use in plant disease control. A search revealed that the fermentation supernatant of Archangium violaceum 3-1, disclosed by the Guangdong Institute of Microbiology, exhibits significant antibacterial activity against Fusarium oxysporum (application number: 202110820724.0); Archangium sp. AC19, disclosed by Nanjing Agricultural University, can prey on various plant pathogens, including Fusarium oxysporum, Fusarium graminearum, and Rhizoctonia solani, and can be used for the control of Fusarium wilt of cucumber and Phytophthora in soybean (application number: 202210755118.X); and Archangium violaceum NST-47, disclosed by Xinjiang Agricultural University, shows good biocontrol effects against pear fire blight and pear canker (application number: 202310637997.0). However, the antibacterial effects vary considerably among different species and strains. Even strains of the same species with different strain types exhibit significant differences in antibacterial activity and mechanisms of action. For example, *Bacillus amyloliquefaciens* shows different antibacterial characteristics against plant pathogens, and related strains have all been patented (e.g., 202411789819.0, 201710053493.9, and 201410503991.5). Furthermore, wheat stem rot is caused by a complex infection of multiple *Fusarium* species, making control even more difficult. There are no reports on the application of *Protozoa violacea* in the biological control of wheat stem rot caused by *Fusarium graminearum*, *Fusarium graminearum*, and *Fusarium graminearum*. Therefore, given the current state of patent protection for biocontrol bacteria, especially *Protozoa violacea* as a class of biocontrol microorganisms with application potential, the isolation, identification, and patent protection of biocontrol bacteria resources with excellent biocontrol effects are of great significance for the green control of plant diseases. Summary of the Invention

[0006] To address the above problems, this invention provides a strain of Archangium violaceum WE12 and its application in the biological control of wheat stem base rot.

[0007] The present invention provides a strain of Archangium violaceum WE12, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), classified and named Archangium violaceum. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is October 29, 2025, and the deposit number is CGMCC No. 36395.

[0008] The present invention also provides a fermentation culture broth of Archangium violaceum WE12 as described in this invention. The fermentation broth is a fermentation culture broth containing cells of Archangium violaceum WE12 prepared according to conventional methods.

[0009] This invention also provides a sterile fermentation broth for Archangium violaceum WE12 as described in this invention. This sterile fermentation broth is obtained by filtering out the bacterial cells from a fermentation culture containing Archangium violaceum WE12 prepared using conventional methods.

[0010] This invention also provides volatile metabolites of Archangium violaceum WE12 as described herein. These volatile metabolites are substances that volatilize into the air produced by culturing Archangium violaceum WE12 using conventional methods.

[0011] This invention also provides a biological agent containing Archangium violaceum WE12 as an active ingredient. The biological agent can be in a common form in the art; for example, it can be a liquid microbial agent prepared from Archangium violaceum WE12 fermentation broth, suitable for controlling wheat stem rot using drip irrigation or spraying; or it can be a solid agent prepared using a solid fermentation substrate of white-spotted beetle sand, suitable for controlling wheat stem rot by pre-sowing basal application. The specific preparation method of the relevant agent can follow conventional methods in the art.

[0012] The present invention also provides a bio-fertilizer comprising Archangium violaceum WE12 as described herein.

[0013] The present invention also provides the application of Archangium violaceum WE12, fermentation broth, sterilized fermentation broth, and volatile metabolites in the preparation of antifungal products, wherein the fungus is selected from the Fusarium genus, and further includes any one or more of Fusarium pseudograss, Fusarium grass, Fusarium chrysogenum, Fusarium oxysporum, Fusarium fusiforme, Fusarium spp., and Fusarium scutellarioides.

[0014] This invention also provides the application of Archangium violaceum WE12, fermentation broth, sterilized fermentation broth, volatile metabolites, and biological agents in the biological control of wheat stem base rot. The pathogen causing wheat stem base rot is a Fusarium strain, specifically one or more of the following: *Fusarium pseudograss*, *Fusarium grass*, *Fusarium xanthophytum*, *Fusarium equisetifolium*, *Fusarium truncatum*, *Fusarium moniliforme*, *Fusarium oxysporum*, and *Fusarium argentis*. Preferably, the pathogen causes wheat stem base rot through infection with one or more of *Fusarium pseudograss*, *Fusarium grass*, and *Fusarium xanthophytum*.

[0015] The present invention also provides the application of the described Archangium violaceum WE12, fermentation broth, biological agent or bio-fertilizer in promoting wheat growth and / or increasing wheat yield.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The biocontrol bacteria Archangium violaceum WE12 provided by the present invention exhibits strong predation ability against a variety of pathogens of wheat stem base rot and has broad-spectrum antibacterial activity.

[0018] (2) The biocontrol agent Archangium violaceum WE12 provided by this invention not only has a good control effect on wheat stem base rot caused by single pathogen infection, but also has a good biocontrol effect on wheat stem base rot caused by multiple infections of dominant pathogens. It solves the problem that existing biocontrol microorganisms have a narrow control spectrum and limited application potential in complex pathogen environments in actual fields.

[0019] (3) The biocontrol agent Archangium violaceum WE12 provided by this invention has multiple antibacterial mechanisms and flexible application methods. The pot efficacy test results show that the Archangium violaceum WE12 fermentation liquid has a control efficacy of 70.33%-82.80% against single infection by Fusarium pseudograss, Fusarium graminearum, and Fusarium xanthophyte, as well as the combined infection of the three pathogens. The control efficacy of Archangium violaceum WE12 solid inoculant is 74.72%-81.52%. Therefore, Archangium violaceum WE12 fermentation liquid can be used to develop liquid microbial inoculants and used for drip irrigation or spraying to control wheat stem rot; solid inoculants can also be prepared using the solid fermentation substrate of white star flower beetle sand and used for basal application before sowing to control wheat stem rot.

[0020] (4) The biocontrol bacteria Archangium violaceum WE12 provided by the present invention can not only control wheat stem base rot, but also promote growth and increase yield.

[0021] (5) The biocontrol bacterium Archangium violaceum WE12 provided by the present invention is an environmentally friendly biocontrol strain. Archangium is widely present in nature and is a "native bacterium" in the soil. It is not pathogenic to crops and is safe for humans, animals, insects and crops, thus solving the problem that microorganisms with biocontrol potential may turn into pathogens.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] Figure 1 The images shown are morphological feature diagrams of strain WE12 in the embodiments of the present invention, where a is a morphological diagram of the bacterial film formed by strain WE12 after being inoculated on VY / 4 solid medium and cultured for 5 days; b is a morphological diagram of the fruiting body of strain WE12; and c is a morphological diagram of strain WE12 cells.

[0024] Figure 2 This is a phylogenetic tree of strain WE12 constructed based on the 16S rDNA and gyrB gene sequences in an embodiment of the present invention, where a is the 16S rDNA gene phylogenetic tree; b is the gyrB gene phylogenetic tree;

[0025] Figure 3 This invention demonstrates the predatory effect of strain WE12 against multiple pathogens causing wheat stem rot in this embodiment of the invention.

[0026] Figure 4 This invention describes the antibacterial effect of the sterilization fermentation filtrate of strain WE12 on the dominant pathogens of wheat stem rot (Fusarium graminearum, Fusarium graminearum, and Fusarium chrysogenum). Specifically, a represents the antibacterial effect of the sterilization fermentation filtrate on the mycelial growth of Fusarium graminearum, Fusarium graminearum, and Fusarium chrysogenum; b represents the inhibition rate of the sterilization fermentation filtrate on the mycelial growth of Fusarium graminearum, Fusarium graminearum, and Fusarium chrysogenum; and c represents the spore lysis rate and spore germination inhibition rate of the sterilization fermentation filtrate on the spores of Fusarium graminearum, Fusarium graminearum, and Fusarium chrysogenum.

[0027] Figure 5 This invention demonstrates the antibacterial effect of the volatile metabolites of strain WE12 on the mycelial growth of *Fusarium graminearum*, *Fusarium graminearum*, and *Fusarium oxysporum* in the embodiments of the present invention.

[0028] Figure 6This invention presents the pot-grown control efficacy of the fermentation broth of strain WE12 against wheat stem base rot in various embodiments; wherein a) is the control effect of the fermentation broth of strain WE12 against wheat stem base rot caused by *Fusarium graminearum* infection; b) is the control effect of the fermentation broth of strain WE12 against wheat stem base rot caused by *Fusarium graminearum* infection; c) is the control effect of the fermentation broth of strain WE12 against wheat stem base rot caused by *Fusarium graminearum* infection; and d) is the control effect of the fermentation broth of strain WE12 against wheat stem base rot caused by a combination of three pathogens.

[0029] Figure 7 This invention presents the potted plant control efficacy of strain WE12 solid inoculant against wheat stem base rot in embodiments of the present invention; wherein a) is the control effect of strain WE12 solid inoculant against wheat stem base rot caused by *Fusarium graminearum* infection; b) is the control effect of strain WE12 solid inoculant against wheat stem base rot caused by *Fusarium graminearum* infection; c) is the control effect of strain WE12 solid inoculant against wheat stem base rot caused by *Fusarium graminearum* infection; and d) is the control effect of strain WE12 solid inoculant against wheat stem base rot caused by a combination of three pathogens.

[0030] Figure 8 This invention illustrates the effect of strain WE12 on wheat growth in an embodiment of the present invention. Detailed Implementation

[0031] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0032] Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0033] LB medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, pH 7.0.

[0034] LBS liquid culture medium: soluble starch 7.0 g / L, tryptone 1.0 g / L, yeast extract 5.0 g / L, MgSO4·7H2O 1.0 g / L, pH 7.2-7.4.

[0035] VY / 4 medium: 2.5 g / L dry yeast, 1.0 g / L CaCl2, 15 g / L agar powder, pH 7.2-7.4.

[0036] PDA medium: 200 g / L potato, 20 g / L glucose, 15 g / L agar, natural pH.

[0037] WCX medium: CaCl2 1.0 g / L, agar powder 15 g / L, pH 7.2, after sterilization, add actinomycete ketone to a final concentration of 25 μg / mL.

[0038] 43% tebuconazole was purchased from Shandong Zouping Pesticide Co., Ltd.

[0039] Example 1: Isolation, purification and identification of strain WE12

[0040] 1.1 Separation and Purification

[0041] Soil samples were collected from chili pepper fields in Kuihe Village, Jiahezi Township, Wusu City, Xinjiang Uygur Autonomous Region, at a depth of 5-10 cm. After air-drying at room temperature, small stones and mulch were removed using a 40-mesh sieve. 20 g of the air-dried soil sample was weighed and placed in a sterile petri dish, then placed in an oven at 60℃ for 30 min. After cooling, actinomycete ketone solution (final concentration 25 μg / mL) was added to saturate the sample. The sample was soaked overnight (≥12 h), and the residual solution was discarded for later use.

[0042] Erwinia amylovora was inoculated into LB liquid medium and cultured overnight (≥12 h) at 30°C with shaking. 1 mL of the bacterial suspension was transferred to a centrifuge tube, centrifuged at 12,000 rpm for 1 min, and the bacterial cells were collected. After rinsing three times with sterile water, 100 μL was retained as the bacterial suspension. Using a sterile inoculation loop, the bacterial suspension was drawn in a crisscross pattern on the surface of WCX medium containing actinomycete ketone (final concentration 25 μg / mL). After drying, 0.5 g of the treated soil sample was placed in the center of each of the four squares and incubated at 30°C. After 48 h, fruiting bodies induced in the WCX medium were picked up using a sterile fine needle under a stereomicroscope and inoculated onto the surface of VY / 4 medium, and incubated at 30°C for 3 days. Once a clear bacterial film has grown on VY / 4 medium, promptly pick off the outer edge of the film and transfer it to a fresh VY / 4 plate for further purification until no contaminating bacteria grow, thus obtaining strain WE12. Inoculate the purified strain WE12 into LB broth and incubate overnight (≥12 h) at 30°C with shaking at 180 rpm. Observe the state of the culture medium; if it is clear and transparent, the strain is pure; if it is turbid, it indicates the presence of other contaminating bacteria. Transfer the pure strain WE12 into 20% glycerol and store at -80°C.

[0043] 1.2 Observation of morphological and cultural characteristics of strain WE12

[0044] After culturing on VY / 4 medium plates for 5 days, strain WE12 formed a dense film with distinct radial lines and ridged edges, making it difficult to pick up. Fruiting bodies were distributed along the edge of the film. Figure 1a). The fruiting body is irregular in shape, relatively hard in texture, initially pinkish-red in color, later turning orange-red, and dark brown after drying. Figure 1 b); the vegetative cells are slender rod-shaped, with slightly pointed ends ( Figure 1 c).

[0045] 1.3 Molecular identification of strain WE12

[0046] Total DNA was extracted from strain WE12 using the TIANamp Bacteria DNA Kit (TIANGEN). The 16S rDNA gene was amplified using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'), and the gyrB gene was amplified using gyrBF (5′-GCGGAAGCGGCCNGSNATGTA-3′) and gyrBR (5′-CCGTCCACGTCGGCRTCNGYCAT-3′). The PCR reaction system consisted of 25 µL: 1.0 µL DNA template, 1.0 µL each of 10 µmol / L primer pairs, 1.5 µL 10 mmol / L dNTPs, 2.5 µL 10×PCR Buffer (2.5 mmol / L MgCl2), 0.5 µL 2.5 U / µL Taq DNA polymerase, and sterile ultrapure water to a final volume of 25 µL. The reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 60 s, for 35 cycles; 72℃ extension for 10 min; and storage at 4℃.

[0047] After the amplification products were detected by 10 g / L agarose gel electrophoresis, the PCR products were cloned and sequenced by Shanghai Sangon Biotech Co., Ltd. The sequencing results of the 16S rDNA and gyrB gene of strain WE12 are shown in SEQ ID No. 1 and SEQ ID No. 2.

[0048] The sequencing results of the 16S rDNA and gyrB gene of strain WE12 were compared in NCBI, and a phylogenetic tree was constructed using the neighbor-joining method in MEGA 5.0 software. Figure 2 (As shown). The results showed that in the phylogenetic tree constructed based on the 16S rDNA sequence, strain WE12 clustered with strains of the genus *Protozoa* on a large branch, but also formed an independent clade. In the phylogenetic tree constructed based on the gyrB gene, strain WE12 and *Archangium violaceum* Cbvi76 DSM 14727... T(KX444678.1) clustered into a small branch. The 16S rDNA gene sequence of strain WE12 is shown in SEQ ID No. 1, and the gyrB gene sequence of strain WE12 is shown in SEQ ID No. 2. Finally, based on morphological characteristics and analysis of the 16S rDNA and gyrB gene sequences, strain WE12 was identified as Archangium violaceum and named Archangium violaceum WE12.

[0049] Strain WE12 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on October 29, 2025, with accession number CGMCC No. 36395, and is classified as Archangium violaceum.

[0050] Example 2: Study on the predatory effect of strain WE12 on the pathogen of wheat stem rot.

[0051] 2.1 Test Methods

[0052] 2.1.1 Preparation of pathogens and myxobacteria

[0053] Mycelial blocks of the pathogens causing wheat stem rot, including *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium oxysporum*, *Fusarium equisetifolium*, *Fusarium truncatum*, *Fusarium moniliforme*, *Fusarium oxysporum*, and *Fusarium argentis*, were inoculated onto fresh PDA plates and incubated at 26°C for activation. When the mycelia had expanded to approximately 1 cm, a 2×2 mm agar block was cut along the edge of the mycelial expansion using a sterilized blade and transferred to another fresh PDA plate. Once the mycelia had expanded to approximately 2 cm, the plate was ready for subsequent experiments.

[0054] After activating strain WE12 on VY / 4 plates for 5 days, an appropriate amount of bacterial film was scraped and inoculated into 3 mL of LBS culture medium. After incubation at 30℃ and 180 rpm for 2 days with shaking, the culture was transferred to 200 mL of LBS culture medium and incubated at 30℃ and 180 rpm for 3 days with shaking to obtain the fermentation broth of strain WE12. The fermentation broth of strain WE12 was centrifuged at 12,000 rpm for 1 min to collect the bacterial cells. The cells were washed three times with sterile water, and any clumps of myxobacterial cells were thoroughly broken up and resuspended in sterile water to prepare OD. 600 =2.0 bacterial suspension.

[0055] 2.1.2 Standoff and Predation Test: Using a sterilized blade, cut 2×2 mm agar blocks along the edge of the activated pathogen hyphae and transfer them to the center of a VY / 4 plate. Incubate at 26℃. When the hyphae have expanded to approximately 0.5 cm, symmetrically inoculate a suspension of strain WE12 along straight lines approximately 1.5 cm from the edge of the hyphae, with a line length of approximately 3 cm and 20 μL inoculated along each line. After air drying, incubate the plates at 28℃. Use solid plates inoculated with the above pathogen alone as a control. Each experiment was repeated in triplicate. After 5 days of standoff, observe and record the collapse of pathogen colonies and measure the colony diameter to calculate the inhibition rate. Inhibition rate (%) = (Correction group pathogen colony diameter - Treatment group pathogen colony diameter) / Control group pathogen colony diameter × 100.

[0056] 2.2 Test Results

[0057] The results of the confrontation experiment showed that the mycelium of strain WE12 could extend into the colonies of eight pathogenic fungi causing wheat stem rot, resulting in the collapse of most of the mycelium and significantly limiting the growth of the pathogenic fungi, while strain WE12 occupied a wider survival area. Figure 3 a). The above results indicate that strain WE12 exhibits good predatory activity against *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium xanthophytum*, *Fusarium equisetifolium*, *Fusarium fusiforme*, *Fusarium moniliforme*, *Fusarium oxysporum*, and *Fusarium argentis*, with inhibition rates of 97.20%, 86.39%, 91.19%, 82.02%, 71.28%, 81.05%, 84.43%, and 75.15%, respectively. Figure 3 (b) It exhibits good broad-spectrum resistance to pathogenic fungi.

[0058] Example 3: Determination of the antibacterial activity of the sterilized fermentation filtrate of strain WE12 against the dominant pathogens of wheat stem rot (Fusarium graminearum, Fusarium graminearum, and Fusarium chrysogenum).

[0059] 3.1 Test Methods

[0060] Strain WE12 was inoculated into LBS liquid medium and cultured at 30℃ and 180 rpm for 3 days to obtain fermentation broth. The fermentation broth was centrifuged at 12,000 rpm for 15 min at 4℃, and the supernatant was collected and filtered through a 0.22 μm microporous membrane to obtain sterilized fermentation filtrate. When VY / 4 medium cooled to approximately 50℃, the sterilized fermentation filtrate was added at a ratio of 40% and mixed thoroughly. Sterile LBS medium was added at a ratio of 40% as a control to VY / 4 medium. *Fusarium graminearum*, *Fusarium graminearum*, and *Fusarium graminearum* mycelial cakes with a diameter of 5 mm were inoculated in the center of the plates and cultured at 26℃ for 5 days. Afterward, the colony diameters of the three pathogenic fungi were photographed and measured to calculate the inhibition rate. Inhibition rate (%) = (Coronary fungal colony diameter of control group - Colony diameter of treated group pathogenic fungi) / Colony diameter of control group × 100.

[0061] Simultaneously, 1 mL of sterilized fermentation filtrate from strain WE12 was mixed with 1 mL of spore suspensions of *Fusarium graminearum*, *Fusarium graminearum*, and *Fusarium xanthophyllus* (1.0 × 10⁻⁶). 6 Mix the spores (spores / mL) thoroughly and co-culture at 26 ℃ and 180 rpm for 24 h. Use an equal volume mixture of sterile LBS culture medium and spore suspensions of *Fusarium graminearum*, *Fusarium graminearum*, and *Fusarium xanthophyte* as a control. Each treatment was repeated in triplicate. After 4 h of co-culture, spore counts were performed using a hemocytometer, and 100 spores were randomly selected to calculate the spore germination rate. Spore lysis rate and spore germination inhibition rate were calculated. Spore lysis rate (%) = (Number of control spores - Number of treated spores) / Number of control spores × 100. Spore germination rate (%) = (Number of germinating spores / 100) × 100; Spore germination inhibition rate (%) = (Spore germination rate of control group - Spore germination rate of treated group) / Spore germination rate of control group × 100.

[0062] 3.2 Test Results

[0063] The results showed that the sterilized fermentation filtrate of strain WE12 had a significant inhibitory effect on the mycelial growth of *Fusarium graminearum*, *Fusarium graminearum*, and *Fusarium xanthophyte*. Figure 4 a), the antibacterial rates were 76.05%, 73.68% and 81.35%, respectively. Figure 4 b). It was also found that the filtrate from the sterilized fermentation of strain WE12 could lyse spores and inhibit spore germination. After co-culturing for 4 h, the spore lysis rates of the WE12 sterilized fermentation filtrate against *Fusarium graminearum*, *Fusarium graminearum*, and *Fusarium oxysporum* were 63.74%, 51.79%, and 61.47%, respectively, and the spore germination inhibition rates were 68.28%, 69.79%, and 63.01%, respectively. Figure 4 c).

[0064] Example 4: Determination of the inhibitory effect of volatile metabolites produced by strain WE12 on the dominant pathogens of wheat stem rot (Fusarium graminearum, Fusarium graminearum, and Fusarium chrysogenum).

[0065] 4.1 Test Methods

[0066] 100 μL of bacterial suspension of strain WE12 (OD) was spread onto a VY4 solid plate. 600 =2.0) were cultured for 3 days, and then activated 5 mm diameter Fusarium pseudograss, Fusarium grass, and Fusarium xanthoides mycelial blocks were inoculated into the center of another PDA plate. The two plates were then sealed together and incubated at 26°C for 5 days. Fusarium pseudograss, Fusarium grass, and Fusarium xanthoides were incubated on sterile VY4 plates as controls. Each treatment was repeated 3 times.

[0067] 4.2 Test Results

[0068] The results showed that the volatile metabolites produced by strain WE12 significantly inhibited the growth of *Fusarium graminearum*, *Fusarium graminearum*, and *Fusarium xanthophyte* hyphae. Compared with the control group, the hyphae of the three pathogenic fungi became significantly sparse and collapsed. Figure 5 ).

[0069] Example 5: Indoor control efficacy of strain WE12 against wheat stem rot

[0070] 5.1 Test Methods

[0071] 5.1.1 Preparation of pathogen inoculum

[0072] Boil millet in boiling water for 3 minutes, quickly rinse with water to cool, and air dry. Dispense 200 g / 500 mL into Erlenmeyer flasks and sterilize. Inoculate each flask with 5 mycelial cakes of *Fusarium graminearum*, *Fusarium graminearum*, or *Fusarium graminearum* (5 mm in diameter). Incubate in a 26°C incubator in the dark, shaking twice daily to ensure even distribution of the pathogens. After 7 days of incubation, prepare *Fusarium graminearum*, *Fusarium graminearum*, and *Fusarium graminearum* inoculums. Simultaneously, mix the prepared *Fusarium graminearum*, *Fusarium graminearum*, and *Fusarium graminearum* inoculums in a 1:1:1 ratio to prepare a composite inoculum.

[0073] 5.1.2 Preparation of fermentation broth and solid inoculum of strain WE12

[0074] Preparation of fermentation broth for strain WE12: A suitable amount of WE12 bacterial film was scraped and inoculated into LBS culture medium. After culturing at 30℃ and 180rpm for 2 days with shaking, the inoculum was transferred to LBS culture medium at a rate of 3% (working volume 200mL / 500mL). After culturing at 30℃ and 180rpm for 3 days with shaking, the fermentation broth of strain WE12 was obtained.

[0075] Preparation of WE12 solid inoculum: White-spotted beetle sand and crushed wheat straw were mixed evenly at a mass ratio of 3:1. After autoclaving, a sterile solid fermentation substrate was obtained. The WE12 fermentation broth was inoculated at a rate of 100 mL / kg, thoroughly mixed, and the moisture content was adjusted to 60% with sterile water. The mixture was incubated at 30℃ for 7 days to obtain the WE12 solid inoculum. After fermentation, a small amount of the WE12 solid inoculum was inoculated onto VY / 4 medium to check for contamination and myxobacterial survival. 1.0 g of the WE12 solid inoculum was inoculated into 9 mL of sterile water and sonicated for 5 minutes (950 W, 30% output power, 3 s sonication intervals of 10 s). The mixture was serially diluted with sterile water, and 100 μL was spread onto VY / 4 plates. After incubation at 30℃ for 5 days, colony counting was performed, and the colony density of myxospores in the WE12 solid inoculum was calculated to be 4.8 × 10⁻⁶. 6 cfu / g.

[0076] 5.1.3 Indoor control efficacy of WE12 fermentation broth against wheat stem rot

[0077] Uncoated wheat seeds (variety: Xindong 18) were soaked in a 2% sodium hypochlorite solution for 3 minutes, rinsed three times with sterile water, then soaked in 75% ethanol for 2 minutes, and rinsed several times with sterile water until the seeds had no pungent odor. Nutrient soil and vermiculite were mixed in a 4:1 ratio and filled into seedling pots (130 mm × 140 mm) to 2 / 3 full. Sow the seeds evenly, cover with a thin layer of soil, then evenly sprinkle 1 g of pathogen inoculum, and cover with another layer of soil about 1 cm thick. After wheat emergence, 10 seedlings were planted per pot. The control experiment began when the wheat reached the one-leaf-one-heart stage.

[0078] This experiment consisted of four groups. Group I determined the control efficacy of the fermentation broth of strain WE12 against wheat stem rot caused by *Fusarium graminearum* infection; Group II determined the control efficacy of the fermentation broth of strain WE12 against wheat stem rot caused by *Fusarium graminearum* infection; Group III determined the control efficacy of the fermentation broth of strain WE12 against wheat stem rot caused by *Fusarium graminearum* infection; and Group IV determined the control efficacy of the fermentation broth of strain WE12 against wheat stem rot caused by a combination of three pathogenic fungi. Each experiment included four treatments: WE12 fermentation broth treatment: 1 mL of WE12 fermentation broth was evenly inoculated along the base of each wheat stem at the one-leaf-one-heart stage using a pipette; 43% tebuconazole treatment: 1 mL of a 500-fold dilution of 43% tebuconazole was evenly inoculated along the base of each wheat stem at the one-leaf-one-heart stage using a pipette; CK: 1 mL of sterile LBS culture solution was evenly inoculated along the base of each wheat stem at the one-leaf-one-heart stage using a pipette; NT: no treatment was given to the wheat control group. Each treatment consisted of three pots, and each treatment was replicated three times.

[0079] 5.1.4 Indoor control efficacy of WE12 strain solid inoculant against wheat stem rot

[0080] This experiment consisted of four groups. Group I determined the efficacy of the WE12 solid inoculant against wheat stem rot caused by *Fusarium graminearum* infection; Group II determined the efficacy of the WE12 solid inoculant against wheat stem rot caused by *Fusarium graminearum* infection; Group III determined the efficacy of the WE12 solid inoculant against wheat stem rot caused by *Fusarium graminearum* infection; and Group IV determined the efficacy of the WE12 solid inoculant against wheat stem rot caused by a combination of three pathogenic fungi. Each experiment included four treatments: WE12 solid inoculant treatment: 1 g of WE12 solid inoculant was evenly sprinkled into the seedling pot at sowing time, covered with a thin layer of soil, and then sown evenly. After covering the seeds with another thin layer of soil, 1 g of pathogen inoculum was evenly sprinkled, followed by a 1 cm layer of soil. Aseptic fermentation substrate treatment: 1 g of aseptic solid fermentation substrate was evenly sprinkled into the seedling pot at sowing time, covered with a thin layer of soil, and then sown evenly. After covering the seeds with another thin layer of soil, 1 g of pathogen inoculum was evenly sprinkled, followed by a 1 cm layer of soil. CK: Wheat was sown, covered with a thin layer of soil, then 1 g of pathogen inoculum was evenly sprinkled, followed by a 1 cm layer of soil. NT: No treatment was given to the wheat group. After emergence, 10 seedlings were thinned per pot, with 3 pots constituting one treatment, and each treatment was replicated 3 times.

[0081] 5.1.5 Disease prevention and control efficacy survey

[0082] The treated wheat was randomly placed in a greenhouse at 25-28℃ and a relative humidity of over 60% for cultivation, and managed under routine conditions. On day 35 after emergence, the disease incidence was assessed. The disease index was determined according to the 7-level grading standard for wheat stem base rot, and the control effect was calculated. Disease incidence (%) = Number of diseased plants / Total number of plants × 100; Disease index = [∑(Number of diseased plants at each level × Level) / (Total number of plants × Highest level)] × 100; Control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100.

[0083] Wheat stem rot disease severity criteria: Grade 0: No symptoms; Grade 1: Embryo disc nodes turn brown; Grade 3: Embryo disc nodes, underground stems, or leaf sheaths all turn brown; Grade 4: Underground stems, leaf sheaths, and lower stems turn brown; Grade 5: Stems show signs of drying and rotting; Grade 6: Plants die due to stem rot; Grade 7: Seedlings wither or seeds rot.

[0084] 5.2 Test Results

[0085] 5.2.1 Control effect of WE12 strain fermentation broth on wheat stem base rot

[0086] The results are shown in Table 1 and Figure 6 As shown, the control efficacy of the fermentation broth of strain WE12 against wheat stem rot caused by *Fusarium graminearum* was 71.28%, against wheat stem rot caused by *Fusarium graminearum* was 82.80%, against wheat stem rot caused by *Fusarium oxysporum* was 70.33%, and against wheat stem rot caused by a combination of the three pathogenic fungi was 80.16%. Moreover, the control efficacy of the fermentation broth of strain WE12 was superior to that of 43% tebuconazole in different experimental groups.

[0087] Table 1. Potted plant control efficacy of WE12 fermentation broth against wheat stem base rot

[0088]

[0089] Note: Data in the table are mean ± standard deviation. Different letters indicate significant differences between groups (P < 0.05).

[0090] 5.2.2 Control effect of WE12 strain solid inoculant on wheat stem base rot

[0091] The results are shown in Table 2 and Figure 7As shown, the WE12 solid inoculant showed a control efficacy of 80.80% against wheat stem rot caused by *Fusarium graminearum* infection, 74.72% against wheat stem rot caused by *Fusarium graminearum* infection, 81.52% against wheat stem rot caused by *Fusarium oxysporum* infection, and 75.56% against wheat stem rot caused by a combination of the three pathogenic fungi. Furthermore, the control efficacy of the WE12 solid inoculant was superior to that of the aseptic fermentation substrate in all experimental groups.

[0092] Table 2. Pot control efficacy of solid inoculant strain WE12 against wheat stem base rot.

[0093]

[0094] Example 6: Effects of strain WE12 on wheat growth

[0095] 6.1 Test Methods

[0096] Fermentation broth, solid inoculant, and sterile solid fermentation substrate for strain WE12 were prepared according to Example 5. The experiment included five treatments: WE12 solid inoculant treatment: 1 g of strain WE12 solid inoculant was evenly sprinkled into the seedling pot before sowing, covered with a thin layer of soil, and then sown; sterile fermentation substrate treatment: 1 g of sterile solid fermentation substrate was evenly sprinkled into the seedling pot before sowing, covered with a thin layer of soil, and then sown; WE12 fermentation broth treatment: 1 mL of strain WE12 fermentation broth was inoculated at the base of each wheat plant using a pipette at the one-leaf-one-heart stage; LBS treatment: 1 mL of sterile LBS culture solution was evenly inoculated at the base of each wheat plant using a pipette at the one-leaf-one-heart stage; NT: untreated wheat. All treatments were not inoculated with the pathogen of wheat stem rot. Ten seedlings were planted per pot, with three pots constituting one treatment, and three replicates were performed. Plant height, taproot length, and whole plant fresh weight were measured 35 days after emergence.

[0097] 6.2 Test Results

[0098] The results are shown in Table 3 and Figure 8 As shown, after applying the solid inoculant strain WE12, the plant height, taproot length, and whole plant fresh weight of wheat were significantly (P<0.05) higher than those of the aseptic fermentation substrate treatment group and the NT treatment group. Furthermore, the plant height and taproot length of the WE12 inoculated fermentation broth treatment group were also significantly higher than those of the LBS culture medium and NT treatment groups. These experimental results indicate that strain WE12 promotes wheat growth.

[0099] Table 3. Effects of strain WE12 on the growth of potted wheat

[0100]

[0101] Note: Data in the table are mean ± standard deviation. Different letters indicate significant differences between groups (P < 0.05).

[0102] Example 7: Field control efficacy of strain WE12 against wheat stem rot.

[0103] 7.1 Test Methods

[0104] 7.1.1 Test Materials

[0105] The wheat variety tested was Hechun 137. The preparation methods of Fusarium oxysporum inoculum, fermentation broth of strain WE12, solid inoculum of strain WE12, and sterile fermentation substrate were as described in Example 5.

[0106] 7.1.2 Overview of the test site

[0107] The field trial was conducted from April to July at the wheat experimental base of the Crop Research Institute of the Xinjiang Academy of Agricultural Sciences in Emin County. The previous crop was maize, and wheat stem rot had not occurred in recent years. The soil in the field was brown calcareous soil with uniform fertility, and drip irrigation facilities were in place. The land was rotary tilled before sowing, no base fertilizer was applied, and white seeds were sown. Land preparation, sowing, management, and harvesting were all carried out uniformly.

[0108] 7.1.3 Experimental Design

[0109] Each experimental plot was 5 m × 1 m, with 6 rows of wheat sown at a row spacing of 15 cm. The experiment consisted of 5 treatments, 3 replicates, and a total of 15 plots, using a randomized block design. The experimental plots for treatments 1-5 were prepared with 70 g / m² wheat during land preparation. 2 Spread the Fusarium wilt inoculum evenly on the soil surface and then lightly till it into the soil.

[0110] Treatment 1 (Strain WE12 solid inoculant treatment): Before sowing, apply strain WE12 solid inoculant at a rate of 14 g / m² evenly in the seed furrow, cover with a thin layer of soil, and then sow.

[0111] Treatment 2 (aseptic fermentation substrate treatment): When sowing, the aseptic solid fermentation substrate is evenly spread in the seed furrow at a rate of 14 g / m, covered with a thin layer of soil, and then sown.

[0112] Treatment 3 (WE12 fermentation broth treatment): After sowing, during the wheat jointing stage, spray the base of the wheat stem with a 10-fold diluted WE12 fermentation broth.

[0113] Treatment 4 (43% tebuconazole treatment): After sowing, during the wheat jointing stage, spray the base of the wheat stem with a 500-fold dilution of 43% tebuconazole.

[0114] Treatment 5 (control): Routine management after sowing, water sprayed at the base of wheat stems during the wheat jointing stage.

[0115] 7.1.4 Survey Period and Methodology

[0116] Disease surveys were conducted during the flowering and grain-filling stages of wheat. Five diagonal sampling points were used in each plot, with 40 wheat plants surveyed at each point. The number of diseased plants was counted to calculate the incidence rate. The disease index was also assessed according to the field wheat stem rot disease grading standard, and the control effect was calculated (calculation method as in Example 5). Simultaneously, during the grain-filling stage, plant height, stem length below the ear, flag leaf area (leaf length × leaf width × 0.83), and area of ​​the second-to-last leaf (leaf length × leaf width × 0.83) were estimated, along with other agronomic traits. The thousand-grain weight was measured at harvest, and actual measurements were taken in each plot (5 m²). 2 The yield was calculated, converted to yield per acre, and the yield increase rate was calculated. Yield increase rate (%) = (treatment yield - control yield) / control yield × 100.

[0117] The grading standards for wheat stem base rot disease refer to the People's Republic of China agricultural industry standard "NY / T 4179-2022 Technical Specification for Monitoring and Forecasting Wheat Stem Base Rot Disease": Grade 0: No browning of leaf sheaths and stems; Grade 1: Obvious browning of lower leaf sheaths, but no browning of stems; Grade 2: Browning of the first internode at the base; Grade 3: Browning of the second internode at the base, but no withered white ears; Grade 4: Browning of the third internode and above at the base, but no withered white ears; Grade 5: Withered white ears or no ears.

[0118] 7.2 Test Results

[0119] Field disease survey results (Table 4) showed that both the WE12 fermentation broth treatment and the solid inoculant treatment significantly reduced the disease incidence and disease index of wheat. Specifically, the WE12 fermentation broth showed control efficacy of 68.53% and 67.54% against wheat stem base rot during the flowering and grain-filling stages, respectively; the WE12 solid inoculant showed control efficacy of 65.07% and 65.64%, higher than the 52% and 44.57% control efficacy of the aseptic fermentation substrate. Furthermore, the control efficacy of both the WE12 fermentation broth and the solid inoculant was significantly higher than that of the chemical agent 43% tebuconazole. The survey also found (Table 5) that the WE12 fermentation broth and the solid inoculant could promote wheat growth and increase yield.

[0120] Table 4. Field control efficacy of strain WE12 against wheat stem rot.

[0121]

[0122] Note: Data in the table are mean ± standard deviation. Different letters indicate significant differences between groups (P < 0.05).

[0123] Table 5. Effects of Protozoa WE12 on wheat growth and yield in the field.

[0124]

[0125] Note: The data in the table are mean ± standard deviation. Different letters indicate that there is a significant difference between groups, P<0.05.

Claims

1. A strain of the purple protozoan Archangium violaceum WE12, characterized in that, It is classified and named Archangium violaceum, with accession number CGMCC No. 36395.

2. The fermentation broth of Archangium violaceum WE12 as described in claim 1.

3. The sterile fermentation broth of Archangium violaceum WE12 as described in claim 1.

4. The volatile metabolites of Archangium violaceum WE12 as described in claim 1.

5. A biological agent comprising Archangium violaceum WE12 as described in claim 1; preferably, the biological agent is a liquid microbial agent or a solid agent prepared using a solid fermentation substrate.

6. A bio-fertilizer comprising Archangium violaceum WE12 as described in claim 1.

7. The use of Archangium violaceum WE12 as described in claim 1, the fermentation broth as described in claim 2, the sterilization fermentation broth as described in claim 3, or the volatile metabolite as described in claim 4 in the preparation of antifungal products, wherein the fungus is selected from the Fusarium genus, preferably any one or more of Fusarium pseudograminearum, Fusarium graminearum, Fusarium culmorum, Fusarium equiseti, Fusarium fujikuroi, Fusarium proliferatum, Fusarium oxysporum, and Fusarium acuminatum.

8. The application of Archangium violaceum WE12 as described in claim 1, the fermentation broth as described in claim 2, the sterilization fermentation broth as described in claim 3, the volatile metabolite as described in claim 4, or the biological agent as described in claim 5 in the biological control of wheat stem base rot.

9. The application according to claim 8, characterized in that, The pathogen causing wheat stem rot is a strain of the genus Fusarium, preferably one or more of the following: Fusarium pseudograminearum, Fusarium graminearum, Fusarium culmorum, Fusarium equiseti, Fusarium fujikuroi, Fusarium proliferatum, Fusarium oxysporum, and Fusarium acuminatum. More preferably, wheat stem rot is caused by one or more of the following: Fusarium pseudograminearum, Fusarium graminearum, and Fusarium culmorum.

10. The application of Archangium violaceum WE12 as described in claim 1, the fermentation broth as described in claim 2, the sterilized fermentation broth as described in claim 3, the volatile metabolites as described in claim 4, or the bio-fertilizer as described in claim 6 in promoting wheat growth and / or increasing wheat yield.

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