Application of pseudomonas syringae psm6 and its phenazine product in preventing and treating tea anthracnose

Abstract

The application discloses a Pseudomonas syringae PSM6 and a phenazine product thereof for preventing and treating tea tree anthracnose, relates to the technical field of biological prevention of plant diseases, and the strain produces phenazine, the phenazine is used for inhibiting conidium germination, appressorium formation and mycelium growth of the tea tree anthracnose pathogen Camellia theae, the half effective concentration value of the phenazine to the Camellia theae is 20.81 μg / mL, the strain is colonized around the stomata of tea tree leaves, the growth, structural integrity, detoxification and virulence function of the pathogen are destroyed through the secretion of the phenazine, and the pathogen is prevented from invading. P. silvicola The PSM6 has good antagonistic activity to the pathogen inside and outside, phenazine is separated and identified as the main antifungal active substance, the EC C. camelliae of the phenazine to the Camellia theae is 20.81 μg / mL, which is stronger than the activity of citronellol (EC 50 = 76.88 μg / mL) and Wuyi mycin (EC 50 = 82.34 μg / mL), and the PSM6 can actively colonize around the stomata of tea trees to continuously secrete the active substance, so that the actual field use amount is greatly reduced. 50 ​

Description

Technical Field

[0001] This invention relates to the field of biological control technology for plant diseases, specifically to the application of Pseudomonas forestii PSM6 and its phenazine products in the control of anthracnose in tea trees. Background Technology

[0002] Tea is an important non-alcoholic beverage economic crop globally, widely cultivated on a large scale in more than 60 countries including my country, India, and Sri Lanka. Anthracnose is a core fungal disease that restricts tea yield and quality, mainly caused by infection with fungi of the genus *Anthracnose*. At least 14 species of anthracnose fungi that can infect tea trees have been reported in my country, among which *Anthracnose cambodiana*, *Anthracnose spp.*, and *Colletotrichum gloeosporioides* are the dominant pathogenic species in various tea-producing areas.

[0003] Anthracnose in tea trees is extremely difficult to control. Key contributing factors include monoculture in tea gardens, a hot and humid climate conducive to pathogen reproduction, the latent infection characteristics of the pathogen, and a complex microbial community structure. Current mainstream field control methods have significant drawbacks: long-term application of fungicides such as carbendazim and pyraclostrobin in chemical control easily leads to drug resistance in pathogens, while also causing pesticide residues, soil and water ecological damage, which does not meet the requirements of green agriculture development; resistance breeding is limited by the long growth cycle of tea trees, the complex molecular mechanisms of disease resistance, and the lengthy breeding cycle, making rapid promotion and application difficult.

[0004] There are significant technological gaps in existing biological control technologies: Current Bacillus-based biocontrol agents only focus on screening for antimicrobial phenotypes, without identifying core antimicrobial active substances and targets, resulting in a vague control mechanism; while antagonistic strains of Pseudomonas exhibit good antimicrobial activity, such as *Pseudomonas pallensii* ZJUCS1001 showing a 71.31% inhibition rate against *Anthracnose chinensis*, the specific active ingredients have not been identified, nor has the pathogen's stress response mechanism been explored; phenazine-1-carboxylic acid and other phenazine-based biofungicides have been registered for use in the control of diseases in other crops, but there are no reports on their application in the control of anthracnose in tea trees or on their toxicity parameters against *Anthracnose chinensis*. Furthermore, while MFS transporters participate in the regulation of multidrug resistance in various pathogenic fungi, their interaction with biocontrol strains and active metabolites in *Anthracnose chinensis* is completely unknown, severely hindering the research and application of precise and green control technologies for tea tree anthracnose. Summary of the Invention

[0005] The purpose of this invention is to provide an application of Pseudomonas forestoides PSM6 and its phenazine products in the prevention and control of anthracnose in tea trees, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a forest pseudomonad PSM6 strain that produces phenazine, wherein the phenazine is used to inhibit the germination of conidia, appressorium formation and hyphal growth of the anthracnose fungus *Anthracis chinensis*.

[0007] Furthermore, the half-maximal effective concentration (IC50) of the phenazine against *Anthrax cambogia* is 20.81 μg / mL.

[0008] Furthermore, the strain colonizes around the stomata of tea leaves and prevents pathogen invasion by secreting phenazine.

[0009] An application of phenazine products from *Pseudomonas forestoides* PSM6 in the prevention and control of anthracnose in tea trees is described. Phenazine produced by the fermentation of *Pseudomonas forestoides* PSM6 is used as the active ingredient to inhibit spore germination, appressorium formation, and mycelial growth of *Anthracnose fungus*, thereby controlling anthracnose in tea trees.

[0010] Furthermore, the phenazine exerts its antibacterial effect by targeting the CcMFS1 protein of Bacillus anthracis; the CcMFS1 protein is a DHA1-type MFS transporter protein, which is located in the cell membrane, septum, and vacuoles of Bacillus anthracis.

[0011] Furthermore, the CcMFS1 protein binds to phenazine via its own hydrophobic pocket and G524 amino acid residue, mediating phenazine efflux and positively regulating the radial growth, appressorium development, stress resistance, and pathogenicity of *Anthracis chinensis*.

[0012] Furthermore, the phenazine can inhibit melanin synthesis in *Anthracis chinensis* and disrupt the integrity of mycelial morphology.

[0013] Furthermore, spraying active agents containing phenazine onto tea leaves can achieve a dual control effect of prevention and treatment of anthracnose in tea trees.

[0014] This invention provides an application of *Pseudomonas forestoides* PSM6 and its phenazine products in the prevention and control of anthracnose in tea trees, which has the following beneficial effects: This invention uses endophytic bacteria P. silvicola Phenidine was isolated and identified as the main antifungal active substance in PSM6, and it is effective against EC50 of C. camelliae. 50 With only 20.81 μg / mL, it has stronger activity compared to citronellol (EC50=76.88 μg / mL) and Wuyi mycin (EC50=82.34 μg / mL). Furthermore, PSM6 can actively colonize around the stomata of tea trees and continuously secrete active substances, significantly reducing the actual amount used in the field. The phenazine of this invention can be produced by fermentation with Pseudomonas aeruginosa, which is much cheaper than chemically synthesized fungicides. Furthermore, the PSM6 fermentation process is simple, has a short cycle, and is suitable for large-scale production. This invention discloses for the first time... C. camelliae The DHA1-type MFS transporter CcMFS1 is a phenazine response target. This protein mediates substrate efflux through a hydrophobic pocket and binding residue G524. Blocking this pathway can restore the pathogen's sensitivity to phenazine. It has strong targeting and does not interfere with host metabolism. In this invention, CcMFS1 not only effluxes phenazines but also positively regulates mycelial growth and pathogenicity. Knocking out or mutating CcMFS1 enhances drug sensitivity while weakening pathogen activity. If pathogens escape inhibition through mutation, they will inevitably pay a fitness cost, thus resulting in slower resistance evolution and better persistence. Attached Figure Description

[0015] Figure 1 Morphological identification, pathogenicity determination and phylogenetic analysis of the pathogen causing anthracnose in tea trees.

[0016] Figure 2 Screening of *Pseudomonas forestosa* PSM6 strains, identification of its colonization ability on tea leaves, and its antagonistic effect against anthracnose.

[0017] Figure 3 The inhibitory effects of different components of PSM6 on spore germination, appressorium development and hyphal growth of *Anthracis chinensis*.

[0018] Figure 4 Results of differential gene analysis of the transcriptome of *Anthracis chinensis* under PSM6 stress.

[0019] Figure 5 The bioinformatics characteristics, subcellular localization, and validation results of the CcMFS1 protein in regulating the growth, resistance, and pathogenicity of Anthracnose in Camellia sinensis were obtained.

[0020] Figure 6 Identification of phenazine, an active ingredient in PSM6 metabolites, and verification of its antibacterial and anti-disease activity against *Anthracis chinensis*.

[0021] Figure 7 Verification of the molecular mechanism by which CcMFS1 protein mediates phenazine efflux and anthrax bacteria stress response and pathogenicity; Figure 8 A schematic diagram illustrating the mechanism by which PSM6 of *Pseudomonas foresta* inhibits *Anthrax chinensis*. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0023] (1) Colletotrichum camelliae It is the pathogen of anthracnose in tea trees (Gougu Nao No. 2). In 2024, the Gougu Nao No. 2 tea tree in Suichuan City, Jiangxi Province ( Ca. sinensis (cv. Gougunao2) Several tea gardens have experienced outbreaks of typical anthracnose. Over 90% of seedlings were infected, and mature leaves showed circular or irregular lesions with grayish-white centers and brown edges. Figure 1Aa). The disease initially appears at the leaf margins, followed by concentric rings of lesions and the formation of small black dots (conidiophores). In severe cases, multiple lesions merge, leading to leaf wilting and abscission. Eleven morphologically consistent isolates (85% isolation frequency) were obtained using tissue isolation. The representative strain JX01 has grayish-white colonies with dense aerial hyphae on the upper surface and gray on the lower surface with a dark green center. Figure 1 Ab). Its conidia are colorless, septate, blunt at both ends, cylindrical, and measure 15.40±3.38×6.69±1.71 μm (n=50); Figure 1 Ac). Conidia germinate to form germ tubes and swollen appressorium (Ac). Figure 1 Ad), the appressorium further darkens, exhibiting an irregular shape with smooth edges, and measures 9.49±1.90×7.83±1.76 μm in size (n=65); Figure 1 Ae). 35 days after inoculation, black, nearly spherical to pear-shaped ascocarps (472.25±225.55×382.19±179.52 μm, n=30) formed, partially embedded in PDA medium. Figure 1 Af). Multiple fusiform, slightly curved, unicellular, colorless and transparent ascospores (13.52±1.88×4.84±0.66 μm, n=65) are clustered within a single ascus. Figure 1 Ag). These colonies and microscopic features are consistent with the genus *Anthracis* (*Bacillus*). Colletotrichum (Consistent)

[0024] Pathogenicity testing showed that 3 days after inoculation, inoculated leaves exhibited the same typical anthracnose symptoms as field plants, including lesions with chlorotic halos. The lesions expanded and merged, causing the leaves to dry out and curl, and begin to fall off after 14 days. Figure 1 B). The morbidity rate in the JX01 inoculation group reached 100%. In contrast, the seedlings treated with water remained healthy. The re-isolated strains were morphologically identical to the inoculated strains. Figure 1 C), indicating that JX01 is the pathogen of anthracnose in tea trees (Gougu Nao No. 2 variety).

[0025] To determine its species-level taxonomic position, phylogenetic analysis was performed using the maximum likelihood (ML) and Bayesian (BI) methods based on the combined fragments of ITS, ACT, GAPDH, and CHS-1. Isolator JX01 was compared with two *Anthracis chinensis* strains. C. camelliae The ) represents a strain forming a tight branch (ML / BI = 99 / 1), which is separate from five other species ( C. kahawae , C. jiangxiense , C. wuxiense , C. psidii and C. tiThey clustered into a larger branch with high support (ML / BI = 95 / 0.99). Figure 1 D). The above results indicate that *Camellia anthracnose* ( Colletotrichum camelliae It is the pathogen that causes anthrax in tea tree species No. 2, known as "Gougu Nao".

[0026] (2) Pseudomonas silvicola PSM6 can inhibit C. camelliae Infection The antagonistic activity of 11 plant endophytic candidate strains (1 *Pseudomonas forestosa* and 10 *Bacillus* strains) against JX01 was evaluated on PDA plates. After 5 days of co-culture, strain PSM6 showed the highest inhibition rate against the pathogen (>80%). Figure 2 A and 2B). Previous studies have confirmed that *Pseudomonas forestosa* ( P. silvicola PSM6 possesses broad-spectrum antifungal activity. Its colonization niche and control effect against anthracnose in tea trees were further investigated. The PSM6 fluorescent strain was sprayed onto leaves. Seven days after inoculation, compared to the weak auto-red fluorescence of chloroplasts in the stomata of the control group, the fluorescence signal around the stomata of the PSM6-mCherry strain-inoculated plants was significantly enhanced, indicating successful colonization. Figure 2 C). In the preventative treatment, PSM6 was pre-sprayed onto the leaves 3 days before inoculation with JX01; in the therapeutic treatment, PSM6 was sprayed 3 days after inoculation with the pathogen. 14 days post-inoculation, no symptoms were observed in the negative control group (treated with water only or PSM6 only). Figure 2 D). In the JX01 inoculation group, almost all leaves (96.67%) were infected; however, the infection rates in the prevention and treatment groups were lower (53.33% and 70%, respectively). Figure 2 D). Compared with the positive control group (77.5%), the disease index of the prevention group and the treatment group decreased to 15.28% and 33.61%, respectively. Figure 2 E). Scanning electron microscopy was used to observe the confrontation between PSM6 and JX01 strains on tea leaves. Unlike the intact hyphae on the leaves of the positive control group, a large number of hyphae around the stomata in the PSM6 treatment group were broken ( Figure 2 F). The above results indicate that *Pseudomonas forestii* PSM6 aggregates around stomata, mitigating the severity of anthracnose in tea trees by preventing pathogen invasion.

[0027] (3) P. silvicola Extracellular metabolites C. camelliae It has antibacterial activity Pseudomonas spp. PseudomonasPSM6 is known for its antifungal secondary metabolites in the control of plant pathogenic fungi. The effects of PSM6 fermentation filtrate, cell culture, and secretory extracts on conidial germination, appressorium formation, and hyphal growth of JX01 were investigated.

[0028] Conidial germination was significantly delayed in all treatment groups (filtrate, bacterial cells, and extract). Six hours after inoculation, the germination rate in the control group was approximately 60%, with some forming appressorium precursors; only relatively long germ tubes were observed in the filtrate group, while neither the bacterial cells nor the extract group showed any germination. Figure 3 A). At 36 h, the germination rate of the PSM6 treatment group (81.18%) was still lower than that of the control group (93.01%). Figure 3 B). In the control group, appressoria darkened over time; in the extract (400 μg / mL) group, appressoria only appeared at 24 h, while no appressoria were observed in other groups at this time. Figure 3 A). Although melanized appressoria eventually formed in each treatment, their number and melanin concentration were significantly lower than those in the control. Figure 3 A, 3B), and all showed swollen appressoria ( Figure 3 A, 3C). In the mycelial growth experiment, JX01 was cultured on PDA plates supplemented with 0.05% filtrate, 0.05% mycelial cells, or 0.02% extract. Growth was severely inhibited on plates supplemented with PSM mycelial cells, and the plates were covered with orange material (…). Figure 3 A), the growth inhibition rate (79.21%) was significantly higher than that of the extract (24.83%) and filtrate (5.81%) groups. Figure 3 D). Scanning electron microscopy showed that PSM6 extract damaged hyphal morphology and mechanical strength; normal hyphae were plump and loose, while the treated groups were shriveled and dense, especially the group with added mycelium. Figure 3 E). This indicates that the extracellular components of PSM6 play an important role in inhibiting conidial germination, mycelial growth, and infection of tea anthracnose fungus.

[0029] (4) P. silvicola and the destruction of its metabolites C. camelliae Growth, metabolism and detoxification processes To investigate the response of *Anthracis chinensis* JX01 to *Pseudomonas forestoides* PSM6 and its metabolites, a comparative transcriptome analysis was performed. Samples were designed based on the accumulation and activity of PSM6 metabolites. Figure 4 A): PSM6-PDA plates were pre-cultured at 28℃ for 5 days, then JX01 mycelia (2.5 days old) were transferred to fresh PDA, PSM6 plates (co-culture, CC), and extract plates (SM), and cultured at 25℃ for 1.5 days. The CC group produced 6338 DEGs (3033 upregulated), and the SM group produced 3062 DEGs (1491 upregulated). Compared with CC, the SM group had 2394 upregulated genes and 2424 downregulated genes. Figure 4B, S2A). Of the 2091 overlapping DEGs shared by CC and SM, 756 and 1094 were simultaneously upregulated and downregulated in the two groups, respectively. Figure 4 B. Table S3). These DEGs involve redox reactions, nutritional metabolism, transcriptional regulation, and biosynthesis (B). Figure 4 C); GO enrichment showed that 88.90% covered small molecule metabolism, organic nitrogen, organic matter, and cellular biosynthesis (Figure S2B). Furthermore, ribosome, amino acid metabolism, DNA replication, peroxisome, and cell cycle pathways were disrupted (Figure S2C). This indicates that PSM6 extracellular metabolites cause global transcriptional disruption of the JX01 gene.

[0030] These DEGs are associated with the aforementioned PSM6-induced growth retardation, melanin deficiency, appressorium defects, and reduced virulence. For example, melanin genes are significantly downregulated, with transcription factor Cmr1 downregulated by 76%–88%, and THNR lower than WT levels by 6%. Figure 4 D). Genes related to cell division, growth, and mitochondrial homeostasis are also disordered. Figure 4 E); Membrane integrity synthesis genes are downregulated, while sphingolipid breakdown, hydrolysis, and glycosyltransferase genes are overexpressed ( Figure 4 E). Numerous detoxification and repair genes were induced, including cytochrome P450, ROS scavengers, and stress response factors (E). Figure 4 F). In summary, PSM6 antagonizes JX01 by impairing its biosynthesis, metabolism, membrane homeostasis, stress resistance, and detoxification capabilities.

[0031] (5) CcMFS1 is a key factor in the radial growth, resistance and pathogenicity of anthracnose fungus in tea trees. In addition to the aforementioned detoxification adaptation factors, 33 MFS members mediating drug transmembrane efflux responded to PSM6 and its metabolites, of which 10 were upregulated ( Cc_11420 The increases were 571.10 and 47.46 times, while the rest remained silent. Figure 5 A). To determine its sensitivity to PSM6 metabolites, 15 samples with significant expression changes were selected. CcMFS The gene was knocked out via homologous recombination (Figure S3). Resistance was tested by inoculating each mutant onto PDA containing 0.025% or 0.05% of the extract. The results showed that Cc_04746, Cc_06088, Cc_01133, and Cc_08422 negatively regulated JX01 resistance to the extract. Cc_11420 The deletion of this gene impaired the radial growth of JX01, and the mutant was more sensitive to the extract than the wild type (Figure S4). This gene was named... CcMFS1 .

[0032] Δ CcMFS1 The mutant colonies are irregular and have a diameter larger than the wild type and EC. CcMFS1Approximately 10 mm ( Figure 5 B, 5C), but polar growth is normal ( Figure 5 B). The inhibition rate of 0.02% extract against the mutant (>41%) was significantly higher than that against the wild type (24.12%) and EC. CcMFS1 (approximately 21%) Figure 5 B, 5D), indicating that CcMFS1 is indispensable for vegetative growth and resistance. SMART and Pfam predictions show that CcMFS1 encodes 611 aa, contains a multidrug-resistant MFS domain, belongs to the DHA1 family, and has 12 transmembrane regions (B, 5D). Figure 5 E). Phylogenetic analysis showed that MFS1 is the most closely related species within the genus *Anthrax*, and its homologous proteins are evolutionarily conserved. Figure 5 F). In subcellular localization, the GFP signal of the CcMFS1-GFP fusion protein is found in the cell membrane, septum, and vacuoles of hyphae, and the signal is consistent in protoplasts. Figure 5 G), indicating that CcMFS1 is located in the membrane structure.

[0033] In the appressorium formation experiment, Δ CcMFS1 The number of appressorium cells is approximately the same as that of wild-type and EC. CcMFS1 Two-thirds of them, some with abnormal shapes ( Figure 5 H, 5I); the addition of extract (200 μg / mL) exacerbated the swelling and irregular morphology of abnormal adherent cells. Figure 5 H, 5J). Pathogenicity assays showed that, CcMFS1 The absence of [certain substances] significantly reduced the diameter of lesions on tea leaves. Figure 5 (K, 5L). In summary, membrane-localized CcMFS1 is crucial for the radial growth, appressorium development, stress resistance, and pathogenicity of *Anthracis chinensis*.

[0034] (6) Phenyzine is the main antifungal active ingredient in the secondary metabolites of Pseudomonas forestosa. Wild type (WT) and Δ CcMFS1 The differential sensitivity of the strains to the extract suggests that PSM6 secretes a potential antifungal active ingredient. To verify this, we used column chromatography, preparative thin-layer chromatography, and LC-MS / MS to separate and identify the extract. A yellow powder (SM-A) was obtained during the petroleum ether / ethyl acetate elution stage. Figure 6 A). Ion characteristics in its LC-MS / MS analysis are similar to those of phenazine (C) in the database. 12 The results were consistent with those of H8N2 (CAS: 92-82-0). Subsequently, the overlapping peak of SM-A and the phenazine standard at 0.89 min in HPLC analysis, and the identical Rf value (~0.51) in TLC analysis, further confirmed this result. Figure 6A). In ESI-MS positive ion mode, a protonated molecular ion peak was observed at m / z = 181.08, with a relative abundance of 100%. Figure 6 B), consistent with the theoretical protonated phenazine mass (181.077). These results indicate that phenazine is one of the secondary metabolites produced by *Pseudomonas forestosa*.

[0035] To evaluate the antifungal activity of phenazine against *Anthracnose cambogia*, colony diameter and appressorium formation rate were measured. On PDA plates supplemented with phenazine, the aerial hyphae of *JX01* became sparse. The activity of phenazine against *JX01* was dose-dependent, EC 100%. 50 =20.81 μg / mL (Regression equation: y=2.954x–3.8943, R²=0.992; Figure 6 C). Similarly, phenazine (20 μg / mL) also inhibited appressorium formation (reduced by 34%) and melanization (10.67% were colorless and transparent). Figure 6 D and 6E). Furthermore, the in vitro efficacy of phenazine (20 μg / mL) was determined by mixing it with a conidial suspension. Compared to the control group, the phenazine-treated group induced smaller lesions on tea leaves (D and 6E). Figure 6 (F and 6G). The above results indicate that PSM6 and its phenazine have good application prospects in the biological control of anthracnose in tea trees.

[0036] (7) Positive regulation of CcMFS1 C. camelliae growth, phenazine resistance and pathogenicity To investigate whether CcMFS1 mediates the response to phenazine in the PSM6 metabolite, its expression profile was analyzed. Treatment with low concentrations of phenazine (5–10 μg / mL) was performed. CcMFS1 Transcription upregulation ( Figure 7 A). At the protein level, CcMFS1 The mutant expresses CcMFS1-GFP (Δ) driven by its own promoter. CcMFS1 _EC CcMFS1-GFP After phenazine induction, GFP fluorescence was enhanced and accumulated on the cell membrane and septum, while the control group showed almost no signal. Figure 7 B), consistent with the transcriptome results, indicates that phenazine is one of the substrates for the CcMFS1 response in the PSM6 extract.

[0037] Given that the CcMFS1 mutant is sensitive to the extract, an overexpression strain (Δ) was constructed in the mutant. CcMFS1 _OE CcMFS1 (Figure S5B). The aerial hyphae of the overexpression strain were more abundant and whiter than those of the wild type. Figure 7 C, S5C). Wild-type inhibition rate was 41.65%, mutant (59.91%) was more sensitive to phenazine, while overexpression strains (approximately 32%) showed the opposite. Figure 7(C, 7D) indicates that CcMFS1 positively regulates mycelial growth and phenazine resistance.

[0038] DHA1-type MFS mediates resistance through substrate efflux. We hypothesize that CcMFS1 is a phenazine efflux pump: its 12 transmembrane regions form hollow hydrophobic channels ( Figure 7 Ea, 7Eb); In molecular docking, the binding free energy of phenazine with CcMFS1 is -7.1 kcal / mol, it is contained in a hydrophobic pocket and forms a 2.7 Å hydrogen bond with G524 (Ea, 7Eb); Figure 7 Ec, 7Ed). To verify, knocking out residues 522-526 (GAGVV) disrupts the pocket, or mutating G524 to glutamate (E), and overexpressing the mutant gene into the mutant (OE). ΔGAGVV OE G524E Although colony diameter and phenazine resistance recovered to wild-type levels ( Figure 7 (D, 7F, 7G), but the positive regulatory function is lost; the mutant protein GFP signal disappears in aerial hyphae ( Figure 7 H), appearing as dots or clumps in the vegetative hyphae (Fig. S5D). This indicates that the localization of CcMFS1 and phenazine efflux depend on intact hydrophobic pockets and G524. Overexpression strains accelerate the expansion of leaf spots on tea leaves ( Figure 7 (I, 7J). In summary, CcMFS1 positively regulates the mycelial growth, phenazine efflux, and pathogenicity of *Anthracis chinensis*.

[0039] It should be noted that, in this document, the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0040] This article uses specific examples to illustrate the principles and implementation methods of the present invention. The above examples are only for the purpose of helping to understand the method and core ideas of the present invention. The above descriptions are only preferred embodiments of the present invention. It should be noted that due to the limitations of textual expression, and the objective existence of infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of the present invention, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of the present invention.

Claims

1. A type of *Pseudomonas forestosa* PSM6, characterized in that, The strain produces phenazine, which inhibits the germination of conidia, appressorium formation, and hyphal growth of *Anthracis chinensis*, the causal agent of anthracnose in tea trees.

2. The *Pseudomonas forestosa* PSM6 according to claim 1, characterized in that, The half-maximal effective concentration (LD50) of the phenazine against *Anthrax cambogia* was 20.81 μg / mL.

3. The *Pseudomonas forestosa* PSM6 according to claim 1, characterized in that, The strain colonizes around the stomata of tea leaves and prevents pathogen invasion by secreting phenazine to disrupt pathogen growth, structural integrity, detoxification, and virulence.

4. The application of a phenazine product of *Pseudomonas forestoides* PSM6 in the control of anthracnose in tea trees, characterized in that... The active ingredient is phenazine produced by fermentation of Pseudomonas forestosa PSM6, which is used to inhibit spore germination, appressorium formation and mycelial growth of Anthracnose in tea trees, and to control anthracnose in tea trees.

5. The application of the phenazine product of *Pseudomonas forestoides* PSM6 for the control of anthracnose in tea trees according to claim 4, characterized in that, The phenazine exerts its antibacterial effect by targeting the CcMFS1 protein of Bacillus anthracis; the CcMFS1 protein is a DHA1-type MFS transporter protein, which is located in the cell membrane, septum and vacuoles of Bacillus anthracis.

6. The application of the phenazine product of *Pseudomonas forestoides* PSM6 for the control of anthracnose in tea trees according to claim 4, characterized in that, The CcMFS1 protein binds to phenazine via its own hydrophobic pocket and G524 amino acid residue, mediating phenazine efflux and positively regulating the radial growth, appressorium development, stress resistance, and pathogenicity of *Anthracis chinensis*.

7. The application of the phenazine product of *Pseudomonas forestoides* PSM6 for the control of anthracnose in tea trees according to claim 4, characterized in that, The phenazine can inhibit melanin synthesis in *Anthracis chinensis* and disrupt the integrity of mycelial morphology.

8. The application of the phenazine product of *Pseudomonas forestoides* PSM6 for the control of anthracnose in tea trees according to claim 4, characterized in that, Spraying active agents containing phenazine onto tea leaves can achieve a dual control effect of prevention and treatment of anthracnose in tea trees.

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