Construction of the spatiotemporal interaction pattern between dominant strain of ustilaginoidea virens heb07 and the pathogen of wheat sharp eyespot

Abstract

The application discloses a Ustilaginoidea virens dominant strain HEB07 and construction of a Ustilaginoidea virens pathogenic bacterium and millet interaction space-time atlas, relates to the technical field of biology, and the strain is screened and verified, the strain has the infection breadth of different millet varieties and the infection pathogenic sensitivity and survival rate of any millet variety, and has high affinity of recombination transformation of a GFP fluorescent label, so as to constitute a gene recombination strain using a green fluorescent protein GFP and explore the Ustilaginoidea virens pathogenic bacterium and millet interaction dominant strain. The application can provide important technical support for cloning research of millet disease-resistant genes, and provide strong scientific basis and technical means for cultivating millet varieties with high resistance to Ustilaginoidea virens.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the construction of a spatiotemporal map of the interaction between the dominant strain of *Pyrrosia lingua* HEB07 and millet, as well as the pathogen of *Pyrrosia lingua* blast disease. Background Technology

[0002] Millet (Setaria italica), belonging to the genus Setaria of the subfamily Sorghum of the family Poaceae, is one of the five grains. After hulling, it is commonly known as millet and is the largest consumer of miscellaneous grains in my country, widely cultivated in the arid and semi-arid regions of northern China. Millet is a C4 photosynthetic crop with a genome size of approximately 430 Mb, exhibiting high genomic synteny with rice, the model crop of the C3 photosynthetic pathway in the Poaceae family. Compared to rice, millet is characterized by high photosynthetic efficiency, drought tolerance, tolerance to poor soil, and strong stress resistance. During its growth, millet is susceptible to various pathogens, which not only affect its normal growth and development but also seriously harm its yield and quality. Among these, millet blast is a major and widespread disease in millet production, exhibiting regional outbreaks in major producing areas of my country. This disease is particularly severe in the summer millet planting zone of the North China Plain (Hebei, Henan, Shandong) and the spring millet cultivation area in the north (Heilongjiang, Shanxi, Inner Mongolia), and has become a significant biological stressor restricting the sustainable development of my country's millet industry. The pathogen of millet blast is *Pyricularia setariae*, a species of *Pyricularia*, which can cause disease throughout the entire growth period of millet. In the middle and late stages of millet growth, the disease incidence rate in affected fields can reach over 70%, leading to large-scale yield reductions or even crop failure. In recent years, the average temperature and precipitation in major millet-producing areas have been higher than normal, and the hot and humid climate has resulted in extremely severe outbreaks of millet blast. Currently, the main control measures for millet blast, besides scientific cultivation management, are chemical control. However, due to the strong variability of *Pyricularia setariae*, long-term use of chemical agents can lead to the development of drug resistance. Furthermore, long-term use of chemical agents can damage the diversity and stability of the soil microbial community, affecting soil health and function. Meanwhile, the use of chemical agents can lead to pesticide residues, posing potential health risks. Therefore, the ideal approach to controlling millet blast is to select and cultivate disease-resistant or tolerant millet varieties and promote their planting. The infection process of *Pyrrosia lingua* mainly includes contact, invasion, and spread stages. Current research on the infection process of *Pyrrosia lingua* in plants focuses primarily on rice, with few reports on its infection process in millet, which greatly restricts the breeding of disease-resistant millet varieties. Traditional methods for detecting pathogen-plant interactions include tissue blotting, GUS staining, and radiolabeled nucleic acid probe methods, all of which are non-in vivo detection methods. However, compared to in vivo observation methods, these non-in vivo detection methods have certain limitations. For example, while tissue blotting can detect the distribution of pathogens in plant tissues, it only provides static information and cannot reflect the dynamic process of pathogen-plant interaction. Although GUS staining can visually display cell expression at the site of pathogen infection, this method may produce false positives and has low sensitivity for some pathogens.Although radiolabeled nucleic acid probes have high specificity and sensitivity, they pose safety risks due to the use of radioactive materials, and the operation process is relatively complex and requires high experimental conditions.

[0003] In the research and control of Magnaphalium blast, pathogenic fungal strains are the core experimental materials for elucidating the pathogenic mechanism, exploring the pathogen-host interaction, screening resistant materials, and developing control technologies. Currently, multiple strains of *Magnaporthe oryzae* have been isolated and identified from different millet-producing areas both domestically and internationally. Simultaneously, some studies, drawing on the research foundation of Magnaphalium blast, are attempting to use closely related *Magnaporthe oryzae* strains (such as the model strain Guy11) to conduct research on Magnaphalium blast. These existing strains have played a certain role in the preliminary identification of Magnaphalium blast pathogenic factors and the preliminary screening of resistant germplasm resources. Furthermore, green fluorescent protein (GFP), as a mature molecular marker tool, has been widely used in the visualization research of plant pathogenic fungi and host interactions. By constructing GFP recombinant strains, it is possible to dynamically track key stages in the pathogen infection process, such as spore germination, appressorium formation, and hyphal expansion. This technology has made breakthrough progress in the study of the interaction mechanisms of major crop diseases such as Magnaphalium blast and wheat scab, providing intuitive technical support for elucidating the disease occurrence patterns. Although existing strains of *Pyrrosia lingua* and molecular marker technology have laid the foundation for research on *Pyrrosia lingua*, significant technical bottlenecks still exist in practical applications, making it difficult to meet the needs of in-depth analysis of the pathogen-millet interaction mechanism and the development of precise control technologies. Specific shortcomings are as follows:

[0004] ① Narrow infection spectrum and insufficient universality of strains: Most existing strains of *Pyrrosia lingua* are indigenous isolates from specific production areas, exhibiting strong host specificity. They can only efficiently infect a few millet varieties with similar genetic backgrounds and cannot be adapted to interaction studies of millet from different ecological regions and with different genetic backgrounds (such as spring millet and summer millet varieties, japonica and glutinous varieties). This limits the representativeness and promotional value of the research results and makes it difficult to support the development of broad-spectrum disease resistance technologies.

[0005] ② Insufficient infection and survival ability, making full-process tracking difficult: Some strains have weak infectivity to the host, low colonization ability and survival rate in millet tissue, and cannot complete the complete infection cycle of "spore germination - appressorium formation - host invasion - hyphal expansion - sporulation and reinfection". Only the local process in the early stage of interaction can be observed, making it difficult to achieve systematic tracking of the entire infection process of pathogens, which restricts the understanding of the whole cycle mechanism of disease occurrence and development.

[0006] ③ GFP recombination transformation affinity is low and labeling reliability is poor: There are significant differences in recombination transformation efficiency between existing strains and GFP fluorescent tags. Some strains have problems such as low transformation efficiency, poor stability of recombinant plasmids, weak fluorescence signal intensity or easy quenching. It is impossible to accurately locate the spatiotemporal distribution of pathogens in millet cells and between cells, resulting in poor visualization and tracking of the interaction process and difficulty in quantifying and analyzing the dynamic interaction relationship between pathogens and the host.

[0007] ④ Lack of a systematic spatiotemporal map of interactions, resulting in unclear control targets: Due to the lack of dominant strains with broad-spectrum infectivity, high survival ability, and high GFP transformation affinity, current research on blast fungus interactions mainly focuses on the interaction characteristics of the early stage of infection (such as the appressorium formation stage) or local tissues. A systematic spatiotemporal map covering the entire process of "pathogen infection - host immune response - disease occurrence and development" has not yet been constructed. This leads to a lack of in-depth understanding of the pathogen infection pathway, host defense mechanisms, and key nodes of the molecular dialogue between the two, resulting in a lack of clear target guidance in the development of control technologies and making it difficult to achieve breakthrough improvements in control effects.

[0008] In summary, screening dominant pathogenic strains of millet blast and using this to explore the interaction between millet blast pathogens and millet, as well as constructing a high-resolution spatiotemporal map of the entire process of the interaction between millet blast pathogens and millet, is of great significance for the prevention and treatment of millet blast. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a dominant strain of *Pyrrosia lingua* HEB07 and the construction of a spatiotemporal map of the interaction between *Pyrrosia lingua* pathogen and millet.

[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows.

[0011] The dominant strain used to construct a spatiotemporal map of the interaction between the blast fungus and millet is *Pyricularia aeruginosa* strain HEB07. After screening and verification, this strain exhibits broad infection range against different millet varieties, high susceptibility and survival rate to any millet variety, and high affinity for GFP fluorescent tag recombination transformation. Therefore, it constitutes a dominant strain for constructing gene recombination strains using green fluorescent protein (GFP) and exploring the interaction between the blast fungus and millet. The strain's accession number is CGMCC No. 42558, deposited at the China General Microbiological Culture Collection Center on January 19, 2026. The strain's classification number is *Pyricularia aeruginosa*. The main biological characteristics of *Grisea* include: belonging to the family Griseaceae (Deuteromycetes) and the genus *Grisea*; mycelium: colorless, transparent, filamentous, and septate; conidiophores: 3-5 clustered together, with 2-8 septa; conidia: colorless, transparent, mature conidia often septate, with a pointed apex and a blunt, rounded base.

[0012] The present invention also includes the use of the *Pyrrosia lingua* strain HEB07, which is used to rapidly and efficiently construct plants and / or tissues carrying millet blast disease in scientific research and production by leveraging the strain's broad infection range and high pathogenicity affinity for different varieties of millet, as well as scientific research and production operations based on millet blast-carrying plants and / or tissues.

[0013] As a preferred technical solution of the present invention, the application particularly includes: constructing a spatiotemporal map of the entire process of interaction between the blast fungus and millet through GFP fluorescent tagging, and constructing a new technical pathway for the prevention and treatment of blast fungus based on this map.

[0014] The present invention also includes the HEB07-GFPox recombinant strain, which is a recombinant strain of HEB07 capable of expressing green fluorescent protein, based on the aforementioned pyridaben strain HEB07, and thus the HEB07-GFPox recombinant strain is obtained.

[0015] The present invention also includes a method for creating the HEB07-GFPox strain, which creates the HEB07-GFPox strain overexpressing the GFP gene by constructing a recombinant strain carrying the pBARGPE1-EGFP vector.

[0016] As a preferred technical solution of the present invention, the specific steps include:

[0017] A. Preparation of conidia of Pyroxburghii;

[0018] B. Preparation of competent cells;

[0019] C. Mix with plasmids containing the target gene;

[0020] D. Add PEG for induction;

[0021] E. Recombinant spores infused with a foreign gene; wherein the foreign gene is the GFP gene;

[0022] F. Recombinant strains were obtained through culture.

[0023] As a preferred technical solution of the present invention, in step A, *Pyrrosia lingua* is obtained by activating and culturing it in CM medium. The *Pyrrosia lingua* strain HEB07, which has multidimensional advantages, was collected from Hebei Province.

[0024] As a preferred embodiment of the present invention, in step E, glufosinate is used as a screening reagent to screen recombinant strains that have successfully integrated the GFP expression plasmid.

[0025] As a preferred embodiment of the present invention, in step F, the HEB07-GFPox strain is colorless and transparent under white light, and its shape is spindle-shaped with septa; bright green fluorescence can be observed under 488nm excitation light.

[0026] The present invention also includes the application of the strain, the recombinant strain, or any of the methods described herein, wherein the application is any one of the following:

[0027] (1) Application in screening or assisting screening of susceptible mutants of millet;

[0028] (2) Application in identifying or assisting in the identification of disease resistance in different millet varieties;

[0029] (3) Application of candidate pesticides in inhibiting key links of blast fungus infection;

[0030] (4) Identification or auxiliary identification of the colonization preference of Blast Angelica glutenosa in specific plant tissues;

[0031] (5) Application in screening or assisting in the screening of millet varieties resistant to millet blast.

[0032] The beneficial effects of the above technical solution are as follows: Overall, this invention utilizes the HEB07 strain of *Pyrrosia lingua*, a dominant blight pathogen collected from Hebei Province and screened and verified to possess multiple advantages, to construct a recombinant strain of *Pyrrosia lingua* tagged with GFP. After inoculating millet leaves, the process of infection of millet leaf cells by the recombinant strain of *Pyrrosia lingua* was observed using a laser confocal microscope. The research of this invention can be used for cloning millet disease resistance genes, studying the molecular mechanism of the interaction between millet and *Pyrrosia lingua*, and breeding disease-resistant varieties. Through the research of this invention, important technical support can be provided for the cloning of millet disease resistance genes, and a new perspective and theoretical basis can be provided for further understanding the interaction between plants and pathogens. Furthermore, the research results of this invention can also be applied to the breeding of disease-resistant varieties, providing a scientific basis and powerful technical means for cultivating millet varieties highly resistant to millet blast.

[0033] Compared with common wild-type strains, the superior strain HEB07 of this invention has multiple advantages, including a wider infection range against different millet varieties, higher susceptibility and survival rate to any millet variety, and high affinity for GFP fluorescent tag recombination transformation. This makes it a superior strain for constructing gene recombination strains using green fluorescent protein (GFP) and exploring the interaction between the blast fungus and millet. This study obtained the superior strain HEB07 of *Pyrrosia lingua* through screening and constructed a spatiotemporal map of the interaction between the blast fungus and millet based on this strain. Its technical solution specifically addresses the core defects of existing technologies and has significant academic value and application prospects. We have summarized its relative technical advantages as follows:

[0034] ① After systematic screening and verification, strain HEB07 possesses broad infection capabilities against millet varieties with different genetic backgrounds and ecological adaptability, breaking the limitation of strong host specificity in existing strains. This characteristic allows the research results to cover most mainstream millet cultivars, significantly improving the representativeness and universality of research on the interaction mechanism between millet blast and wheat. It provides standardized and unified core experimental materials for screening broad-spectrum disease-resistant germplasm resources and developing cross-ecological zone millet blast control technologies. ② Ensuring survival throughout the infection process and supporting complete interaction analysis: Strain HEB07 exhibits high infection sensitivity and stable in vivo survival rate against any millet variety, efficiently completing the entire infection cycle from spore germination, appressorium differentiation, host invasion, to mycelial colonization and expansion within tissues, sporulation, and re-infection. This advantage ensures that the research can fully capture the dynamics of the entire chain of pathogen-host interaction, providing a key guarantee for analyzing the pathogenic strategies of pathogens at different infection stages and the laws of host immune response, filling the technical gap that existing research can only focus on local stages. ③ Optimize GFP recombination transformation efficiency for precise visual tracking: The HEB07 strain exhibits high recombination transformation affinity with the GFP fluorescent tag, enabling efficient construction of fluorescently labeled recombinant strains. Furthermore, the recombinant strains demonstrate high fluorescence signal intensity and good stability. This characteristic facilitates precise localization of the pathogen within different tissues and cell structures of millet, as well as real-time dynamic tracking of pathogen morphological changes and expansion pathways during infection, providing a reliable technical means for quantifying and analyzing the spatiotemporal characteristics of the interaction. ④ Based on the superior characteristics of the HEB07 strain, this study, for the first time, constructed a spatiotemporal map of the interaction between the blast fungus and millet, covering the entire process from initial pathogen infection to host immune activation and the manifestation of disease symptoms. This map clearly defines the key time nodes of pathogen infection (peak spore germination period, critical invasion period, and accelerated hyphal expansion period), spatial distribution characteristics (preferred infection sites, inter-tissue expansion pathways), and the spatiotemporal dynamics of the host defense response.

[0035] Furthermore, it is conceivable that the HEB07 strain can rapidly and efficiently construct standardized millet blast-carrying plants and tissues, providing an efficient experimental system for scientific research, including cloning resistance genes, verifying the function of pathogenic factors, and analyzing interactive molecular networks. Simultaneously, in the production field, this strain can be used for the rapid identification and screening of resistant varieties, the evaluation and optimization of control agents' activity, and the precise determination of control timing, significantly improving the efficiency and accuracy of millet blast research and production applications. Moreover, this study provides clear theoretical basis and technical guidance for developing fungicides targeting pathogenic factors, conducting immune pathway-based disease-resistant genetic engineering breeding, and formulating precise timing control technologies, which has significant practical implications for ensuring high and stable millet yields and promoting the high-quality development of the millet industry.

[0036] Furthermore, the introduction of the GFP gene offers at least the following advantages: GFP emits bright green fluorescence when excited by a laser of a specific wavelength; against a dark background, only the pathogen glows, resulting in a very high signal-to-noise ratio, allowing for clear differentiation from host tissues, even single hyphae or tiny appressoriums can be clearly observed and recorded; the entire infection process can be observed in real-time on living plants using equipment such as confocal laser scanning microscopes, including: spore attachment and germ tube formation, appressorium formation and maturation, how infection spikes penetrate plant cell walls, the expansion and colonization of infection hyphae within plant cells, the dynamics of hyphae shuttling between cells, etc.; additionally, image analysis software can be used to precisely quantify the intensity and area of ​​the fluorescence signal, enabling us to quantify the spread rate of the pathogen in plant varieties with different resistance levels and analyze the pathogen's colonization preferences in specific plant tissues such as leaf veins and guard cells. Attached Figure Description

[0037] Figure 1 A schematic diagram for preparing conidia of the pyridostigma strain HEB07. In the diagram: a. Induction of conidia production from pyridostigma strain HEB07 on oat medium; b. Observation of conidia production from pyridostigma strain HEB07 under an optical microscope.

[0038] Figure 2 This is a schematic diagram of the fluorescence of the HEB07-GFPox recombinant strain. In the diagram: a. Expression of GFP in the conidia of the HEB07-GFPox recombinant strain; b. Expression of GFP in the vegetative hyphae of the HEB07-GFPox recombinant strain.

[0039] Figure 3 This diagram illustrates the infection process of HEB07-GFPox conidia on the Ci846 millet variety. In the diagram: a. Ci846 leaves inoculated with HEB07-GFPox conidia, with samples taken at different times and the infection process observed using a laser confocal microscope; b. Phenotype of Ci846 leaves 72 h after inoculation with HEB07-GFPox conidia.

[0040] Figure 4 This is a schematic diagram illustrating the expression analysis of disease resistance-related PRR, NLR, and SA response genes.

[0041] Figure 5 This is a schematic diagram illustrating the infection process of HEB07-GFPox conidia on wild-type Yugu1 and EMS mutants bsm-1 and bsm-2.

[0042] Figure 6 This is a schematic diagram of the isolation and culture experiment of Bacillus thuringiensis.

[0043] Figure 7 This is a schematic diagram of the purification experiment for Bacillus thuringiensis.

[0044] Figure 8 This is a schematic diagram of the experiment on the culture and preservation of conidia of Pythium oryzae.

[0045] Figure 9 This is a schematic diagram of the experiment for strain activation and spore suspension preparation.

[0046] Figure 10 This is a schematic diagram of the inoculation test of *Pseudomonas glutenosa* on detached leaves.

[0047] Figure 11 This is a schematic diagram of the strain screening results. Detailed Implementation

[0048] The following embodiments illustrate the present invention in detail. All raw materials and equipment used in the present invention are commercially available products and can be directly obtained through market purchase.

[0049] It should be understood that, when used in this application specification and the appended claims, the term "comprising" indicates the presence of the described features, integrals, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or a collection thereof.

[0050] It should also be understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0051] As used in this application specification and the appended claims, the term "if" may be interpreted, depending on the context, as "when," "once," "in response to determination," or "in response to detection." Similarly, the phrase "if determined" or "if detected [the described condition or event]" may be interpreted, depending on the context, as meaning "once determined," "in response to determination," "once detected [the described condition or event]," or "in response to detection [the described condition or event]."

[0052] Furthermore, in the description of this application and the appended claims, the terms "first," "second," "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0053] References to "one embodiment" or "some embodiments" as described in this specification mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized.

[0054] Example 1

[0055] The pathogenic blight fungus *Pyrrosia lingua* strain HEB07 (Genetic Resource Source Disclosure Registration Form), *Escherichia coli* DH5α strain (Genetic Resource Source Disclosure Registration Form), millet variety Ci846 (Genetic Resource Source Disclosure Registration Form), and vector pBARGPE1-EGFP (Genetic Resource Source Disclosure Registration Form) were all preserved in our laboratory. KOD-Plus high-fidelity enzyme was ordered from TOYOBO, and Super yeast (Genetic Resource Source Disclosure Registration Form) competent cells and transformation kit (Plus) was ordered from Coolaber. LB liquid medium, CM medium, and oat medium were prepared in our laboratory.

[0056] Example 2

[0057] The HEB07 strain of *Pyrrosia lingua* was activated and cultured on CM medium. After 3 days, it was transferred to oat medium and cultured for another 3 days at 28°C. Aerial mycelia were scraped off from the surface of the oat medium, and sporulation was carried out under continuous light. After 3 days of culture, spores were distributed throughout the oat medium. The phenotypic characteristics of the spores were observed and recorded under an optical microscope. The spores on the medium were scraped off, rinsed with water, filtered into Erlenmeyer flasks, and used for subsequent experiments.

[0058] The strain identified as *B. glutenosa* strain HEB07, after screening and verification, demonstrated a wide range of infection against different millet varieties, high susceptibility and survival rate to any millet variety, and high affinity for GFP fluorescent tag recombination transformation. This makes it a superior strain for constructing recombinant strains using green fluorescent protein (GFP) and exploring the interaction between *B. glutenosa* pathogens and millet. To prepare the *B. glutenosa* recombinant strain expressing GFP protein, it is first necessary to culture the HEB07 strain to produce conidia.

[0059] After activating the HEB07 strain, it was transferred to oat medium. After 3-4 days of cultivation, the mycelium would completely cover the medium. At this point, all the mycelium on the surface of the medium was scraped off, and cultivation continued for another 3-4 days until the surface of the medium turned grayish-black. Figure 1 a). Scrape off the grayish-black surface of the culture medium to prepare a suspension. Filter the suspension through gauze to obtain a spore suspension. Take a small amount and drop it onto a millet leaf. Observe it under an optical microscope. You can see that the spores are translucent, pear-shaped, and generally have two septa ( Figure 1 b).

[0060] Example 3

[0061] Extensive screening and experiments in the early stages have shown that the *Bacillus oryzae* strain HEB07 is capable of infecting most millet varieties, exhibiting strong susceptibility to infection and high survival rates, and also demonstrating good affinity for genetic modification. This study used the HEB07 strain as experimental material to construct a recombinant strain carrying the pBARGPE1-EGFP vector.

[0062] The successfully constructed GFP-tagged recombinant *Pyrostemum griseus* spores and hyphae were observed under 488 nm excitation light using a laser confocal microscope. First, the *Pyrostemum griseus* spores and hyphae were observed and photographed under white light to obtain basic information on their morphology and structure. Then, the field of view was switched to 488 nm excitation light; at this point, the GFP-tagged *Pyrostemum griseus* spores and hyphae emitted green fluorescence, and the fluorescence images were recorded. Finally, the images from the white light field and the fluorescence images from the excitation light field were merged to obtain an image simultaneously showing the morphology and structure of *Pyrostemum griseus* spores and hyphae, as well as the distribution of GFP fluorescence.

[0063] The conidia and hyphae obtained from the transformation were observed using a laser confocal microscope. The results showed that under 488nm excitation light, the conidia of *Pseudomonas aeruginosa* (…) Figure 2 a) and the mycelium of *Pseudomonas aeruginosa* ( Figure 2 b) All showed strong green fluorescence, indicating that the GFP expression element had been successfully integrated into the genome of the HEB07 strain and achieved stable expression.

[0064] The conidia of *Pseudomonas aeruginosa* appear colorless and transparent under white light, and are spindle-shaped with septa. Mature spores have a pointed apex and a rounded base with small protrusions, and most have two septa. Successfully constructed GFP recombinant strains exhibit bright green fluorescence under 488nm excitation light. Figure 2During fungal transformation, due to differences in genes and different insertion sites of expression vectors, the loss of exogenous genes in transformants often occurs, leading to the loss of GFP fluorescence. After multiple generations of screening, a recombinant strain HEB07-GFPox that produces strong green fluorescence and has good genetic stability was obtained, which can be used for subsequent infection experiments and observation studies.

[0065] Example 4

[0066] Agrobacterium-mediated genetic transformation, using reagents including:

[0067] (1) MM medium: glucose 4 g, K2HPO4 4.1 g, KH2PO4 2.9 g, MgSO4·7H2O 1.2 g

[0068] Add g of CaCl2, 0.02 g of (NH4)2SO4, and 1 g of distilled water, stir well, and bring the volume to 2 L. Heat at 121 °C.

[0069] Sterilize by high-temperature autoclaving for 20 minutes. When using, add 1 mL of filtered and sterilized Z-salts to every 200 mL of MM culture medium.

[0070] (2) IM medium: MM medium, 8.7 g of 98% MES, 5 g of glycerol, adjust pH to 5.4 with HCl, bring volume to 1 L, add 15 g of agar to solid medium, autoclave at 113℃ for 20 min. When using, add acetylsuccine to a final concentration of 200 μM.

[0071] (3) Oat culture medium: 150 mL of fresh tomato juice was obtained by filtration and juicing. 30 g of oat flakes were boiled for 5 min. After cooling, 150 mL of tomato juice and 30 g of agar were added. Distilled water was added to make up to 1 L. The mixture was then sterilized by high temperature and high pressure steam at 121 °C for 20 min.

[0072] (4) Z-Salts: ZnSO4·7H2O 0.1 g, MnSO4·H2O 0.1 g, CuSO4·5H2O 0.1 g, H3BO3 0.1 g, NaMoO4·H2O 0.1 g, add distilled water and stir evenly, then make up to 100 mL, filter to sterilize, dispense and store at -20℃ for later use.

[0073] (5) CM medium (1 L): 3 g of acid-hydrolyzed casein, 3 g of enzyme-hydrolyzed casein, 6 g of yeast extract, 10 g of sucrose, add distilled water and stir well to make up to 1 L. Add 16 g of agar to the solid medium and sterilize at 121 ℃ high temperature and high pressure for 20 min.

[0074] The conversion steps include:

[0075] (1) The constructed vector with GFP fluorescent tag was transformed into Agrobacterium EHA105 strain (Genetic Resource Source Disclosure Registration Form). After two days of shaking culture in liquid medium at 28 ℃, an appropriate amount of bacterial culture was transferred to 20 mL MM medium (containing 50 μg / mL kanamycin, 100 μg / mL rifampin, and 100 μL Z-salts) and continued to be shaken at 28 ℃ for two more days.

[0076] (2) Collect the above Agrobacterium cells, centrifuge at 5000 rpm for 2 min, discard the supernatant, resuspend the cells in IM medium, adjust the bacterial concentration to OD600 = 0.16, and culture at 28 ℃ and 150 rpm with shaking until OD600 = 0.4-0.6.

[0077] (3) Simultaneously prepare conidia of Bacillus thuringiensis. Take the strain that has grown on oat culture medium for about 10 days, scrape off the aerial mycelium, and culture it under light at 28 ℃ for about 3 days.

[0078] (4) Add 5 mL of ultrapure water to the above oat culture medium, gently scrape the spores with a sterile cotton swab, filter out the hyphae through a sterile filter cloth to obtain a gray-black spore suspension, and adjust the concentration to 1×10⁻⁶. 5 pcs / mL

[0079] (5) Mix the Agrobacterium tumefaciens in step (2) and the conidia suspension in step (4) in a 1:1 ratio. Take 100 μL of the mixture and spread it evenly on cellophane containing 40 μM acetylsuccinone in IM medium. Invert the cellophane at 28 ℃ and incubate for 2 days.

[0080] (6) Transfer the cellophane to CM medium (containing 200 μg / mL hygromycin or 400 μg / mL LG418, 50 μg / mL streptomycin, 200 μg / mL cephalosporin and 20 μg / mL 5-FU (not added in the overexpression system)) and incubate upside down at 28 ℃ in the dark for 2 days.

[0081] (7) Remove the cellophane after two days and continue to incubate upside down until transformants grow.

[0082] (8) Select transformants and transfer them to CM resistant plates, and purify them by 3-5 re-screenings.

[0083] (9) Extract the genome of the purified transformants for gene identification, select positive transformants for transfer to oat culture medium, and preserve the strain using the filter paper method.

[0084] Example 5

[0085] The observation and construction of the full-time spatiotemporal map of cell infection in millet leaves by the HEB07-GFPox recombinant strain are detailed below.

[0086] ① The leaves of the susceptible millet variety Ci846 were infected with the HEB07-GFPox recombinant strain, and the cell infection process in the millet leaves was observed. The concentration of the spore suspension was adjusted to 3×10⁻⁶. 4 The concentration of HEB07-GFPox conidia was sprayed onto the millet model variety Ci846 using a spray bottle for inoculation. The infection process of millet cells by HEB07-GFPox conidia was observed using a laser confocal microscope. It was found that 3 hours after infection, under 488nm excitation light, the germ tubes of HEB07-GFPox conidia began to germinate and grow from one end of the conidia; by 6 hours, the germ tubes continued to elongate; when infection progressed to 12 hours, typical appressorium structures gradually formed at the ends of the germ tubes. The formation of these appressoriums is an important indicator of successful pathogen infection, helping the pathogen to better adhere to the plant cell surface and laying the foundation for subsequent infection. At 24 hours, hyphae began to grow and spread rapidly in millet leaf cells, indicating that the pathogen had established a certain foundation for survival and reproduction within the plant cells. Finally, at 48 hours, the hyphae continued to spread and infect adjacent cells, showing that the pathogen infection had a certain degree of diffusion and continuity. Figure 3 a). When infected for 72 hours, millet leaves develop a typical phenotype of millet blast lesions, characterized by a grayish-white center, a brown inner ring, and a yellow halo on the outermost edge. Figure 3 b).

[0087] ② Transcriptional analysis of disease resistance-related genes. Precise tracing of the infection process based on the HEB07-GFPox marker strain ( Figure 3Then, by sampling at different time points (0, 3, 6, 12, 24, 48 hours), we systematically analyzed the dynamic expression patterns of key immune marker genes in millet during the response to *Pseudomonas aeruginosa* infection. Plant pattern recognition receptors (PRRs), as the first line of defense in sensing pathogen-associated molecular patterns (PAMPs), limit pathogen colonization in the early stages of infection (12-24 hours) by triggering PAMP-triggered immunity (PTI). Meanwhile, intracellular nucleotide-binding leucine-rich repeat (NLR) proteins activate effector-triggered immunity (ETI) by recognizing pathogen effectors, inducing hypersensitivity responses and other potent defenses. Salicylic acid (SA), as a core defense hormone, regulates systemic resistance. Precise tracing of the infection process was achieved using the HEB07-GFPox labeling system. Figure 4 We selected PRR genes (SiSERK1, SiSIT3, SiASLRK), NLR genes (SiPiSH, SiRPM1, SiZAR1), and SA pathway marker genes (SiPR1, SiPR2, SiNPR3) for qRT-PCR detection. Figure 4 As shown, the PRR gene was rapidly upregulated within 12-24 hours after infection, followed by significant activation of the NLR gene at 48 hours, indicating a cascade signaling from PTI to ETI; SA-related genes remained highly expressed within 12-48 hours. These results confirm that millet resists blast fungus infection through the PRR-NLR-SA cascade pathway, and further demonstrate that sampling from the HEB07-GFPox strain can accurately identify the interaction process between millet and blast fungus.

[0088] ③ Screening for susceptible mutants of millet using the HEB07-GFPox recombinant strain. The millet variety Yugu1 (Genetic Resource Source Disclosure Registration Form) exhibits good resistance to millet blast. Previously, the laboratory treated Yugu1 seeds with the chemical mutagen ethyl methane sulfonate (EMS), obtaining over a thousand mutant offspring. These mutants were then inoculated with the HEB07-GFPox recombinant strain, and the infection process of millet blast was observed. Two susceptible mutants, bsm-1 (blast-sensitive mutant-1) and bsm-2 (blast-sensitive mutant-2), were screened. Figure 5 As shown, 6 hours after infection, HEB07-GFPox spore germ tubes began to germinate in bsm-1 and bsm-2 leaves; 24 hours later, the germ tubes further elongated and formed appressoriums; and 48 hours later, hyphae grew within leaf cells of bsm-1 and bsm-2 and began to infect adjacent cells. Conversely, HB07 spores inoculated into Yugu1 leaves mostly died after 24 hours. This indicates that the bsm-1 and bsm-2 mutants are sensitive to HEB07-GFPox. Figure 5 ).

[0089] Example 6 (Supplementary content: Isolation, purification, culture, preservation and infection process of Blast Rice strain)

[0090] I. Collection of leaves infected with blast disease

[0091] Millet leaves infected with *Pseudomonas aeruginosa* were collected from millet-growing areas in multiple provinces including Heilongjiang, Shandong, Shanxi, Henan, Beijing, Liaoning, Hebei, Jilin, and Shaanxi, as well as Inner Mongolia Autonomous Region. The sample collection process is as follows:

[0092] 1. Sampling time and location selection. Collect samples in the early morning before the dew dries (when leaf moisture content is high and pathogen spores are active); select leaves at the boundary between diseased and healthy tissue (the edge of a typical spindle-shaped lesion), retaining a small amount of healthy tissue (to avoid contamination by saprophytic fungi).

[0093] 2. Sample pretreatment. Place the leaves into sterile centrifuge tubes (50ml) or breathable paper bags (avoid condensation); each tube / bag contains 1-2 diseased leaves, labeled with sampling information (location, variety, date).

[0094] 3. Storage and Transportation. Place one sterile filter paper and 2-3 silica gel desiccants inside the container (to control humidity); transport to the laboratory at 4°C in an ice pack.

[0095] II. Isolation of Blast Infectious Disease

[0096] 1. Lesion treatment: In a clean bench, cut infected leaf tissue along the edge of the blast fungus lesion;

[0097] 2. Surface cleaning: Rinse the leaf tissue surface with sterile water to remove dirt;

[0098] 3. Surface disinfection: Immerse the tissue in 75% alcohol for 30 seconds, then rinse once with sterile water;

[0099] 4. Drying and inoculation: Blot dry the tissue surface with sterile filter paper and place it on the prepared CM culture medium;

[0100] 5. Incubation: Incubate in a constant temperature incubator at 25-28℃ for approximately 7 days. See appendix. Figure 6 .

[0101] III. Purification of Pythium oryzae

[0102] 1. Initial screening: After 7 days of incubation, observe the plates. The target blast fungus colonies are initially white or grayish-white, with mycelium that is cottony or fluffy and grows densely.

[0103] 2. Purification Culture: Transfer the target colony region to a new CM medium plate for secondary purification; if non-target strains still grow after the second purification, perform a third purification; if all colonies are single colonies formed by the target strain, proceed with subsequent preservation. See Appendix. Figure 7 .

[0104] IV. Culture and Preservation of Pythium oryzae Conidia

[0105] 1. Sporulation induction: Take the strain that has undergone secondary purification and culture for one week, and use a flame-sterilized blade to cut off pieces of mycelium-covered mycelium, then transfer them to new oat medium plates. Incubate at 25-28℃ for about one week, until the mycelium completely covers the medium;

[0106] 2. Promote sporulation: Gently scrape off some of the mycelium from the surface of the culture medium, keep the medium moist, and place it under light conditions to induce sporulation;

[0107] 3. Spore collection and preparation: After culturing for about 3 days under suitable temperature and humidity, conidia will be produced in large quantities and cover the surface of the culture medium;

[0108] 4. Dry storage: Remove the humidifier and continue to place in a dry environment for about 3 days until the substrate is completely dry. This dried substrate can be stored for a long time at a low temperature (e.g., 4°C).

[0109] 5. Activation and Use: For subsequent activation and storage of the strain, use PDA medium. See Appendix. Figure 8 .

[0110] V. Strain activation and spore suspension preparation

[0111] 1. Spore collection: Use a sterile scraper to scrape the spores off the dried or fresh sporulation plate. Rinse the spores with an appropriate amount of sterile water and filter them through a sterile filter cloth into an Erlenmeyer flask (adjust the number of filtrations according to the number of plates scraped; usually, 2 filtrations are required for every ≥4 plates scraped).

[0112] 2. Centrifugation concentration: Aliquot the spore filtrate into 50ml centrifuge tubes and centrifuge (recommended conditions: 4℃ or room temperature, 800rpm, 1 minute; if no strict requirements are specified, centrifugation should be performed until spores precipitate).

[0113] 3. Collection of bacterial culture: Discard the supernatant, resuspend the spore precipitate at the bottom of each centrifuge tube with a small amount of sterile water, and combine them into 1-2 centrifuge tubes (usually the amount of spores from 4 plates can be collected into 1 tube).

[0114] 4. Spore counting and concentration adjustment: Take 10 μl of the combined spore suspension and add it to a hemocytometer; observe and count under a microscope, with a target concentration of 0.9-1.0 × 10⁻⁶. 6 / ml; add 2% gelatin solution, mix well and bring the volume to 50ml to obtain the desired spore suspension;

[0115] 5. Storage: Store the prepared spore suspension at 4°C for later use in subsequent inoculation. See Appendix. Figure 9 .

[0116] VI. Inoculation of detached leaves with Blast Angelica glutenosa

[0117] 1. Prepare a 10 μg·mL⁻¹ 6-benzylaminopurine aqueous solution at pH 7.0. Dispense approximately 30 mL into sterile 9 cm diameter petri dishes.

[0118] 2. Select healthy millet seedlings that have grown to the six-leaf stage, and cut 6 cm long leaf segments from the same part (such as the second to last leaf);

[0119] 3. Using the tip of a 10 μL pipette, gently puncture the leaf epidermis every 1.5 cm to create tiny wounds, making 3 wounds per leaf segment. Place the punctured leaf segments into a culture dish containing 6-benzylaminopurine solution, allowing them to float on the surface, with 6 leaf segments per dish;

[0120] 4. Inoculate 10 μL of the spore suspension prepared in step five at each epidermal wound; incubate the inoculated petri dishes in the dark for 24 hours; then transfer them to a photoperiod of 16 hours light / 8 hours dark; typically, spindle-shaped lesions can be observed about 4 days after inoculation. See Appendix Figure 10 .

[0121] VII. Results of Strain Screening

[0122] Through the above inoculation process, strain HEB07 was obtained. This strain has the following significant advantages: broad-spectrum activity and high pathogenicity, rapid sporulation rate, good spore activity, convenient laboratory culture, strong resistance to contamination, not easily infected by contaminating microorganisms during culture and multiple subcultures, good environmental adaptability, stable and typical infection phenotype after laboratory inoculation (presenting clear spindle-shaped lesions); compared with common wild-type strains, the superior strain HEB07 has multiple advantages, including a wider infection range against different millet varieties, higher susceptibility and survival rate to any millet variety, and high affinity for GFP fluorescent tag recombination transformation. Therefore, it constitutes a superior strain for constructing gene recombination strains using green fluorescent protein (GFP) and exploring the interaction between millet blast pathogens and millet. See Appendix. Figure 11 Based on the above advantages, strain HEB07 was selected as the starting strain for subsequent green fluorescent protein (GFP) labeling modification to observe the dynamic process of *Pythium oryzae* infection in millet.

[0123] VIII. Culture medium formulation:

[0124] CM culture medium (1L)

[0125] Element volume Yeast paste 6g peptone 6g sucrose 10g Agar Power 15g <![CDATA[ddH2O]]> to 1L

[0126] Oat culture medium (1L)

[0127] Element volume oat 40 g Agar Power 20 g <![CDATA[ddH2O]]> to 1L

[0128] PDA medium (1L)

[0129] Element volume Potato 200g glucose 20g Agar Power 15g <![CDATA[ddH2O]]> to 1L

[0130] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0131] As demonstrated in the examples above, millet blast is caused by infection with *Pyricularia setariae*, a species of fungus in the genus *Pyricularia*, and is an explosive, epidemic fungal disease of millet. In recent years, with climate change and the decreasing disease resistance of popular millet varieties, millet blast has become a major disease affecting millet production, seriously threatening yield and quality. Clarifying the interaction process between *Pyricularia setariae* and millet is of great significance for the control of millet blast. Using the blast pathogenic strain HEB07, a recombinant strain expressing green fluorescent protein (GFP) was constructed. Using laser confocal microscopy under 488 nm laser excitation, we dynamically observed the infection process of the HEB07 recombinant strain. Experimental results showed that the infection process of this strain exhibited a distinct temporal pattern: in the initial stage (0-12 h), conidia germinated on the host surface and formed appressoriums with infectious function; in the middle stage (12-24 h), appressoriums differentiated to produce infection nails, which then expanded within the plant tissue through polar growth at the hyphal tips; in the later stage (after 24 h), the hyphal network gradually established, ultimately leading to the formation of typical lesions. These findings provide fundamental data for cloning millet disease resistance genes, exploring molecular mechanisms, and breeding disease-resistant varieties.

[0132] The technology of this invention allows for clear observation of the infection process and distribution of pathogens within millet grains, providing a direct basis for investigating the colonization patterns of *Pseudomonas aeruginosa* spores. Furthermore, by studying key stages in the pathogen infection process, such as attachment, invasion, reproduction, and spread, this research is expected to identify critical nodes and targets in the infection, leading to the development of highly targeted and effective fungicides, providing strong technical support for effectively controlling the occurrence and spread of millet blast. (Based on a precise infection timeline using HEB07-GFPox markers...) Figure 3 The PRR gene was significantly upregulated 12-24 hours after infection, corresponding to the critical stage of pathogen penetration into the epidermis. This early PTI response may activate the MAPK phosphorylation cascade by recognizing the conserved PAMP of *Pythium oryzae*, thus delaying hyphal expansion. The subsequent explosive expression of the NLR gene at 48 hours indicates that the secretion of pathogen effectors triggered ETI defense. The sustained high expression of SA-related genes from 12-48 hours confirms that millet resists *Pythium oryzae* infection through the PRR-NLR-SA cascade pathway. Furthermore, this study successfully screened EMS-induced mutants bsm-1 and bsm-2, which significantly enhance susceptibility to *Pythium oryzae*, by constructing the HEB07-GFPox marker strain, providing important materials for elucidating the basic immune mechanism of rice and mining disease resistance gene resources.

[0133] Furthermore, in-depth research into this interaction mechanism will help identify genes and markers closely related to disease resistance. These genes and markers will become key factors in breeding millet varieties with durable disease resistance. Utilizing these genes and markers, we can more effectively improve and breed disease-resistant millet varieties, cultivating varieties capable of resisting pathogen attacks, thereby effectively reducing the impact of millet blast on millet yield and quality, and making a positive contribution to ensuring food security.

[0134] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A dominant strain for constructing a spatiotemporal map of the interaction between the blast fungus and millet, characterized in that: The strain is HEB07 of *Pyrrosia lingua*. After screening and verification, this strain has the broad range of infection to different varieties of millet, the sensitivity and survival rate of infection to any millet variety, and the high affinity for GFP fluorescent tag recombination transformation. Therefore, it constitutes the superior strain for constructing gene recombination strains using green fluorescent protein GFP and exploring the interaction between the blast fungus and millet. The strain has the accession number CGMCC No.42558 and is deposited at the China General Microbiological Culture Collection Center on January 19, 2026. The classification number of this strain is Pyricularia grisea. Its main biological characteristics include: a deuteromycete belonging to the family Pyriculariaceae and the genus Pyricularia; mycelium: colorless and transparent, filamentous, septate; conidiophores: 3-5 conidiophores forming bundles, with 2-8 septa; conidia: colorless and transparent, mature conidia often septate, with a pointed apex and a blunt, rounded base.

2. The use of the *Pyrrosia lingua* strain HEB07 according to claim 1, characterized in that: The intended use is to leverage the strain's broad infection range and high pathogenicity affinity for different millet varieties to rapidly and efficiently construct millet blast-carrying plants and / or tissues in scientific research and production, as well as to conduct scientific research and production operations based on millet blast-carrying plants and / or tissues.

3. The use of the *Pyrrosia lingua* strain HEB07 according to claim 2, characterized in that: The applications include, in particular, constructing a spatiotemporal map of the entire process of interaction between the blast fungus and millet using GFP fluorescent tags, and constructing new technical pathways for the prevention and treatment of blast fungus based on this map.

4. The HEB07-GFPox recombinant strain, characterized by: Based on the *Pyrrosia lingua* strain HEB07 described in claim 1, a recombinant strain of HEB07 capable of expressing green fluorescent protein was constructed through gene recombination, thus obtaining the HEB07-GFPox recombinant strain.

5. A method for creating the HEB07-GFPox strain, characterized in that: By constructing a recombinant strain carrying the pBARGPE1-EGFP vector, a HEB07-GFPox strain overexpressing the GFP gene was created.

6. The method for creating the HEB07-GFPox strain according to claim 5, characterized in that: The specific steps include: A. Preparation of conidia of Pyroxburghii; B. Preparation of competent cells; C. Mix with plasmids containing the target gene; D. Add PEG for induction; E. Recombinant spores infused with a foreign gene; wherein the foreign gene is the GFP gene; F. Recombinant strains were obtained through culture.

7. The method for preparing HEB07-GFPox strain according to claim 6, characterized in that: In step A, *Pyrrosia lingua* was obtained by activating and culturing it in CM medium. The *Pyrrosia lingua* strain used was HEB07, which was collected from Hebei Province and has multidimensional advantages.

8. The method for preparing HEB07-GFPox strain according to claim 6, characterized in that: In step E, glufosinate was used as a screening reagent to screen recombinant strains that successfully integrated the GFP expression plasmid.

9. The method for preparing HEB07-GFPox strain according to claim 5, characterized in that: In step F, the HEB07-GFPox strain appears colorless and transparent under white light, and its shape is spindle-shaped with septa; bright green fluorescence can be observed under 488nm excitation light.

10. The application of the strain of claim 1, the recombinant strain of claim 4, or the method of any one of claims 5-9, wherein the application is any one of the following: (1) Application in screening or assisting screening of susceptible mutants of millet; (2) Application in identifying or assisting in the identification of disease resistance in different millet varieties; (3) Application of candidate pesticides in inhibiting key links of blast fungus infection; (4) Identification or auxiliary identification of the colonization preference of Blast Angelica glutenosa in specific plant tissues; (5) Application in screening or assisting in the screening of millet varieties resistant to millet blast.

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