Fusarium solani fluorescence labeled strain and application thereof

Abstract

The invention relates to the technical field of microbial genetic transformation and phytopathology, in particular to a fusarium solani fluorescence labeled strain and application thereof. The bacterial strain is classified and named as Fusarium solani (Fss9-YFP), and the preservation number of the bacterial strain is CGMCC (China General Microbiological Culture Collection Center) NO: 42172. The fusarium solani fluorescence labeled strain is obtained by integrating a YFP fluorescent protein expression cassette sequence driven by a TEF-1alpha promoter into a protoplast of a wild type fusarium solani Fss1 strain. The method has the advantages that the fluorescently labeled strain Fusarium solani (Fss9-YFP) is stable in inheritance and good in fluorescence expression, and after the Fusarium solani (Fss9-YFP) is inoculated to soybean seedlings, germination and infection ways of spores are directly observed by adopting a microscope, so that distinguishing of disease-resistant soybean varieties, real-time tracing of soybean fusarium root rot, analysis of disease-resistant mechanisms and the like are facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial genetic transformation and plant pathology, and particularly relates to a fluorescently labeled Fusarium solani strain and application thereof. BACKGROUND

[0002] Fusarium root rot is a widely distributed and serious soil-borne fungal disease, which has a significant impact on the quality and yield of soybeans. Currently, Fusarium root rot caused by Fusarium solani, Fusarium oxysporum, Fusarium fujikuroi, Fusarium proliferatum, Fusarium graminearum and the like has been listed as a class of crop diseases of soybeans in China. There are many species of Fusarium, and they have the characteristics of complex infection. In addition, they are soil-borne, which makes it more difficult to prevent and control Fusarium root rot of soybeans. As of now, at least 24 species of Fusarium have been identified in China, including Fusarium solani (F. solani), Fusarium oxysporum (F. oxysporum), Fusarium fujikuroi (F. fujikuroi), Fusarium proliferatum (F. proliferatum), Fusarium graminearum (F. graminearum) and the like. Among them, F. solani is one of the main pathogenic fungi causing soybean root rot, but its pathogenic mechanism and interaction mechanism with soybeans have not been elucidated. Fusarium solani Fusarium oxysporum Fusarium fujikura Fusarium proliferatum Fusarium graminearum Fusarium solani

[0003] Due to the difficulty of directly observing the infection process of pathogenic fungi in the soil environment, fluorescently labeled strains have become an important technical means for analyzing the pathogenic mechanism, which not only can track the disease process, but also helps to analyze the function of genes. This process relies on an efficient protoplast transformation system. Protoplast gene transformation technology has been widely used in the study of pathogenic mechanisms of various plant pathogens, such as banana Fusarium oxysporum, corn Fusarium graminearum, wheat Fusarium graminearum, tomato Fusarium oxysporum and the like. Researchers use fluorescently labeled strains to monitor the infection process of pathogenic fungi in real time, thereby revealing the development of the disease process. However, there is still a lack of systematic research on the infection mechanism of F. solani, especially its interaction with soybeans. SUMMARY

[0004] The present application provides a fluorescently labeled F. solani strain and application thereof to solve the above problems.

[0005] The first object of the present application is to provide a fluorescently labeled F. solani strain, which is classified and named as F. solani Fusarium solani , with the accession number CGMCC NO: 42172, preserved at the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Beijing City, China, and preserved on August 18, 2025.

[0006] ​​​​​​Preferably, the fluorescently labeled Fusarium solani pisi strain is obtained by using the vector pSHUT4-eYFP as a template to amplify the YFP fluorescent protein expression cassette sequence driven by the TEF-1α promoter, and then integrating the sequence into the protoplast of the wild-type Fusarium solani pisi Fss1 strain.

[0007] Preferably, the YFP fluorescent protein expression cassette sequence is amplified by using the following specific primers: YFPa-F: 5'-CACCAAGTCCATAAGCCACA-3'; YFPa-R: 5'-GATGCCACGAATTCCAATCT-3'.

[0008] Preferably, the protoplast of the wild-type Fusarium solani pisi Fss1 strain is prepared by the following method: S1. preparing the conidial suspension of the wild-type Fusarium solani pisi isolate Fss1; S2. protoplast preparation; specifically including the following sub-steps: S21. adjusting the conidial suspension of Fusarium solani pisi to a concentration of 1 x 10 6 ~1 x 10 7 cfu ml -1 , adding it into the CMC medium, and culturing at 23-27°C and 150-250 rpm for 2-4 days; filtering the obtained bacterial solution, centrifuging the spores at 3500-4500 rpm for 8-15 min, discarding the supernatant, resuspending with sterile water, repeating the centrifugation, and obtaining the spores; S22. adding the spores into the YEPD medium, culturing at 23-27°C and 150-250 rpm for 10-15 h, filtering, and collecting the mycelium; S23. preparing the enzymatic solution, adding it into the mycelium, and enzymatically digesting at 28-32°C and 70-100 rpm for 2-5 h; filtering the enzymatic solution, rinsing with a 0.7M NaCl solution, centrifuging the filtrate at 0-4°C and 2500-3500 rpm for 8-15 min, discarding the supernatant, resuspending with the STC solution, mixing well, and repeating the centrifugation once.

[0009] Preferably, step S1 specifically includes the following sub-steps: S11. cutting the root tissue at the junction of the soybean disease lesion and healthy tissue; removing the bacteria from the root tissue, and culturing the bacteria-removed tissue in the PDA medium at 23-27°C for 3-4 days; S12. taking the edge mycelium, inoculating it in the PDA medium to recover growth, taking the edge mycelial cake into the PD liquid medium, and culturing at 23-27°C and 150-250 rpm for 1.5-3 days under shaking conditions to obtain a suspension, i.e., the conidial suspension of the wild-type Fusarium solani pisi isolate Fss1.

[0010] Preferably, in step S2, the CMC culture medium contains 14-16 g of sodium carboxymethyl cellulose, 0.8-1.5 g of yeast extract, 0.8-1.5 g of ammonium nitrate, 0.8-1.5 g of potassium dihydrogen phosphate, and 0.4-0.6 g of magnesium sulfate heptahydrate per liter; The YEPD culture medium contains 8-15 g of peptone, 2-4 g of yeast extract, and 15-25 g of glucose per liter, and the pH is 7.5; The STC solution contains 20-25 g of sorbitol, 0.5-1 g of CaCl2, and 1 mL of Tris-HCl solution per 100 mL, and the rest is water.

[0011] Preferably, in step S21, the culture is carried out at 25℃ and 200 rpm for 3 days, and the spores are enriched by centrifugation at 4000 rpm for 10 min; in step S22, the culture is carried out at 25℃ and 200 rpm for 12 h, the mycelium is filtered with gauze and collected; in step S23, the enzymolysis is carried out at 30℃ and 85 rpm for 3 h; and the filtrate is centrifuged at 4℃ and 3000 rpm for 10 min.

[0012] The second object of the present application is to provide an application of the fluorescently labeled Fusarium solani pisi strain in real-time tracing of Fusarium root rot of soybean.

[0013] Preferably, the application comprises: preparing the Fusarium solani pisi Fusarium solani inoculum; immersing the roots of 3-5 day-old soybean seedlings in the Fusarium solani pisi Fusarium solani inoculum, and culturing at 23-27℃ under 16 h light / 8 h dark conditions, regularly sampling, observing the YFP fluorescence signal by fluorescence microscopy or confocal microscopy, analyzing the pathogen infection dynamics, and realizing real-time tracing.

[0014] The third object of the present application is to provide an application of the fluorescently labeled Fusarium solani pisi strain in distinguishing resistant and susceptible soybean varieties.

[0015] Compared with the prior art, the present application can achieve the following beneficial effects: The yellow fluorescent protein YFP is introduced into wild type Fusarium solani fsp. psi Fss1 by using a protoplast transformation method, and the obtained Fusarium solani fsp. psi Fss9-YFP maintains the same colony morphology and pathogenicity as the wild type Fss1, the fluorescent strain can be stably inherited, and the fluorescence expression is good, so that the stable Fusarium solani fsp. psi Fss9-YFP can be obtained by using the fluorescent strain transformation method provided in the application. In addition, the application of the fluorescent marker strain of Fusarium solani fsp. psi in real-time tracing of soybean Fusarium root rot, distinguishing resistant and susceptible soybean varieties, and studying disease control mechanism, etc. is provided. By using the conidial liquid culture method provided, the obtained fluorescent strain is inoculated into soybean seedlings, and the spore germination and infection pathway are directly observed by using a fluorescence microscope or a confocal microscope, so that the infection process of Fusarium solani fsp. psi in the soybean root system can be easily mastered, and the disease resistance mechanism can be analyzed. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is a schematic diagram of a Fusarium solani fsp. psi fluorescent marker strain vector and a fluorescent strain construction process according to an embodiment of the application; A is a Fusarium solani fsp. psi fluorescent marker strain vector; B is a PEG-mediated protoplast transformation system flow chart.

[0017] Figure 2 It is a stable expression result of a yellow fluorescent protein (YFP fluorescent protein) in Fusarium solani fsp. psi according to an embodiment of the application; in the figure, A is a fluorescent detection of conidium and mycelium of the Fusarium solani fsp. psi YFP fluorescent marker strain, the excitation wavelength is 488 nm, the emission wavelength is 510 nm, Bright represents a bright field, GFP represents a green fluorescent protein, and Merge represents an image obtained by superimposing and merging the bright field and the green fluorescent image; B is a 5-day-old colony phenotype of a wild type isolated strain Fss1 of Fusarium solani fsp. psi and the fluorescent marker strain Fusarium solani fsp. psi Fss9-YFP, the scale bar = 1 cm; and C is a colony diameter statistics of the wild type isolated strain Fss1 of Fusarium solani fsp. psi and the fluorescent marker strain Fusarium solani fsp. psi Fss9-YFP for 3-6 days.

[0018] Figure 3 It is an influence of the fluorescent strain on the pathogenicity of the soybean root according to an embodiment of the application; A is a plant without inoculation, serving as a control, Control represents a control; B is a material inoculated with the wild type Fss1, WT represents a wild type; and C is a material inoculated with the fluorescent strain Fusarium solani fsp. psi Fss9-YFP provided in the application.

[0019] Figure 4 It is an invasion difference of the fluorescent strain in the root system main root maturation zone of resistant and susceptible soybean varieties according to an embodiment of the application; A is a root system cross section superimposition of a resistant material; B is a fluorescent field corresponding to A; C is a root system cross section superimposition of a susceptible material; and D is a fluorescent field corresponding to C. DETAILED DESCRIPTION

[0020] Hereinafter, embodiments of the present application will be described with reference to the accompanying drawings. In the following description, the same modules are denoted by the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, detailed descriptions thereof will not be repeated.

[0021] In order to make the objectives, technical solutions, and advantages of the present application clearer, further detailed descriptions will be made to the present application in combination with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, and do not constitute a limitation on the present application.

[0022] The present application provides a fluorescently labeled Fusarium solani strain, the strain classification name is Fusarium solani Fusarium solani , the strain name is Fss9-YFP, the preservation number is CGMCC NO:42172, and the preservation address is No. 1, Beichen West Road, Chaoyang District, Beijing, China. The preservation date is August 18, 2025. The preparation method of the above-mentioned fluorescently labeled Fusarium solani strain is as follows: S1. Preparation of Fusarium solani conidial liquid; comprising: S11. Cutting root tissue at the junction of soybean disease-like lesion and healthy tissue; removing bacteria from the root tissue, and placing the bacteria-removed tissue in PDA medium and culturing at 23-27℃ for 3-4 days; S12. Taking the edge mycelium, inoculating in PDA medium to restore growth, taking the edge mycelium cake in PD liquid medium, and culturing at 23-27℃, 150-250rpm under shaking conditions for 1.5-3 days to obtain a suspension, i.e. Fusarium solani conidial liquid; Specifically, the Fusarium solani conidial liquid is the conidial liquid of wild-type Fusarium solani isolate Fss1; the size of the root tissue is about 5mmx5mm; The PDA medium contains 200g peeled potato pieces, 20g glucose, and 15g agar powder per liter, and chloramphenicol resistance is added; In step S11, the bacteria-removed tissue is placed in the PDA medium containing chloramphenicol resistance and cultured at 25℃ for 3-4 days; The shaking conditions of step S12 are 25℃, 200rpm.

[0023] S2. Preparation of protoplasts; comprising: S21. Adjusting the Fusarium solani conidial liquid to a concentration of 1x10 6 cfu ml -1Add the bacterial culture to CMC medium and incubate at 23-27℃ and 150-250 rpm for 2-4 days. Filter the obtained bacterial solution and centrifuge at 3500-4500 rpm for 8-15 min to enrich the spores. Discard the supernatant and resuspend the spores in sterile water. Repeat the centrifugation to obtain the spores. S22. Add spores to YEPD medium and incubate at 23-27℃ and 150-250 rpm for 10-15 h. Filter and collect mycelia. S23. Prepare the enzymatic hydrolysate, add it to the mycelium, and enzymatically hydrolyze at 28-32℃ and 70-100 rpm for 2-5 h; filter the enzymatic hydrolysate and wash with 0.7M NaCl solution, centrifuge the filtrate at 0-4℃ and 2500-3500 rpm for 8-15 min, discard the supernatant; resuspend in STC solution, mix well, and repeat the centrifugation once to obtain the protoplasts of wild-type Fusarium solani strain Fss1; Specifically, each liter of CMC culture medium contains 14-16g sodium carboxymethyl cellulose, 0.8-1.5g yeast extract, 0.8-1.5g ammonium nitrate, 0.8-1.5g potassium dihydrogen phosphate, and 0.4-0.6g magnesium sulfate heptahydrate; YEPD medium contains 8-15g peptone, 2-4g yeast extract, and 15-25g glucose per liter, with a pH of 7.5. The enzymatic hydrolysate includes 0.08-0.15 g of snail enzyme, 0.08-0.15 g of wall-lysing enzyme, 0.03-0.07 g of wall-breaking enzyme, NaCl, and water. The NaCl concentration is 0.7 M, and the water concentration is 10 mL. STC solution contains 20-25g sorbitol, 0.5-1g CaCl2, 1mL Tris-HCl solution per 100mL, with the remainder being water; Step S21 involves culturing at 25℃ and 200rpm for 3 days, followed by centrifugation at 4000rpm for 10min to enrich spores; In step S22, the mycelium is cultured at 25℃ and 200rpm for 12h, filtered through gauze and collected. In step S23, the enzyme was hydrolyzed at 30℃ and 85 rpm for 3 hours; the filtrate was centrifuged at 4℃ and 3000 rpm for 10 minutes.

[0024] S3. Protoplast transformation: take protoplast and add YFP fluorescent protein expression box amplification product, mix, ice bath 15~25min;Add 2 times the volume of PEG solution of protoplast, mix, ice bath 8~15min, repeat once;Add 8 times the volume of PEG solution of protoplast, mix, incubate at room temperature for 15~25min in the dark;The mixed solution is added to the lower layer of RM medium, and the growth is recovered at 23~27℃ for 10~15h, and then the upper layer of RM medium is covered on it, and the upper layer of RM medium is grown at 23~27℃, until the single colony grows on the surface of the upper layer of RM medium, which is Fusarium solani fsp. pisi Fss9-YFP; Specifically, the PEG solution contains 25~35g PEG 4000, 0.2~0.3g anhydrous CaCl2, 0.5mL Tris-HCl solution per 50mL, and the rest is water; The lower layer of RM medium contains 23~25g sucrose, 0.08~0.15g yeast extract, 1.3~2g agar powder, 0.08~0.15g acid hydrolyzed casein per 100mL; The upper layer of RM medium contains 33~35g sucrose, 0.08~0.15g yeast extract, 0.8~1.2g agar powder, 0.08~0.15g acid hydrolyzed casein, 8~15mg G418 per 100mL.

[0025] The application also provides the application of the above-mentioned fluorescently labeled Fusarium solani fsp. pisi strain in real-time tracing of soybean Fusarium root rot, distinguishing resistant and susceptible soybean varieties, and studying disease control mechanisms.

[0026] Specifically, the application of the fluorescently labeled Fusarium solani fsp. pisi strain in real-time tracing of soybean Fusarium root rot includes: preparing Fusarium solani fsp. pisi Fss9-YFP inoculum;Dipping the roots of 3~5-day-old soybean seedlings into the Fusarium solani fsp. pisi Fss9-YFP inoculum, and culturing at 23~27℃ with 16h light / 8h dark, periodically sampling, observing YFP fluorescent signal by fluorescence microscope or confocal microscope, analyzing pathogen infection dynamics and achieving real-time tracing.

[0027] Preferably, the preparation method of the Fusarium solani fsp. pisi Fss9-YFP inoculum is: adding Fusarium solani fsp. pisi Fss9-YFP into a mixed solution containing 1 / 5 concentration of MS medium and 10% concentration of soil leachate mixture, to prepare Fusarium solani fsp. pisi Fss9-YFP inoculum with a spore concentration of 1×10 6 ~1×10 7 cfu mL -1 ; Wherein, the volume ratio of MS medium to soil leachate mixture is 9:1; MS medium contains 0.8-0.9 g M524 per liter, PH=5.7; Preparation method of soil leaching solution mixture: 100 g of nutrient soil was added to 900 mL of sterile water, shaken for 2 h, filtered, 0.5 g of glucose was added, and sterilized at 121 ℃ for 1 h.

[0028] Specifically, the application of the fluorescently labeled Fusarium solani f. sp. glycines strain in distinguishing resistant and susceptible soybean varieties includes: by infecting the root of soybean seedlings, it is observed that the soybean variety with mycelium spreading to the inner cortex of the root is a susceptible variety, and the soybean variety with mycelium still in the outer cortex of the root is a resistant variety.

[0029] Example 1 The present embodiment provides a method for isolating and purifying Fusarium solani f. sp. glycines, which is specifically as follows: About 5 mm x 5 mm of root tissue was cut at the junction of the soybean lesion and healthy tissue using a sterile scalpel; the bacteria on the root tissue were removed, and the tissue after removing the bacteria was placed in PDA medium containing chloramphenicol resistance and cultured at 25 ℃ for 3-4 days. Subsequently, the edge mycelium of the tissue was inoculated in PDA medium for growth, and the edge mycelium cake was taken to PD liquid medium and cultured at 25 ℃, 200 rpm vibration for 2 days to obtain Fusarium solani f. sp. glycines; the conidial suspension was diluted to 1 x 10 2 cfu mL -1 , 50 μL of which was coated on one PDA medium. After obtaining the purified strain by single spore isolation technology, ITS sequence was used for sequencing identification. After Blast comparison of the ITS region sequence with the NCBI database, the species of the isolated strain was determined, and the Fusarium solani f. sp. glycines isolated strain Fss1 (wild type) was screened and obtained for subsequent experiments.

[0030] Example 2 The establishment of the fluorescently labeled Fusarium solani f. sp. glycines strain is specifically as follows: 1. Construction of fluorescent labeling vector In the present embodiment, the fluorescent labeling vector pSHUT4-eYFP of Fusarium solani f. sp. glycines was used as a template, and the TEF-1α promoter-driven YFP fluorescent protein expression cassette sequence (SEQ ID NO: 1) was amplified in the vector by site-specific primers. Figure 1 TEF-1α: eYFP ); the specific primers are as follows: YFPa-F: 5'-CACCAAGTCCATAAGCCACA-3'; YFPa-R: 5'-GATGCCACGAATTCCAATCT-3'; 2. Preparation and transformation of protoplasts The prepared and transformed method of protoplast was used to integrate the amplified sequence (shown in SEQ ID NO. 1) into the genome of Fusarium solani f. sp. pisi strain Fss1 (shown in SEQ ID NO. 2) by homologous recombination; the prepared and transformed method of protoplast is as follows: Figure 1 (1) Preparation of culture medium and solution (1) Preparation of culture medium and solution Carboxymethyl Cellulose (CMC) medium components: 15 g carboxymethyl cellulose, 1 g yeast extract, 1 g ammonium nitrate, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, and constant volume to 1 L; Yeast Extract Peptone Dextrose (YEPD) medium components: 10 g peptone, 3 g yeast extract, 20 g glucose, constant volume to 1 L, PH=7.5; Enzymatic solution: 0.1 g snailase, 0.1 g lywallzyme and 0.05 g lycomyces; STC solution: 21.86 g sorbitol, 1 mL 1 mol / L Tris-HCl solution, pH=7.5, 0.555 g CaCl2, and constant volume to 100 mL with water; PEG solution: 30 g PEG 4000, 0.5 mL 1 mol / L Tris-HCl, pH=7.5, 0.279 g anhydrous CaCl2, and constant volume to 50 mL with water; RM lower layer medium components: 23.96 g sucrose, 0.1 g yeast extract, 1.5 g agar powder, 0.1 g acid hydrolysis casein, constant volume to 100 mL; RM upper layer medium components: 34.2 g sucrose, 0.1 g yeast extract, 1 g agar powder, 0.1 g acid hydrolysis casein, constant volume to 100 mL, and 100 μg / mL -1 G418; PDA medium: containing 100 μg / mL -1 G418.

[0031] (2) Preparation of protoplast The conidia liquid of Fusarium solani was adjusted to a concentration of 1×10 6 cfu / mL -1, take 100 μL into 50 mL CMC medium, cultivate at 25°C, 200 rpm for 3 days; the obtained bacterial liquid is filtered, centrifuged at 4000 rpm for 10 min to enrich spores, the supernatant is discarded and resuspended with sterile water, centrifuged once again to obtain spores. The spores are added into 50 mL YEPD medium, cultivated at 25°C, 200 rpm for 12 h. Filtered with gauze, the mycelium is collected, washed with 0.7M NaCl solution and then transferred into a flask for standby. Prepare enzyme solution, add the mycelium, and enzymolysis at 30°C, 85 rpm for 3 h; then filter the enzyme solution with lens paper and wash with 0.7M NaCl solution, centrifuge the filtrate at 4°C, 3000 rpm for 10 min, discard the supernatant; resuspend with 1 mL STC solution, mix gently with 1 mL sharp gun head, and repeat centrifugation once. Resuspend with appropriate amount of STC solution, count under a microscope, and dilute to 1×10 6 ~1×10 7 cfu mL -1 , and place on ice for standby.

[0032] (3) Protoplast transformation Take 100 μL of the above protoplast and add 5 μg of YFP fluorescent protein expression cassette amplification product, mix gently with a sharp blue gun head, and ice bath for 20 min. Then add 200 μL of PEG solution, mix gently, and ice bath for 10 min, repeat once. Then add 800 μL of PEG solution, mix gently, and incubate at room temperature in the dark for 20 min. Add the above mixture to 20 mL of RM lower medium, cultivate at 25°C for 12 h to recover growth. Cover 10 mL of RM upper medium on top, cultivate at 25°C, and grow single colonies on the surface of the RM upper medium.

[0033] (4) Identification of transformants Pick the mycelium of single colonies and inoculate on PDA medium, cultivate at 25°C; after new mycelium grows, use the above-mentioned protoplast transformation method to obtain transformants. Then use the CTAB method to extract the DNA of the transformants, and use primers to verify the YFP fluorescent expression cassette of the transformants; the primers are as follows: YFPb-F: 5'-CGGTTCTTCCAGCGCTTCTTCA-3' YFPb-R: 5'-GGAAATACCATGCTACGTTAAC-3'; The results show that 13 of them are positive transformants, 20 are negative transformants, and the positive rate is 39.4%.

[0034] For the verification of successful transformants, the bacterial cake was taken at the edge of its colony, inoculated on PDA medium, and continuously subcultured to the sixth generation. It was found through confocal microscope observation that YFP fluorescent protein was successfully expressed in the transformants; both spores and mycelium showed strong green fluorescence Figure 2 A), and finally a fluorescently labeled strain stably expressing YFP was obtained; Figure 2 Bright of medium A represents bright field, GFP represents green fluorescent protein, and Merge represents the image obtained by superimposing the bright field and green fluorescent images.

[0035] The transformant strain was preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC NO: 42172, and the classification name Fusarium solani f. sp. pisi Fusarium solani , the strain was named Fss9-YFP, and the preservation date was August 18, 2025.

[0036] In order to determine whether the fluorescent labeling affects the growth of the strain, the fluorescently labeled Fusarium solani f. sp. pisi Fss9-YFP and the wild type strain Fss1 (WT) were cultured in PDA medium and observed, and the colony diameters were counted on the 3rd to 6th day. The observation results showed that there was no significant difference in the colony size of the fluorescently labeled Fusarium solani f. sp. pisi Fss9-YFP and the wild type strain (Fig. Figure 2 B). The diameter counting results showed that there was no significant difference in the growth curves of the fluorescently labeled Fusarium solani f. sp. pisi Fss9-YFP and the wild type strain Fss1, and both showed linear growth (Fig. Figure 2 C). The above results showed that the expression of YFP fluorescent protein had no significant effect on the growth of the strain.

[0037] Example 3 MS medium: 0.86 g M524, PH = 5.7, constant volume to 1 L; Soil leachate mixture: 100 g of nutrient soil was added to 900 mL of sterile water, shaken for 2 h, and then filtered with gauze. 1 L of filtrate was added to 0.5 g of glucose, and sterilized at 121°C for 1 h; Conidial liquid culture method: according to the method for preparing conidial liquid in Example 1, the blood cell counting plate was used to count under a microscope, and then the conidial suspension was diluted to 1×10 6 cfu mL -1 , and each test tube was divided into 80 mL of the mixture. Well-grown 4-day-old soybean seedlings were selected, washed, fixed in the test tube, and the roots of the seedlings were immersed below the liquid level. Cultivation was carried out at 25°C under a light cycle of 16 h light / 8 h darkness, and the tissues of 3 soybean seedlings were sliced by hand every day, and observed using a confocal microscope.

[0038] Example 4 Application of the fluorescently labeled strain of F. solani pisi, as follows: (1) To verify the effect of fluorescent labeling on the pathogenicity of the strain, the conidial suspension of Example 3 was used to inoculate the soybean variety G22C017 with the fluorescently labeled strain of F. solani pisi Fss9-YFP and the wild-type strain Fss1 (WT). Phenotypic observation was performed at 4 dpi. The observation found that the pathogenicity of F. solani pisi Fss9-YFP and Fss1 did not show significant difference. Compared with the uninfected plants, both F. solani pisi Fss9-YFP and Fss1 could significantly inhibit the development of lateral roots, and brown lesions appeared in the mature zone of the main roots Figure 3 ).

[0039] (2) Observation of the disease process of F. solani pisi infection in soybean roots. The observation results showed that 8 h after inoculation, the conidia of F. solani pisi began to adhere to the epidermis of the main roots and lateral roots in small amounts, firmly colonized on the epidermal cells, and then differentiation of infection pegs at the hyphal tips penetrated the epidermal cells of the root system; 24 h after inoculation, the fluorescence signal of the pathogen was significantly enhanced in the mature zone of the main roots and the base of the lateral roots, and the hyphae expanded longitudinally and transversely along the epidermis of the main root mature zone and the base of the lateral roots, eventually forming a hyphal layer around the entire root system; microscopic observation 96 h after inoculation found that the hyphae broke through the epidermal cells of the main root mature zone and the base of the lateral roots, successfully invading the cortex tissue of the soybean roots. And in the main root mature zone and the base of the lateral roots, the hyphae regularly extended along the intercellular space of the cortex tissue, gradually expanding to the inside of the stele.

[0040] (3) Application of fluorescent F. solani pisi in the comparison of infection processes in different resistant varieties of soybean. Also using the spore suspension inoculation method, a systematic histopathological comparative analysis was performed on resistant and susceptible soybean varieties at the key infection time point of 96 h after inoculation with F. solani pisi. The observation found that the fluorescence signal of the resistant variety in the main root mature zone was significantly less than that of the susceptible variety, indicating that the amount of hyphal infection of the former was significantly less than that of the latter. And the infection site of the susceptible variety was more invasive than that of the resistant variety, the hyphae of the susceptible variety had spread to the inner cortex, while the hyphae of the resistant variety were still in the outer cortex Figure 4 ). Microstructure analysis further revealed significant differences in the degree of infection between the two varieties, which may be key factors leading to differences in disease resistance between varieties.

[0041] It should be understood that the various forms of the flow shown above can be reordered, added to, or deleted from without departing from the scope of the present disclosure. For example, the steps recited in the present disclosure can be executed in parallel or in sequence, or in a different order, as long as the desired results of the technical solutions of the present disclosure can be achieved, which is not limited herein.

[0042] The foregoing detailed description has set forth various embodiments of the devices and / or processes via the use of specific terminology. However, embodiments thereof can be practiced with the exact description not being presented in detail. The term "device" should be understood to encompass devices operating in various modes, such as active mode, sleep mode, hibernate mode, and the like. The terms "coupled" and "connected," along with their derivatives, can be used. It should be understood that these terms are not intended as synonyms for each other. Rather, particular circuitry that can be said to be coupled or connected can be coupled and connected via some transmission medium.

Claims

1. A fluorescently labeled strain of Fusarium solani, characterized in that: The strain classification name is Fusarium solani f. sp. pisi Fusarium solani , the accession number is CGMCC NO: 42172, and the depositing center is China General Microbiological Culture Collection Center, the depositing address is No. 1, Xibaixili, Chaoyang District, Beijing, and the depositing date is August 18, 2025.

2. The fluorescently labeled strain of Fusarium solani according to claim 1, characterized in that: The fluorescent Fusarium solani strain is obtained by integrating a TEF-1α promoter-driven YFP fluorescent protein expression cassette sequence into a protoplast of a wild-type Fusarium solani Fss1 strain.

3. The fluorescently labeled strain of Fusarium solani according to claim 2, characterized in that: The YFP fluorescent protein expression cassette sequence is amplified by the following specific primers: YFPa-F: 5'-CACCAAGTCCATAAGCCACA-3'; YFPa-R: 5'-GATGCCACGAATTCCAATCT-3'.

4. The fluorescently labeled strain of Fusarium solani according to claim 2, characterized in that: The protoplast of the wild-type Fusarium solani Fss1 strain is prepared by the following method: S1. Prepare conidial suspension of wild-type Fusarium solani isolate Fss1; S2. Protoplast preparation; specifically including the following sub-steps: S21. Adjusting Fusarium solani f. sp. pisi conidial liquid to a concentration of 1 x 10 6 7 -1 cfu / ml, adding into CMC medium, culturing at 23-27°C, 150-250 rpm for 2-4 days; the obtained bacterial liquid is filtered, and then centrifuged at 3500-4500 rpm for 8-15 min to enrich spores, the supernatant is discarded, and the spores are resuspended with sterile water, and centrifuged repeatedly to obtain spores;​​ S22. Add spores to YEPD medium, cultivate at 23-27°C, 150-250 rpm for 10-15 h, filter and collect mycelium; S23. Prepare enzyme solution, add to the mycelium, enzymolysis at 28-32°C, 70-100 rpm for 2-5 h; filter the enzyme solution and rinse with 0.7M NaCl solution, centrifuge the filtrate at 0-4°C, 2500-3500 rpm for 8-15 min, discard the supernatant; resuspend with STC solution, mix well, repeat centrifugation once.

5. The fluorescently labeled strain of Fusarium solani according to claim 4, characterized in that: The step S1 specifically includes the following sub-steps: S11. Cut the root tissue at the junction of soybean disease lesions and healthy tissue; remove the bacteria from the root tissue, and place the bacteria-free tissue in PDA medium and cultivate at 23-27°C for 3-4 days; S12. Take the edge mycelium, inoculate in PDA medium to restore growth, take the edge mycelium cake in PD liquid medium, cultivate at 23-27°C, 150-250 rpm for 1.5-3 days, obtain the suspension, which is the conidial suspension of wild-type Fusarium solani isolate Fss1.

6. The fluorescently labeled strain of Fusarium solani according to claim 4, characterized in that: In the step S2, the CMC medium contains 14-16 g carboxymethyl cellulose sodium, 0.8-1.5 g yeast extract, 0.8-1.5 g ammonium nitrate, 0.8-1.5 g potassium dihydrogen phosphate, 0.4-0.6 g magnesium sulfate heptahydrate per liter; The YEPD medium contains 8-15 g peptone, 2-4 g yeast extract, 15-25 g glucose per liter, and the pH is 7.5; The enzyme solution includes 0.08-0.15 g snailase, 0.08-0.15 g lywallzyme, 0.03-0.07 g lycomatozyme, 0.7M NaCl and 10 mL water; The STC solution contains 20-25 g sorbitol, 0.5-1 g CaCl2, 1 mL Tris-HCl solution per 100 mL, and the rest is water.

7. The fluorescently labeled strain of Fusarium solani according to claim 6, characterized in that: The step S21 is incubated at 25℃, 200rpm for 3 days, and the spores are enriched by centrifugation at 4000rpm for 10min; in step S22, the mycelium is collected by filtering with gauze after incubation at 25℃, 200rpm for 12h; in step S23, the enzyme is hydrolyzed at 30℃, 85rpm for 3h; and the filtrate is centrifuged at 4℃, 3000rpm for 10min.

8. The use of the fluorescently labeled Fusarium solani strain of claim 1 in real-time tracing of soybean Fusarium root rot.

9. The use of a fluorescently labeled strain of Fusarium solani f. sp. glycines according to claim 8 for real-time tracking of F. solani f. sp. glycines in soybean F. solani f. sp. glycines root rot, characterized in that: The application includes: preparing fusarium solani Fusarium solani Inoculum; 3-5 day old soybean seedling roots were immersed in fusarium solani Fusarium solani Inoculum, cultured at 23-27°C, 16h light / 8h dark, sampled regularly, observed YFP fluorescence signal by fluorescence microscope or confocal microscope, analyzed pathogen infection dynamics and realized real-time tracking.

10. The use of the fluorescently labeled Fusarium solani strain of claim 1 in distinguishing between resistant and susceptible soybean varieties.

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