Nucleic acid aptamer for specifically recognizing fusarium oxysporum as well as screening method and application of nucleic acid aptamer
By combining a nucleic acid aptamer that specifically recognizes Fusarium oxysporum with gold nanoparticles to form a colorimetric probe, the problems of long detection time and low sensitivity in existing Fusarium detection methods have been solved, achieving rapid, low-cost, and highly specific detection results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-24
- Publication Date
- 2026-04-21
AI Technical Summary
Existing Fusarium detection methods are time-consuming, have low sensitivity, and cannot be used for rapid on-site detection. Furthermore, antibody detection methods suffer from poor stability and high costs.
By employing nucleic acid aptamers that specifically recognize Fusarium oxysporum and combining nanotechnology, the Cell-SELEX screening technique was used to screen out nucleic acid aptamers that specifically bind to Fusarium from a random ssDNA library. These aptamers were then combined with gold nanoparticles to form a colorimetric probe for rapid detection.
It enables Fusarium detection to be completed within 50 minutes, significantly shortening the detection time, reducing costs, and improving the specificity and sensitivity of the detection. It is suitable for rapid estimation in emergency environments, and the aptamers are non-toxic, have small molecular weights, and are easy to synthesize and label.
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Abstract
Description
Technical Field
[0001] This invention relates to biotechnology, specifically to a nucleic acid aptamer that specifically recognizes Fusarium oxysporum, its screening method, and its application. Background Technology
[0002] Fusarium is a globally significant plant pathogenic fungus, causing wilt and Fusarium head blight, ranking fourth and fifth respectively among the world's top ten plant fungal diseases. Fusarium head blight in wheat caused by *Fusarium graminearum* and wilt in cucurbits and solanaceous crops caused by *Fusarium oxysporum* result in substantial economic losses to agricultural production. These diseases can occur from the seedling stage to maturity, exhibiting diverse symptoms: for example, affected tobacco plants show stunted growth, browning of stems, and root rot; wheat displays "three rots and one blight" (stem rot, ear rot, basal rot, and seedling blight). In recent years, with changes in planting systems, the incidence of Fusarium root rot in tobacco has reached as high as 30% in major producing areas such as Yunnan and Henan, rising from a secondary disease to a major one. Fusarium has a wide host range, capable of both single-infection and co-infection with other pathogens, greatly increasing the difficulty of control.
[0003] Some Fusarium detection methods typically involve isolating and culturing Fusarium in a culture medium, followed by morphological observation and molecular biology techniques (such as polymerase chain reaction (PCR)) to detect the pathogen in the samples. Chen Gaohang et al. collected tobacco root rot samples from Enshi (Lichuan, Xuan'en, Xianfeng, Hefeng, Jianshi, Laifeng, and Badong), isolated the pathogens, and identified them as *Fusarium spp.*, *Fusarium trifidum*, and *Fusarium oxysporum* through morphological observation and internal transcribed spacer (ITS) sequence analysis. Chen Zhimin et al., through pathogen isolation and identification, clarified that the main root rot disease of tobacco in Fujian is *Fusarium tumefaciens* root rot. Molecular biological identification by Liu Lijia et al. revealed that strain NC-11 of tobacco plants infected with Fusarium root rot in Xiangcheng County, Xuchang City, Henan Province, was highly similar to Fusarium oxysporum (MN417202.1, China). Phylogenetic analysis showed that NC-11 clustered with Fusarium oxysporum strain MN417202.1. Lu Xingxing et al. used multiplex PCR to achieve specific and simultaneous detection of three types of root rot pathogens in tobacco: Tobacco Black Shank, Tobacco Black Rot, and Fusarium. Jun Zi Zhu et al. conducted morphological observation and molecular biological identification of the pathogen causing tobacco root rot in Hunan Province and found that its similarity to Fusarium strains was as high as 99.70%. Lei Yu et al. identified Fusarium oxysporum as the pathogen in the first case of tobacco Fusarium root rot caused by Fusarium solani in Lincang City, Yunnan Province. Although these methods are feasible, they are time-consuming, have low sensitivity, and cannot be used for rapid on-site detection.
[0004] In recent years, researchers have focused on developing nanoanalytical techniques for detecting pathogens based on bio-affinity probes such as antibodies. However, antibodies suffer from drawbacks such as poor stability, high cost, and cumbersome functionalization. Aptamers (also known as "chemical antibodies") are oligonucleotides obtained through in vitro screening that can bind to specific ligands (such as proteins, antibiotics, toxins, and bacteria) with high affinity and strong specificity. Compared with antibodies, aptamers have significant advantages: simple structure, short preparation time, and low cost; easy molecular modification and functionalization; and chemical stability, making them less prone to denaturation and inactivation. Although nucleic acid aptamers have attracted widespread attention from researchers in chemistry, biology, materials science, and other disciplines, and their application research in food analysis, environmental monitoring, new drug development, and disease treatment is booming, current research on "nucleic acid aptamer screening for Fusarium" is still in its early stages both domestically and internationally. Therefore, utilizing the specific molecular recognition capabilities of nucleic acid aptamers, combined with nanotechnology and biotechnology, to establish a rapid detection technology for Fusarium is expected to provide a simple, rapid, and accurate diagnosis of Fusarium root rot. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention discloses a nucleic acid aptamer that specifically recognizes Fusarium oxysporum, its screening method, and its application.
[0006] The technical solution of the present invention specifically includes: a nucleic acid aptamer for recognizing Fusarium, wherein the nucleic acid aptamer is at least one of Fu01, Fu02, and Fu03, and the sequences of the nucleic acid aptamers Fu01, Fu02, and Fu03 are as follows:
[0007] Fu01:
[0008] 5'-TGACACCGTACCTGCTCTAAGCACGCCAGGGACTAT-3'(SEQ ID NO.1)
[0009] Fu02:
[0010] 5'-ATAGTCCCTGGCGTGCTTGGCCAGAGCAGGTACGGTGTCA-3'(SEQ ID NO.2)
[0011] Fu03:
[0012] 5'-TGACACCGTACCTGCTCTACCCAAGCACGCCAGGGACTAT-3' (SEQ ID NO. 3).
[0013] Under the conditions of 25℃, 147mM Na+, and 5mM Mg2+, the secondary structures of the nucleic acid aptamers Fu01, Fu02, and Fu03 are as follows: Figure 1As shown.
[0014] The method for screening nucleic acid aptamers described herein is based on the in vitro Cell-SELEX screening technology for nucleic acid aptamers. Using Fusarium surface proteins as targets, nucleic acid aptamers that specifically bind to Fusarium are screened from a random ssDNA library.
[0015] The method for screening nucleic acid aptamers of Fusarium includes the following steps:
[0016] Fusarium bacteria were incubated with an ssDNA library for reverse selection to remove non-specifically bound ssDNA and enhance aptamer specificity. The eluent was obtained by high-speed centrifugation, the DNA was precipitated, and the DNA concentration was determined.
[0017] The above DNA was subjected to ePCR and purification to obtain the dsDNA required for the next round of screening.
[0018] The obtained dsDNA is used in the next round and all subsequent rounds of screening.
[0019] After ten rounds of screening, the DNA samples were tested. Those that passed were repaired at the ends and fitted with A-tails. Sequencing adapters were ligated to both ends of the fragments, and the samples were purified without PCR amplification. A PCR-free library was prepared. High-throughput paired-end sequencing was performed using the Illumina platform, sequencing 150 bp from each end. Sequences were extracted from the 5' and 3' ends of the target sequence (6 bases each) to obtain the aptamer sequence that specifically binds to Fusarium.
[0020] The 5' or 3' end of at least one of the nucleic acid aptamers Fu01, Fu02, and Fu03 is chemically modified with a fluorescent group, an amino group, biotin, digoxigenin, or polyethylene glycol.
[0021] The 5' end of at least one of the nucleic acid aptamers Fu01, Fu02, and Fu03 is modified with a FAM group.
[0022] The application of the described nucleic acid aptamer in the preparation of detection reagents or kits for Fusarium.
[0023] The application of the described nucleic acid aptamer in the preparation of a drug that inhibits Fusarium-related diseases.
[0024] The application of the described nucleic acid aptamer in establishing a detection method for Fusarium.
[0025] The detection method described is a colorimetric detection method.
[0026] (1) Preparation of gold nanoparticle solution
[0027] Add 1 mL of 1% tetrachloroauric acid solution to 100 mL of distilled water and boil; quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 20 min; continue stirring until naturally cooled to room temperature; a stable gold nanoparticle solution is obtained.
[0028] (2) Preparation of nucleic acid aptamer solution
[0029] The DNA sequence of the nucleic acid aptamer is shown in Fu01; a 100 μM solution of the nucleic acid aptamer was prepared using distilled water.
[0030] (3) Preparation of nucleic acid aptamer-gold nanoparticle colorimetric probe
[0031] The gold nanoparticle solution and the nucleic acid aptamer solution were mixed at a volume ratio of 100:1 and incubated at 37°C for 10 min to obtain the colorimetric probe.
[0032] When the ultraviolet absorption wavelength of gold nanoparticles shifts from 520 nm to 524 nm, it indicates that the nucleic acid aptamer in the colorimetric probe has been successfully adsorbed onto the gold nanoparticles.
[0033] The beneficial technical effects of this invention are reflected in the following aspects:
[0034] 1. Experimental verification shows that, compared with traditional techniques (chain displacement reaction, polymerase chain reaction amplification, and enzyme-linked immunosorbent assay), the latter requires 12 hours to 3 days to detect some common model strains and has specificity cross-problems; while the nucleic acid aptamer-gold nanoparticle colorimetric probe of this invention can complete the detection in only 50 minutes, significantly shortening the detection time. Taking polymerase chain reaction amplification as an example, although it can theoretically amplify the target rapidly, it is extremely sensitive to trace amounts of exogenous DNA in the environment (such as template DNA remaining from previous reactions, nucleic acids carried by operators, and contamination of experimental consumables). Even a very small amount of contamination can be amplified exponentially, leading to false positive results. It requires strict adherence to laboratory zoning (reagent preparation area, sample processing area, amplification analysis area) and anti-contamination operations (such as ultraviolet irradiation and UNG enzyme degradation of old products labeled with dUTP), which significantly increases the complexity and cost of operation.
[0035] 2. Tests have shown that the linear regression equation established under standard conditions can be directly applied to the detection of Fusarium oxysporum in soil without the need to re-culture fungi to establish a standard curve. The established linear regression equation can be applied directly, saving time and making it suitable for quickly estimating the preliminary concentration of Fusarium in common samples under emergency conditions.
[0036] 3. In summary, the Fusarium nucleic acid aptamer prepared in this invention is a novel nucleic acid aptamer. Furthermore, it can be combined with gold nanoparticles to form a colorimetric probe, and verification has confirmed the effectiveness and feasibility of using the Fusarium nucleic acid aptamer prepared in this invention. In the field of nucleic acid aptamers, the number of known effective nucleic acid aptamers is currently limited. The design of the Fusarium nucleic acid aptamer prepared in this invention considers not only the binding rate with Fusarium but also the specific binding rate with other common, potentially coexisting fungi and bacteria.
[0037] Compared with the prior art, the advantages of the present invention are as follows:
[0038] 1. This nucleic acid aptamer is non-toxic, has a small molecular weight, good permeability, and is easy to synthesize and label.
[0039] 2. The synthesis cost of this nucleic acid aptamer is lower than that of antibody preparation, and the cycle is shorter with better reproducibility.
[0040] 3. This nucleic acid aptamer can bind to Fusarium with high affinity and high specificity, and its Kd values are 6.13 nM (FuO1), 3.71 nM (FuO2), and 12.72 nM (FuO3), which shows promise for application in the diagnosis of Fusarium infection.
[0041] 4. This aptamer can be used to monitor the presence of Fusarium in the environment (such as water quality and soil) and food, to prepare Fusarium detection reagents, and as a drug carrier for targeted drug delivery. It has broad application prospects in multiple fields such as agriculture, medicine, food safety, and environmental monitoring, and has important scientific, social and economic value. Attached Figure Description
[0042] Figure 1 This is a secondary structure diagram of the nucleic acid aptamer of the present invention.
[0043] Figure 2 This is a flow cytometry result of the screening library and Fusarium binding in this invention.
[0044] Figure 3 This is a diagram showing the specificity analysis results of the nucleic acid aptamer of this invention.
[0045] Figure 4 This is a diagram of the Cell-SELEX screening strategy for the nucleic acid aptamers of this invention.
[0046] Figure 5 This is a schematic diagram illustrating the application principle of colorimetric detection in this invention.
[0047] Figure 6 This is the UV-Vis absorption spectrum of the nucleic acid aptamer and the gold nanoparticles before and after adsorption, as detected by colorimetry in this invention.
[0048] Figure 7This is the ultraviolet-visible absorption spectrum of different concentrations of Fusarium in this invention, detected by colorimetric assay.
[0049] Figure 8 This is a standard curve diagram of different concentrations of Fusarium detected by colorimetric assay in this invention.
[0050] Figure 9 This is a specific detection diagram of various bacteria using colorimetric detection in this invention. Detailed Implementation
[0051] The present invention will now be described in detail with reference to the embodiments.
[0052] The following detailed description, in conjunction with specific embodiments, illustrates the in vitro screening of Fusarium nucleic acid aptamers and the specificity of the nucleic acid aptamers with Fusarium.
[0053] The relevant reagent formulations involved in the implementation of this invention are as follows:
[0054] (1) PDA solid culture medium (100mL): 2.6g potato dextrose agar,
[0055] (2) PDB liquid culture medium (50mL): 1.3g potato glucose broth.
[0056] The experimental strains involved are as follows:
[0057] Fusarium oxysporum, Mucor circinelloides, Trichoderma viride, Aeromonas veronii, and Bacillus subtilis are all strains preserved in our laboratory.
[0058] The main experimental reagents involved are as follows:
[0059]
[0060] 1) Example 1: In vitro screening of nucleic acid aptamers
[0061] Centrifuge the Fusarium at 10,000 rpm for 2-3 minutes, remove the upper culture medium, wash 1-2 times with 1×SELEX Buffer, and centrifuge again.
[0062] The first round of binding elution and DNA purification: The oligonucleotide library was dissolved in an EP tube using 10×SELEX buffer, and pre-treated Fusarium was added and incubated at room temperature for 1 h. After centrifugation, the supernatant was discarded, and 1×TE buffer was added to the metal bath for 10 min, followed by centrifugation. The resulting supernatant was used as the elution buffer, and the DNA was precipitated and its concentration was measured. A 300 μL oil surfactant mixture was generated (vortexed and stored on ice), and a 50 μL PCR aqueous phase was prepared. These were mixed and vortexed at maximum speed for 5 min, then divided into three replicates of 100 μL / tube. PCR was then performed. The three replicates of ePCR were pooled and vortexed with butanol. Orange-SFX buffer was added to the solution, and the mixture was vortexed for 2 min, followed by centrifugation at 12000 rpm for 2 min for phase separation. The lower aqueous phase was retained and mixed with ethanol. Place the purification column in a receiving tube. Add 30 μL of Buffer SF to the membrane at the center of the bottom of the purification column at room temperature. Transfer the first round of ePCR mixture to the column, up to a maximum of 600 μL each time. Incubate at room temperature for 2 min, centrifuge and discard the waste liquid. Add 600 μL of Wash-SFX buffer, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and reassemble the column. Add another 350 μL of Wash-SFX buffer, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and reassemble the column. After elution, open the cap and incubate for 10 min to allow the alcohol to evaporate completely. Add 50-100 μL of Elution-SFX buffer to the membrane at the center of the purification column, incubate at room temperature and centrifuge, then discard the column. Cap the collection tube to obtain purified DNA. Store at -20℃.
[0063]
[0064] Subsequent rounds of purification and ePCR: Mix 1-3 μg dsDNA with 50-100 μL [10x] SELEX buffer, and add nuclease-free water to a final volume of 500-1000 μL. Add to the treated fungus and incubate at 37°C with gentle shaking for 30 min. Wash twice with SELEX buffer. Incubate with magnetic beads in 2% [w / v] SDS at 70-94°C. Quickly transfer the supernatant without magnetic beads to a new reaction tube. Precipitate the DNA and determine the DNA concentration. Perform ePCR and DNA purification.
[0065] After 10 rounds of screening, the final product was obtained. After the samples passed inspection, end repair and A-tailing were added. Sequencing adapters were ligated to both ends of the fragment, and the fragment was purified without PCR amplification. A PCR-free library was prepared. High-throughput paired-end sequencing was performed using the Illumina platform, sequencing 150 bp from each end. Sequences were extracted from the 5' and 3' ends of the target sequence (6 bases each) to obtain the nucleic acid aptamer sequence that specifically binds to Fusarium.
[0066] The 5' end of the nucleic acid aptamer Fu01 was modified with a FAM fluorescent group. The nucleic acid aptamer was synthesized by Shanghai Sangon Biotech Co., Ltd.
[0067] To evaluate the specificity of the nucleic acid aptamer for *Fusarium oxysporum*, four other common pathogenic bacteria were selected. Suspensions of *Fusarium oxysporum*, *Bacillus subtilis*, *Aeromonas hydrophila*, *Trichoderma viride*, and *Mucor* at different concentrations were prepared and incubated with nucleic acid aptamers modified with a FAM fluorescent group at their 5' ends. Fluorescence signals were measured, and the fluorescence quenching rate for each bacterium was calculated. Results are as follows: Figure 3 As shown, the fluorescence quenching rate of Fusarium oxysporum is significantly higher than that of other bacteria, indicating that the nucleic acid aptamer Fu01 of the present invention has an advantage in Fusarium recognition and can recognize fungal targets through its unique structure.
[0068] Example 2 describes the steps for using Fu01 to prepare a colorimetric probe:
[0069] 1) Preparation of gold nanoparticle solution
[0070] Add 12 μL of 1% tetrachloroauric acid solution to 100 mL of distilled water and boil; quickly add 2.5 mL of 1% trisodium citrate aqueous solution and continue boiling for 20 min; continue stirring until naturally cooled to room temperature; a stable gold nanoparticle solution is obtained.
[0071] 2) Preparation of Fusarium nucleic acid aptamer solution
[0072] The DNA sequence of the nucleic acid aptamer is shown in Fu01; a 100 μM solution of the nucleic acid aptamer was prepared using distilled water.
[0073] 3) Preparation of nucleic acid aptamer-gold nanoparticle colorimetric probes
[0074] The gold nanoparticle solution and the nucleic acid aptamer solution were mixed at a volume ratio of 100:1 and incubated at room temperature for 20 min to obtain a colorimetric probe for further use.
[0075] Fusarium cultured to the logarithmic growth phase was filtered through a filter to obtain Fusarium cells. These cells were then ground with liquid nitrogen, weighed in an EP tube, and mixed with sterile water. The cells were then broken up using an ultrasonic cell disruptor to ensure uniform distribution of Fusarium cells in the liquid, thus preparing a 0.01 g / mL bacterial test solution.
[0076] The prepared gold nanoparticle solution was subjected to ultraviolet absorption spectroscopy with a colorimetric probe. (See [link to relevant documentation]). Figure 6 The black curve represents the UV absorption spectrum of Au NPs without Fusarium aptamer binding, while the red curve represents the UV absorption spectrum of a mixed solution of Au NPs and Fusarium aptamer binding. A shift in the UV absorption wavelength of Au NPs from 520 nm to 524 nm indicates successful modification of the gold nanoparticles with the Fusarium aptamer.
[0077] Meanwhile, different concentrations of Fusarium oxysporum were incubated with the colorimetric probe at room temperature for 10 min to obtain their ultraviolet absorption spectrum curves, see [link to relevant documentation]. Figure 7 A standard curve was plotted by taking the relative UV absorption intensity at 524 nm as the ordinate of each UV absorption spectrum curve and the concentration of *Fusarium oxysporum* as the abscissa. (See [reference needed]). Figure 8 The regression equation is y = 0.1652x + 0.9063, and the correlation coefficient (R2) is 0.977.
[0078] Next, a colorimetric reaction will be performed:
[0079] The gold nanoparticle solution and nucleic acid aptamer solution were mixed at a volume ratio of 100:1 (total volume 101 μL) and incubated at room temperature for 20 min. Then, 20 μL of a Fusarium oxysporum (0.01 g / mL) test solution containing bacteria was added, and the mixture was incubated for another 10 min, with the solution color recorded. Finally, 9 μL of a 500 mM NaCl solution was added, and the solution color was recorded after 30 s. The results are as follows: Figure 9 As shown in (A, a), the solution changes from light red to blue-purple.
[0080] Next, *Fusarium oxysporum* was replaced with *Mucor*, *Trichoderma viride*, *Aeromonas vesiculosus*, and *Bacillus subtilis*, respectively, and 0.01 g / mL bacterial test solutions were prepared for each. These solutions underwent the same treatment and colorimetric reactions. The results are as follows: Figure 9 As shown, only after adding Fusarium oxysporum did the solution change from light red to blue-purple, while adding other bacteria did not cause any color change.
[0081] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.
Claims
1. A nucleic acid aptamer for recognizing Fusarium, characterized in that, The nucleic acid aptamer is at least one of Fu01, Fu02, and Fu03, and the sequences of the nucleic acid aptamers Fu01, Fu02, and Fu03 are as follows: Fu01: 5'-TGACACCGTACCTGCTCTAAGCACGCCAGGGACTAT-3'(SEQ ID NO.1) Fu02: 5'-ATAGTCCCTGGCGTGCTTGGCCAGAGCAGGTACGGTGTCA-3'(SEQ ID NO.2) Fu03: 5'-TGACACCGTACCTGCTCTACCCAAGCACGCCAGGGACTAT-3' (SEQ ID NO. 3).
2. A screening method for identifying nucleic acid aptamers of Fusarium, characterized in that, The Cell-SELEX screening technology based on nucleic acid aptamers targets Fusarium surface proteins and screens nucleic acid aptamers that specifically bind to Fusarium from a random ssDNA library; specifically, it includes the following steps: Fusarium spp. was incubated with an ssDNA library for reverse selection to remove non-specifically bound ssDNA and enhance aptamer specificity. The eluent was obtained by high-speed centrifugation, DNA was precipitated and its concentration was determined, and the DNA was subjected to ePCR and purification to obtain the dsDNA required for the next step. After the DNA sample was tested and qualified, end repair and A-tailing were added, and sequencing adapters were ligated to both ends of the fragment and purified. No PCR amplification was performed in between to prepare a PCR-free library. High-throughput paired-end sequencing was performed using the Illumina platform, sequencing 150 bp from each end. Sequences were extracted from the 5' and 3' ends of the target sequence (6 bases each) to obtain the nucleic acid aptamer sequence that specifically binds to Fusarium spp.
3. The nucleic acid aptamer for recognizing Fusarium according to claim 1, characterized in that, The 5' end of at least one of the nucleic acid aptamers Fu01, Fu02, and Fu03 is modified with a FAM group.
4. The nucleic acid aptamer for recognizing Fusarium according to claim 1, characterized in that, The application of the described nucleic acid aptamer in the preparation of detection reagents or kits for Fusarium.
5. The nucleic acid aptamer for recognizing Fusarium according to claim 1, characterized in that, The application of the described nucleic acid aptamer in the preparation of a drug that inhibits Fusarium-related diseases.
6. The nucleic acid aptamer for recognizing Fusarium according to claim 1, characterized in that, The application of the described nucleic acid aptamer in establishing a detection method for Fusarium.
7. The nucleic acid aptamer for recognizing Fusarium according to claim 1, characterized in that, The 5' end of at least one of the nucleic acid aptamers Fu01, Fu02, and Fu03 is modified with a FAM group.
8. The nucleic acid aptamer for recognizing Fusarium according to claim 6, characterized in that, The detection method is a colorimetric method; the gold nanoparticle solution and the nucleic acid aptamer solution are mixed at a volume ratio of 100:1, with a total volume of 101 μL, and incubated at room temperature for 20 min. Then, 20 μL of the test solution containing Fusarium bacteria with a concentration of 0.01 g / mL is added, and the mixture is incubated for a total of 10 min. The color of the solution is recorded. Add 9 μL of 500 mM NaCl solution, and record the solution color after 30 seconds: it changes from light red to blue-purple. Next, *Fusarium oxysporum* was replaced with *Mucor*, *Trichoderma viride*, *Aeromonas vesiculosus*, and *Bacillus subtilis*, respectively, and test solutions containing the bacteria at a concentration of 0.01 g / mL were prepared. These solutions underwent the same treatment and colorimetric reactions. The results are shown in Figure 9. Only after adding *Fusarium oxysporum* did the solution change from light red to blue-purple; adding other bacteria did not cause any color change.