Method for preventing and treating root rot of cardamine violifolia

Abstract

The invention belongs to the technical field of plant disease control, and particularly relates to a method for controlling root rot of cardamine violifolia. The technical problem to be solved by the invention is to clarify the pathogenic bacteria of cardamine violifolia and provide a new choice for preventing and treating cardamine violifolia root rot. The invention also provides a method for preventing and treating the root rot of cardamine violifolia. The method comprises the following step: treating the cardamine violifolia by adopting at least one of hymexazol (T1), oxaconazole (T2) and propiconazole (T9). According to the application, the key pathogenic bacteria of the root rot of the Enshi cardamine violifolia are determined to be Aspergillus Goddavidii (A2), Mucor circinelloides Luusitanius variant (A3) and Blumeria tuberosa (A6) for the first time. According to the invention, bactericides, namely propiconazole, hymexazol and oxadiazole, which have a remarkable inhibition effect on three pathogenic bacteria are screened from nine conventional bactericides, and the mixed use effect of propiconazole, hymexazol and oxadiazole is better; a new scheme is provided for preventing and treating the root rot of cardamine violifolia.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant disease control, and particularly relates to a method for preventing and treating root rot of Cardamine violifolia. BACKGROUND

[0002] Cardamine violifolia is a cruciferous herb with the functions of food and medicine. It has super selenium accumulation ability in Enshi, Hubei, and has become a research hotspot in the fields of agriculture and food. The selenium content of wild Cardamine violifolia can reach 1000 mg kg -1 DW, and the selenium content of artificially cultivated Cardamine violifolia is close to 10000 mg kg -1 DW. The soil selenium enrichment coefficient is as high as 68 times. The protein content of leaf and seed is 17.8% and 17.5% respectively, which is significantly higher than that of common vegetables such as lettuce and Chinese cabbage. Cardamine violifolia has the functions of selenium supplementation and nutrition, and has great application potential in the fields of organic selenium extraction and functional vegetable development. At present, the plant has been artificially cultivated on a large scale, but the disease problem has gradually become a key factor restricting the development of the industry.

[0003] Root rot is a major soil-borne disease in the large-scale cultivation of Cardamine violifolia. It occurs frequently in spring and summer with high temperature and high humidity, and is more serious in low-lying waterlogged and heavy soil plots. Root rot is usually caused by the synergistic infection of multiple pathogenic fungi. Fungi are the core pathogenic group, and common pathogenic fungi include Rhizoctonia, Fusarium and Phoma. After the pathogenic fungi invade through the root wound, they can cause vascular necrosis and root rot, and further cause wilting of the aboveground part and even whole plant death, which seriously affects the growth and yield of the plant.

[0004] Bactericides are widely used in the prevention and treatment of crop diseases and pests and the protection of crop growth due to their convenience and effectiveness. In a broad sense, bactericides can cover all agents that can inhibit pathogenic microorganisms; and in a narrow sense, they mainly refer to agents for killing fungi. In the chemical control of Fusarium root rot, commonly used agents include chlorothalonil, carbendazim, diclomezine, iprobenfos, chloropicrin, myclobutanil, hymexazol, etc. These are mostly chemical agents. Although these bactericides have ideal effects on the prevention and treatment of root rot, environmental pollution, enhanced pathogen resistance and toxicity residues have gradually become prominent with the overuse of chemical bactericides. Long-term single use of a certain agent has led to resistance risk in some production areas, so it is urgent to optimize the dosage and improve the application strategy to delay the development of resistance. In addition, as a potential functional food raw material, the residual amount of water celery is very strict, which limits the use of traditional agents. Under such circumstances, screening of new bactericides with high efficiency, low toxicity and environmental friendliness, or development of targeted agents based on the biological characteristics of pathogenic fungi, is of great importance to the sustainable development of the water celery industry. SUMMARY

[0005] The technical problem to be solved by the present application is to identify the pathogenic bacteria of P. cordata, and to provide a new option for preventing and treating root rot of P. cordata.

[0006] The technical solution of the present application is the application of a substance that inhibits Costa Rica Aspergillus, Mucor Lusitanicus variant and / or B. berninum Fusarium in the prevention and treatment of root rot of P. cordata.

[0007] Specifically, the substance is at least one of propiconazole, hymexazol and difenoconazole.

[0008] The concentration of propiconazole is 0.625-50 mg / L.

[0009] Preferably, the concentration of propiconazole is 2.5-40 mg / L.

[0010] More preferably, the concentration of propiconazole is 10-40 mg / L.

[0011] Most preferably, the concentration of propiconazole is 30 mg / L.

[0012] Further, the concentration of hymexazol is 10-50 mg / L.

[0013] Preferably, the concentration of hymexazol is 20-40 mg / L.

[0014] More preferably, the concentration of hymexazol is 30 mg / L.

[0015] Particularly, the concentration of the uniconazole is 10-50 mg / L.

[0016] Preferably, the concentration of the uniconazole is 20-40 mg / L.

[0017] More preferably, the concentration of the uniconazole is 30 mg / L.

[0018] The application also provides a method for preventing and treating root rot of Cardamine hirsuta, comprising the following steps: treating Cardamine hirsuta with at least one of propiconazole, hymexazol and uniconazole.

[0019] Specifically, the pathogenic bacteria of the root rot are Costa Rica aspergillus, Lusitanicus variant of Mucor circinelloides and / or Fusarium bactridioides.

[0020] Preferably, the concentration of the propiconazole is 2.5-40 mg / L.

[0021] Preferably, the concentration of the propiconazole is 2.5-40 mg / L.

[0022] More preferably, the concentration of the propiconazole is 10-40 mg / L.

[0023] Most preferably, the concentration of the propiconazole is 30 mg / L.

[0024] Further, the concentration of the hymexazol is 10-50 mg / L.

[0025] Preferably, the concentration of the hymexazol is 20-40 mg / L.

[0026] More preferably, the concentration of the hymexazol is 30 mg / L.

[0027] Particularly, the concentration of the uniconazole is 10-50 mg / L.

[0028] Preferably, the concentration of the uniconazole is 20-40 mg / L.

[0029] More preferably, the concentration of the uniconazole is 30 mg / L.

[0030] The application also provides a fungicide comprising at least one of propiconazole, hymexazol and uniconazole.

[0031] Preferably, the concentration of the propiconazole is 2.5-40 mg / L.

[0032] Preferably, the concentration of the propiconazole is 2.5-40 mg / L.

[0033] More preferably, the concentration of the propiconazole is 10-40 mg / L.

[0034] Most preferably, the concentration of propiconazole is 30 mg / L.

[0035] Further, the concentration of hymexazol is 10-50 mg / L.

[0036] Preferably, the concentration of hymexazol is 20-40 mg / L.

[0037] More preferably, the concentration of hymexazol is 30 mg / L.

[0038] In particular, the concentration of oxpoconazole is 10-50 mg / L.

[0039] Preferably, the concentration of oxpoconazole is 20-40 mg / L.

[0040] More preferably, the concentration of oxpoconazole is 30 mg / L.

[0041] Advantages of the present application: The present application first determines that the key pathogenic bacteria of Enshi Steller's root rot are Costa Rica Aspergillus (A2), Lusitanicus variant of Mucor circinelloides (A3) and Bemba Fusarium (A6). The present application also screens three fungicides, propiconazole, hymexazol and oxpoconazole, from nine conventional fungicides, which have a significant inhibitory effect on the three pathogenic bacteria. In particular, the fungicidal effect of propiconazole is the best, and the half maximal effect concentration (EC50) of propiconazole fungicide on Costa Rica Aspergillus (A2) is 21.5214 mg / L; the half maximal effect concentration (EC50) on Lusitanicus variant of Mucor circinelloides (A3) is 55.0963 mg / L; and the half maximal effect concentration (EC50) on Bemba Fusarium (A6) is 2.2504 mg / L.

[0042] Further experiments found that the inhibitory effect of the combination of propiconazole, hymexazol and oxpoconazole on the three pathogenic bacteria is significantly better than that of single agent. The half maximal effect concentration (EC50) of each fungicide in the combination of propiconazole, hymexazol and oxpoconazole on Costa Rica Aspergillus (A2) is 7.313 mg / L; the half maximal effect concentration (EC50) on Lusitanicus variant of Mucor circinelloides (A3) is 12.2983 mg / L; and the half maximal effect concentration (EC50) on Bemba Fusarium (A6) is 0.1781 mg / L. Among them, the inhibition rate is increased by 62.62%, 77.53% and 20.85% compared with the use of propiconazole alone at a low concentration of 10 mg / L.

[0043] And, the present application also makes it clear that the medicament plays a role by damaging the cell membrane, which can provide theoretical and technical support for the precise prevention and control of the root rot of the C. stelleriana and the production of healthy seedlings. Based on the foregoing results, the present application also provides a method for preventing and treating the root rot of the C. stelleriana. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 It is a symptom chart of the root rot of the C. stelleriana, and the scale is 1 cm.

[0045] Figure 2 It is a colony morphological characteristic of different root rot strains.

[0046] Figure 3 It is a pre-experiment (14 d) of the stab wound in the pathogenicity detection of the root rot, and the scale is 1 mm. The red dotted line frame indicates the disease site.

[0047] Figure 4 It is a pathogenicity verification of the root rot and a mycelium and spore morphological chart of the root rot fungus. A-H are the disease conditions of the C. stelleriana infected by the Costa Rica Aspergillus (A2), the Lusitanicus variant of Mucor circinelloides (A3) and the Fusarium bactridioides (A6), and the scale is 2 mm (D and H); I-K are the mycelium and spore morphological charts of the pathogenic fungi taken by the inverted fluorescence microscope, and the scale is 20 μm; L-Q are the mycelium morphological charts of the pathogenic fungi taken by the scanning electron microscope, and the scale is 50 μm.

[0048] Figure 5 It is a phylogenetic tree constructed based on the rDNA ITS-RPB2 double sequences.

[0049] Figure 6 It is the primary screening of different fungicides. A, B: the inhibitory effect of different fungicides on the mycelium of the A6 pathogenic fungus, and different lowercase letters indicate significant differences (P<0.05); C: the mycelium growth of the pathogenic fungus after 5 d under the treatment of different fungicides, and the scale is 1 cm.

[0050] Figure 7 It is the influence of different concentrations of propiconazole, hymexazol and difenoconazole on the mycelium growth diameters of the three pathogenic fungi.

[0051] Figure 8 It is the secondary screening of different fungicides. A: the mycelium growth of different pathogenic fungi after 5 d under the treatment of T1, T2 and T9 fungicides, and the scale is 1 cm; B: the influence of T1, T2 and T9 fungicides on the colony diameters of the pathogenic fungi; C: the inhibitory effect of T1, T2 and T9 fungicides on the mycelium of the pathogenic fungi, and different lowercase letters in the same column indicate significant differences (P<0.05).

[0052] Figure 9Toxicity of different concentrations of the fungicide to the pathogen. A: Mycelial growth of different pathogens after 5 days of treatment with T10 fungicide. Scale bar, 1 cm; B: Effect of T10 fungicide on the colony diameter of the pathogen; C: Inhibition of T10 fungicide on the mycelium of the pathogen. Different lowercase letters in the same column indicate significant differences (P < 0.05).

[0053] Figure 10 Effect of different fungicides on the cell membrane damage of the pathogen of root rot.

[0054] Figure 11 Effect of the fungicide on the cell membrane damage of the pathogen of root rot. DETAILED DESCRIPTION

[0055] The medium used in the following examples is a potato dextrose broth (PDB) medium (g / L). The formula is as follows: PDB powder 26 g, tertiary water 1000 mL, pH value 5.3±0.2. The potato dextrose agar (PDA) medium is prepared by adding 15 g / L agar to the potato dextrose broth medium.

[0056] The following examples use Excel 2019 software to process experimental data, and GraphPad Prism 10 software to analyze and plot experimental data, P<0.05 significant difference.

[0057] Root rot is a major soil-borne disease in the large-scale cultivation of P. delavayi, and it occurs more frequently in spring and summer with high temperature and humidity, and it is more severe in low-lying waterlogged and heavy soil plots. Root rot is usually caused by multiple pathogenic fungi, with fungi being the core pathogenic group. After the pathogen invades through the root wound, it can cause vascular necrosis and root rot, and even cause the aboveground part to wither and even the whole plant to die, which seriously affects plant growth and yield. In this application, 9 strains of fungi were purified from 8 root rot samples by tissue isolation method, combined with morphological observation, Koch's postulate pathogenicity verification and rDNA ITS-RPB2 double sequence molecular identification, it was first determined that Aspergillus costaricensis (A2), Mucor circinelloides f. lusitanicus (A3) and Fusarium pernambucanum (A6) were the key pathogenic fungi causing root rot of P. delavayi in this region. Among them, the spore stalks of A2 and A3 strains are arranged in a wheat ear shape, and the conidia are spherical and grape-like, and the conidia of A6 strain are arch-shaped and sickle-shaped.

[0058] Through the primary screening and rescreening of 9 conventional fungicides, it was found that there were obvious differences in the inhibition effect of single fungicide on the three pathogenic fungi: propiconazole (T9) had the best inhibition effect on A6 (EC50=2.2504 mg / L), but weaker inhibition effect on A2 (EC50=21.5214 mg / L) and A3 (EC50=55.0963 mg / L); hymexazol (T1) and oxathiaplat (T2) only showed good inhibition effect on A6 (EC50 were 5.7328 mg / L and 17.9445 mg / L, respectively), and had no significant inhibition effect on A2 and A3. Based on this, propiconazole, hymexazol and oxathiaplat were compounded to form a new fungicide T10, and the EC50 of T10 on A2, A3 and A6 were reduced to 7.313 mg / L, 12.2983 mg / L and 0.1781 mg / L, respectively. Compared with the use of propiconazole alone, the inhibition rate of T10 at a low concentration of 10 mg / L was increased by 62.62%, 77.53% and 20.85% respectively, and the inhibition effect was significantly better than that of single fungicide, achieving broad-spectrum and efficient inhibition of the three pathogenic fungi.

[0059] Cell membrane is an important place for material exchange and has a protective effect, so propidium iodide (PI) cannot normally penetrate the intact cell membrane. However, after treatment with fungicides, PI can enter the cell membrane of damaged or dead cells and embed in double-stranded DNA, emitting red fluorescence under a fluorescence microscope, and the fluorescence intensity reflects the degree of cell membrane damage. Therefore, the roots of the three pathogenic fungi of root rot were treated with different concentrations of fungicides, and then stained with PI solution and observed under a fluorescence microscope. The results of PI fluorescence staining showed that T1, T2, T9 and the compounded fungicide T10 all played an inhibitory role by damaging the cell membrane integrity of the pathogenic fungi. The mycelium treated with the fungicides showed obvious red fluorescence under the fluorescence microscope, and the fluorescence intensity increased with the increase of the concentration of the fungicides, confirming that cell membrane damage was the core mechanism of the fungicides to inhibit the growth of the pathogenic fungi.

[0060] Example 1 Isolation and purification of root rot fungi of C. crenata

[0061] Pteroxygonum giraldii infected by root rot disease was collected from Enshi, Hubei. Pteroxygonum giraldii root rot disease is a typical soil-borne disease, which frequently occurs in spring and summer in southern regions. During this period, rain is frequent, air humidity increases significantly, and temperature also gradually rises. These environmental factors provide very favorable conditions for the growth, reproduction, and transmission of the pathogen. In a high-humidity and high-temperature environment, the pathogen mainly invades through the wounds of the plant roots, thus initiating the process of pathogenesis. After the plant is infected, the young root parts usually exhibit a waterlogged brown appearance at the early stage of the disease. Subsequently, the epidermis and xylem of the root gradually turn black, and the pathogen continuously spreads and spreads upwards. As the disease progresses, the epidermis of the root appears to be soft and rotten, and under high-humidity conditions, a small amount of grayish-white mold can be clearly observed attached to it. In the later stages, the vascular bundle of the root is blocked, the color turns black, the root system loses vitality, and its absorption capacity is severely weakened or even completely lost; the color of the root-stem junction turns brown or even black, and the phloem and xylem of the diseased part turn black-brown. Due to the invasion of the pathogen at the root-stem junction, water and nutrients cannot be normally absorbed and transported, the leaves of the aboveground part of the plant begin to turn yellow, the degree of yellowing gradually increases, and the leaves start to fall off. In severe cases, the whole plant wilts and dies. Figure 1 ).

[0062] The root rot infected plant samples were first washed with clean water to remove the surface soil, and then about 1 cm long tissues at the diseased and healthy interface were cut. In a sterile operation table, the tissues were sequentially immersed in 75% alcohol for 30 s, 10% hydrogen peroxide for 30 s, and washed with sterile water for 3 times. Then, the surface water of the root was absorbed with sterile filter paper, the diseased part was cut with a sterile scalpel, and the cut was placed on PDA medium added with cefotaxime (concentration 0.1%), and cultured in an inverted culture box at (28±0.5)℃ for 5-7 days. When different morphological colonies grew on the plate, different morphological colonies were purified. The mycelium at the edge of different morphological colonies was picked with a sterile inoculation needle and placed on a new culture medium and cultured in a constant temperature incubator at (28±0.5)℃. After 3 subcultures, part of the strains were used for experiments, and the other part was preserved by modified test tube slant method and glycerol method. Test tube slant method: add appropriate amount of PDA in the test tube, place it obliquely, and wait for the PDA to solidify into a slant (the highest point of the slant does not exceed 1 / 2 of the height of the test tube), inoculate the slant with a bacterial cake, and store it at 4℃ after the strain is full. Glycerol method: the mycelium and spores are preserved in 15%-20% glycerol. The specific operation is as follows: 100 μL of PDA liquid medium is taken to the fungus culture dish with a pipette gun, the inoculation shovel is sterilized by flame and cooled, then the mycelium and spores are scraped, 500 μL of glycerol is taken to transfer the mycelium and spores to a 2 mL sterile centrifuge tube, stirred evenly to make the final concentration of glycerol 15%-20%, and finally stored at -60℃ for standby.

[0063] The pathogenic fungi separated and purified were punched with a sterile gun head (φ=6 mm) and re-inoculated to new PDA medium in the form of bacterial cake, and the growth conditions, colony color, morphological characteristics, etc. of the pathogenic fungi were observed and recorded by taking pictures after incubation at (28±0.5)℃ for 7 days. The mycelium of the pathogenic bacteria was photographed by scanning electron microscope. In the experiment, 8 root rot symptom samples of P. decursivum were collected, and 9 fungal strains were obtained after isolation and purification. In PDA medium, there were obvious differences in the morphology of different strain samples, the aerial mycelium of the isolated colonies was in the form of villi or thin villi, the colony surface color was white or beige, and the colony back was mostly colorless (Figure 2).

[0064] Example 2 Pathogenicity test of pathogenic fungi

[0065] 1. Pathogenicity pre-experiment and determination

[0066] According to Angel's wound inoculation method, and with appropriate improvement, the pathogenicity pre-experiment and subsequent pathogenicity determination were carried out. Healthy P. decursivum seedlings were selected as plant materials, and the isolated fungi were subjected to in vivo back-inoculation test.

[0067] Wounding pre-experiment: Firstly, the preserved purified strains were reactivated and subcultured. The strains were reactivated and subcultured in a (28±0.5) °C incubator and cultured in a dark environment at (28±0.5) °C for 7 days. Subsequently, the base of the stems and roots of healthy G. punctata seedlings that had germinated for 14 days was wounded with a toothpick. Two wounding points were set for each plant. A sterile gun head with a diameter of 6 mm was used to take a bacterial cake from the culture medium, and a clean toothpick was used to move the bacterial cake to the wound site. Then, the wound site was wrapped with a wet cotton ball, and finally, it was wrapped with sticky plastic wrap to keep it moist. Three seedlings inoculated with each fungus were used as a treatment group, and all the isolated fungi were inoculated. Plants inoculated with clean PDA cakes were used as controls, and the moisture retention method was the same as that of the treatment group. After 14 days, the incidence of disease in the treatment and control groups was observed and recorded.

[0068] Root inoculation with fungus block pre-experiment: A sterile gun head with a diameter of 6 mm was used to take a bacterial cake, and the roots of healthy G. punctata seedlings were placed on the bacterial cake. Finally, the roots were kept moist with wet filter paper. Three seedlings inoculated with each fungus were used as a treatment group, and all the isolated fungi were inoculated. Plants inoculated with clean PDA cakes were used as controls, and the moisture retention method was the same as that of the treatment group. After 12 days, the incidence of disease in the treatment and control groups was observed and recorded.

[0069] The results of the pathogenicity determination of the strain pre-experiment are as follows: Wounding pre-experiment Figure 3 The results showed that after inoculation with strains A2, A3, and A6, the stems and roots of G. punctata showed blackening and root breakage, while the other strains showed no significant changes compared to the CK. Therefore, strains A2, A3, and A6 were selected for subsequent root irrigation pathogenicity experiments (results see Figure 4 A-H).

[0070] 2. Pathogenicity determination

[0071] Pot experiments were conducted to determine the pathogenicity of each strain according to the Koch's postulates. Healthy seedlings of two different varieties of G. punctata and G. plicata were cultivated as plant materials. The five root rot isolates that showed obvious disease in the pre-experiment were cultured on PDA medium at (28±0.5) °C for 48 h. Single colonies were picked and inoculated into 50 mL of PDA liquid medium, which was incubated at 28 °C and 160 r / min for 48 h. The bacterial suspension was centrifuged at 4000 r / min for 15 min, and the bacterial pellet was diluted with deionized water to 10 6 CFU / mL to prepare a bacterial suspension. The bacterial suspension was irrigated into normally growing potted plants, and the plants were normally managed and cultured for 7 and 14 days before root excavation for disease investigation.

[0072] As Figure 4 A-D and Figure 4As shown in E-H, after 7 d and 14 d of the experiment, the A2, A3 and A6 pathogenic bacteria treatment of the P. cordata neck has obvious blackening and breaking phenomenon. According to the Koch's rule, it is clear that the three strains are the pathogenic bacteria causing the root rot of P. cordata.

[0073] The mycelium and spore morphology of the three pathogenic bacteria were observed under an inverted microscope and a scanning electron microscope, and the results are shown in Figure 4 I-Q.

[0074] The A2 pathogenic bacteria has white mycelium at the initial stage, which gradually changes to cream to light yellow, with velvet texture and sparse spore formation; the conidial head is radial, the conidial phialide is short and smooth, the top is swollen to form a spherical or subspherical vesicle, the vesicle surface is covered with double-layer small phialides, the conidia are chain-shaped, spherical, with small spines on the surface, and the color is gray-green to black-brown; according to its morphological characteristics and referring to the Fungal Classification Manual, it is preliminarily identified as Aspergillus.

[0075] The A3 pathogenic bacteria has white velvet mycelium at the initial stage, grows rapidly, gradually changes to yellow-brown, and has cotton-like texture, and can form black spore sacs at the later stage; the mycelium is non-septate, has multiple branches, and has winding property; the spore sac is spherical, breaks to release spore sac spores after maturation, the spore sac spores are elliptical or subspherical, the surface is smooth, and the color is gray-brown to dark brown; under a microscope, typical winding structures and a large number of spore sac spores can be observed, the spore sac spore phialides are like wheat spikes and arranged in an ellipse, and the conidia are arranged in spherical grape-like shape. According to its morphological characteristics and referring to the Fungal Classification Manual, it is preliminarily identified as Mucor.

[0076] The A6 pathogenic bacteria has white velvet mycelium at the initial stage, which gradually changes to white rice color, and then to yellow-green white interlaced color at the later stage; under a microscope, it is found that the conidia are in the shape of arch-shaped sickle. According to its morphological characteristics and referring to the Fungal Classification Manual, it is preliminarily identified as Fusarium spp.

[0077] Example 3: Molecular identification of the strains

[0078] The genomic DNA of the root rot strain was extracted by CTAB method and stored at -20°C for standby. The rDNA ITS sequence region was amplified by using universal primers ITS1 and ITS4 for fungal identification. The PCR reaction conditions were as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 48°C annealing for 40 s, 72°C extension for 1 min, a total of 35 cycles; finally 72°C extension for 10 min. The primers fRPB2-5f and fRPB2-7cR (Hatvani L, Homa M, Chenthamara K, Cai F, Kocsubé S, Atanasova L, Mlinaric-Missoni E, Manikandan P, Revathi R, Dóczi I, et al. Agricultural systems as potential sources of emerging human mycoses caused by Trichoderma: a successful, common phylotype of Trichoderma longibrachiatum in the frontline [J]. FEMS Microbiology Letters, 2019, 366(21): fnz246) were used for amplification of the RPB2 sequence region, and the PCR reaction conditions were as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 1 min, 55°C annealing for 90 s, 72°C extension for 90 s, a total of 35 cycles; finally 72°C extension for 7 min. After the amplification products were stored at 4°C for a short time, they were sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing. The measured gene sequences were uploaded to the National Microbial Science Data Center NMDC (http: / / nmdc.cn / ) and the sequence number was obtained, and the BLAST function of the NCBI website was used for comparison, and sequences with a similarity of more than 95% were downloaded. Subsequently, the MEGA12 software was used to construct a phylogenetic tree by using the Neighbor-Joining method.

[0079] The rDNA ITS sequence and the RPB2 sequence obtained by sequencing were connected head to tail to construct a phylogenetic tree, and the phylogenetic tree (Fig. 1) was obtained by using the Neighbor-Joining method. Figure 5As shown in Table 1, strains A4, A5 and A6 have high homology with Fusarium pernambucanum (reference sequence GenBank accession number: URM 7599), the similarity is as high as 99%, the coverage of the reference sequence in GeneBank is 100%, and the comparison percentage is 99.54%, 96.54% and 96.54% respectively. In combination with the morphological characteristics, it is identified as Fusarium pernambucanum; strains A1, A3, A8 and A9 have high homology with Mucor circinelloides f. lusitanicus (reference sequence GenBank accession number: CBS 108.17), the similarity is as high as 98%, the coverage of the reference sequence in GeneBank is 100%, 100%, 100% and 99% respectively, and the comparison percentage is 98.03%, 99.88%, 99.54% and 99.88% respectively. In combination with the morphological characteristics, it is identified as Mucor circinelloides f. lusitanicus. Strain A7 has high homology with Fusarium luffae (reference sequence GenBank accession number: CGMCC3.3.19497), the similarity is as high as 97%, the coverage of the reference sequence in GeneBank is 98%, and the comparison percentage is 95.87% respectively. In combination with the morphological characteristics, it is identified as Fusarium luffae. Strain A2 has high homology with Aspergillus costaricensis (reference sequence GenBank accession number: CBS 115574), the similarity is as high as 99%, the coverage of the reference sequence in GeneBank is 100%, and the comparison percentage is 96.55% respectively. In combination with the morphological characteristics, it is identified as Aspergillus costaricensis. In combination with the morphological and molecular biological characteristics, the 9 strains are identified as 4 species: A4, A5 and A6 belong to Fusarium pernambucanum; A1, A3, A8 and A9 are Mucor circinelloides f. lusitanicus, A7 belongs to Fusarium luffae, and A2 is Aspergillus costaricensis.

[0080] Table 1 BLAST comparison information list of Pteridium aquilinum root rot pathogen isolates and GenBank reference sequences

[0081] .

[0082] Example 4: Virulence test of different bactericides against pathogens

[0083] 1. Initial screening of bactericides

[0084] Based on preliminary pathogenicity testing and identification results, pathogen A6 is more virulent than the other two fungi. Fusarium spp. is currently the most prevalent pathogenic group for root rot in many crops in my country and has been identified as the dominant pathogen in the vast majority of surveys. Therefore, fungicides were screened based on pathogen A6 as the primary pathogen.

[0085] Based on the identification results and pathogenicity tests, nine commonly used fungicides (Table 2) were selected: hymexazol (T1), oxadixyl (T2), prochloraz (T3), metalaxyl-mancozeb (T4), chlorothalonil (T5), carbendazim (T6), metronidazole (T7), kasugamycin-quinoline copper (T8), and propiconazole (T9) for virulence testing against A6 pathogens. According to the active ingredient of each fungicide, the corresponding mass of the fungicide was accurately weighed and dissolved in sterile water to prepare a 5000 mg / L stock solution for later use. Then, 0.5 mL of the stock solution was added to 49.5 mL of PDA medium to prepare drug-containing medium with a concentration of 50 mg / L. A drug-free PDA medium was used as a control group, and each treatment was repeated in triplicate. Using a 6 mm diameter sterile pipette tip, holes were punched at the edge of colonies that had grown for 5 days to obtain mycelial cakes, which were then inoculated into the center of each medium. After culturing for 5 days in the dark at 28°C, the colony diameter was measured using the cross-cross method, and the inhibition rate was calculated.

[0086] The formula for calculating the inhibition rate (%) is: [(CK colony diameter - treated colony diameter) / (CK colony diameter)] × 100%.

[0087] Table 2 Types of bactericides

[0088] .

[0089] Nine fungicides were initially screened (for A6 pathogenic bacteria) using the mycelial growth rate method, and the results are as follows: Figure 6 As shown, at a fungicide concentration of 50 mg / L, the colony diameters treated with fungicides T1, T2, T3, T8, and T9 showed significant differences compared to the control, while other fungicide treatments showed no significant differences compared to the control. The T9 treatment achieved an inhibition rate of 93.99%, indicating the best inhibitory effect against A6 pathogens, while the T8 treatment significantly promoted the growth of A6 pathogens. Therefore, fungicides T1, T2, and T9, with inhibition rates exceeding 30%, were selected for a secondary screening experiment against A2, A3, and A6 pathogens.

[0090] 2. Secondary screening of fungicides

[0091] The fungicides (T1 hymexazol, T2 oxpoconazole, T9 propiconazole) with inhibition rate more than 30% in the primary screening were further subjected to secondary screening against A2, A3 and A6 strains. These fungicides were prepared into a series of concentrations: the first concentration was 50 mg / L, the second concentration was 40 mg / L, the third concentration was 30 mg / L, the fourth concentration was 20 mg / L, the fifth concentration was 10 mg / L, and lower concentration PDA test plates (0.625 mg / L, 1.25 mg / L, 2.5 mg / L, 5 mg / L). A 6 mm diameter plug of the pathogen was inoculated in the center of each plate, and incubated at (28 ± 0.5) °C in the dark for 5 days. The growth of the mycelium was recorded every other day. The conventional PDA plate was used as a control, and the experiment had 5 treatments with 3 replicates for each treatment. The mycelial diameter was measured by the cross method, and the inhibition rate was calculated. The virulence regression equation of each fungicide, the inhibition median concentration (EC50 value) of different fungicides and the correlation coefficient (R) were calculated by Excel and DPS data processing system.

[0092] The results of the secondary screening of fungicides are as follows: among the three fungicides tested, T9 (propiconazole) had a certain inhibitory effect on the three pathogens ( Figure 7 ), and with the increase of the concentration of the fungicide, the mycelial diameter became smaller and smaller, and the inhibition rate became larger and larger ( Figure 8 , Table 4 and Table 5). The EC50 values (Table 3) for A2, A3 and A6 pathogens were 21.5214 mg / L, 55.0963 mg / L and 2.2504 mg / L, respectively. However, the EC50 values for A2 and A3 pathogens were greater than 20 mg / L, and the inhibitory effect was not good. In addition, the EC50 values of T1 (hymexazol) and T2 (oxpoconazole) fungicides for A6 pathogens were less than 20 mg / L, and the inhibitory effect on this pathogen was good. However, different concentrations of these fungicides had no significant inhibitory effect on A2 and A3 pathogens. The order of the effect of the three fungicides on A2, A3 and A6 pathogens was: propiconazole > hymexazol > oxpoconazole.

[0093] Table 3 Virulence regression equation of different fungicide treatments on mycelial growth of three pathogens

[0094] .

[0095] Table 4 Inhibitory effect of T9 fungicide on pathogen mycelium .

[0096] Note: Different lowercase letters in the same column indicate significant difference (P < 0.05).

[0097] Table 5 Inhibition of mycelial growth of pathogenic fungi by T1 and T2 fungicides

[0098] .

[0099] Note: The same column, different lowercase letters represent significant differences (P <0.05).

[0100] Example 5 Toxicity test of fungicide mixture on pathogenic fungi

[0101] Select fungicides with inhibition rate of more than 30% for mixing and compounding as a new type of fungicide T10: propiconazole + hymexazol + difenoconazole, and conduct toxicity test on pathogenic fungi.

[0102] When A2 and A3 are pathogenic fungi, the concentration of each fungicide in the used fungicide mixture is as follows: the concentration gradient of hymexazol and difenoconazole is 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L and 50 mg / L, and the concentration gradient of propiconazole is: 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L and 50 mg / L.

[0103] When A6 is a pathogenic fungus, the concentration of each fungicide in the used fungicide mixture is as follows: the concentration gradient of hymexazol and difenoconazole is 0.625 mg / L, 1.25 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L and 50 mg / L, and the concentration gradient of propiconazole is: 0.625 mg / L, 1.25 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L and 50 mg / L.

[0104] The toxicity regression equation of each fungicide, the inhibition medium concentration (EC50 value) of different fungicides and the correlation coefficient (R) are calculated by Excel and DPS data processing system.

[0105] Since the three fungicides have poor inhibitory effect on A2, A3 pathogenic fungi, in order to find an effective fungicide to inhibit A2, A3 pathogenic fungi, three fungicides were selected for compounding and further testing the virulence of the compounded fungicide on three pathogenic fungi. The results showed that the EC50 value of the compounded fungicide for A2, A3, A6 pathogenic fungi was 7.313 mg / L, 12.2983 mg / L, and 0.1781 mg / L, respectively. The inhibition rate of T10 compounded fungicide on A2, A3, A6 pathogenic fungi was 98.65%, 88.45%, and 98.41% at 30 mg / L. Compared with the use of propiconazole alone at a low concentration of 10 mg / L, the inhibition rate increased by 62.62%, 77.53%, and 20.85%, respectively; compared with the use of T9 propiconazole fungicide alone, the growth of the three pathogenic fungi was more effectively inhibited Figure 9 , Table 6 and Table 7).

[0106] Table 6 Virulence regression equation of compounded fungicide on mycelial growth of three pathogenic fungi .

[0107] Table 7 Inhibition of T10 fungicide on pathogenic fungal mycelium .

[0108] Note: The same column and different lowercase letters represent significant differences (P < 0.05).

[0109] Example 6 PI fluorescence staining analysis

[0110] Referring to the PI staining method of Wang Xiaogang (Wang Xiaogang, Luo Donglan, Zhao Zhibing, et al. Isolation and identification of pitaya pathogenic fungi and antibacterial research of thymol [J]. Journal of Food Safety Quality Detection, 2023, 14 (17): 223-230.), slight modifications were made on this basis. The edge mycelium growing on the PDA plate after 5 days of treatment with different fungicide concentrations was washed with phosphate buffer solution (Phosphate buffer saline, PBS, 0.1 mol / L, pH 7.0) for 3 times, and the washed mycelium was transferred to a clean glass slide. Then, 50 μL of PI dye (1 mg / mL) was added and stained in the dark for 30 min, and then the excess PI dye was washed with PBS again, covered with a cover glass, and prepared into an observation slide, which was observed under a fluorescence microscope.

[0111] PI fluorescence staining combined with fluorescence microscopy is used to identify whether the cell is dead. The cell membrane is an important place for the exchange of substances inside and outside the cell, and has the function of protecting the internal structure and function of the cell, so under normal circumstances, PI cannot penetrate the intact cell membrane. However, after T1, T2, T9, T10 four fungicides are treated, PI can enter the cell membrane of damaged or dead cells and embed in double-stranded DNA, and excite red fluorescence under fluorescence microscope, and the fluorescence intensity reflects the degree of cell membrane damage. Based on this, the present application treats three kinds of pathogenic fungi of root rot with different concentrations of fungicides, and combines PI solution staining and fluorescence microscope observation. The results show that the mycelium without four kinds of fungicide treatment does not emit red fluorescence, while the mycelium of the pathogenic fungi treated emits a larger area of red fluorescence region, and with the increase of the concentration of the fungicide, the red fluorescence region also increases accordingly, which shows that the four kinds of fungicides have obvious damage to the integrity of the cell membrane of the pathogenic fungi Figure 10 and Figure 11 ).

Claims

1. Application of substances that inhibit Aspergillus costa cambogia, Mucor spp. Lusitanicus variant and / or Fusarium pernambuco in the control of root rot in Viola yedoensis.

2. The application according to claim 1, characterized in that: The substance is at least one of propiconazole, hymexazol, and oxadiazon.

3. The application according to claim 2, characterized in that: It is at least one of the following: The concentration of propiconazole is 0.625~50 mg / L; The concentration of the hymexazol is 2.5~40 mg / L; The concentration of the oxadiazine is 20-40 mg / L.

4. The application according to claim 3, characterized in that: It is at least one of the following: The concentration of propiconazole is 30 mg / L; The concentration of the hymexazol is 30 mg / L; The concentration of the oxadiazine is 30 mg / L.

5. A method for preventing and controlling root rot in Viola yedoensis, characterized in that: The process includes the following steps: treating *Corydalis violaceus* with at least one of propiconazole, hymexazol, or oxadiazon.

6. The method according to claim 5, characterized in that: The pathogens causing the root rot are Aspergillus costatus, Mucor lucida (Lusitanicus variant), and / or Fusarium pernambuco.

7. The method according to claim 4, characterized in that: It is at least one of the following: The concentration of propiconazole is 0.625~50 mg / L; The concentration of the hymexazol is 10-50 mg / L; The concentration of the oxadiazine is 10-50 mg / L.

8. A bactericide, characterized in that: Including at least one of propiconazole, hymexazol, and oxadiazon.

9. The bactericide according to claim 7, characterized in that: It is at least one of the following: The concentration of propiconazole is 0.625~50 mg / L; The concentration of the hymexazol is 10-50 mg / L; The concentration of the oxadiazine is 10-50 mg / L.

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