Application of nanomolecular material fe3o4 in inhibiting plant soil-borne pathogen fusarium graminearum

Abstract

The application discloses application of nanometer molecular material Fe3O4 in inhibiting plant soil-borne pathogenic fungus Fusarium graminearum. The 5nm nanometer molecular material Fe3O4 can not only inhibit sporulation and conidium germination of the Fusarium graminearum, but also can efficiently induce the Fusarium graminearum mycelium and spore to produce deformity, thereby inhibiting pathogenicity of the Fusarium graminearum. In addition, the experimental results also show that the 5nm nanometer molecular material Fe3O4 can induce a large amount of accumulation of ROS of the mycelium and spore of the Fusarium graminearum, and further induce a large amount of apoptosis and death of the mycelium and spore. Further, it is proved that the inhibition of the 5nm nanometer molecular material Fe3O4 on the pathogenicity of the Fusarium graminearum is realized by inducing a large amount of accumulation of ROS, cell apoptosis and cell death of the conidium and mycelium. The finding shows that the nanometer molecular material Fe3O4 has great application prospect in preventing and treating plant diseases in agricultural production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant protection, and relates to application of 5nm nanomaterial Fe3O4 in inhibition of plant pathogenic fungus Fusarium graminearum. BACKGROUND

[0002] The nanomaterial Fe3O4 is a small molecule substance, and has attracted much attention of researchers due to its excellent physical and chemical properties and wide application prospect. At present, the known application fields mainly include biological medicine, magnetic fluid and magnetic recording material, pollutant treatment and photoelectric catalysis. The magnetic Fe3O4 nanomaterial has the advantages of no toxic side effects and good biocompatibility, and is widely applied in many biomedical fields such as tumor magnetic heat therapy, magnetic resonance imaging technology, targeted drug carrier and magnetic separation. The magnetic Fe3O4 nanoparticle can improve drug efficacy, reduce drug amount and enhance therapeutic effect as a slow-release targeted drug carrier. The targeted drug reaches the lesion directly under the guidance of an external magnetic field. The transmission path of the drug can be tracked by nuclear magnetic resonance imaging of Fe3O4. At present, the molecular mechanism research on the biological function of the nanomaterial Fe3O4 in agriculture has not been involved. The 5nm nanomaterial Fe3O4 is a small molecule substance, and it has not been found that the nanomaterial Fe3O4 has antibacterial activity. SUMMARY

[0003] The present application aims at the above-mentioned deficiencies of the prior art, and provides application of 5nm nanomaterial Fe3O4 in inhibition of plant pathogenic fungus Fusarium graminearum.

[0004] The nanometer Fe3O4 is purchased from a company (Sigma-AIdrich). The nanometer Fe3O4 is applied in inhibition of the plant pathogenic fungus Fusarium graminearum.

[0005] As a preferred embodiment of the present application, the particle size of the nanometer Fe3O4 is 4-10nm.

[0006] As a further preferred embodiment of the present application, the particle size of the nanometer Fe3O4 is 4-5nm, preferably 5nm.

[0007] As a preferred embodiment of the present application, the nanometer Fe3O4 is applied in inhibition of sporulation and germination of conidia of Fusarium graminearum.

[0008] As a preferred embodiment of the present application, the nanometer Fe3O4 is applied in efficient induction of abnormality of hyphae and spores of Fusarium graminearum, so as to inhibit pathogenicity of the Fusarium graminearum.

[0009] As a preferred embodiment of the present application, the nanometer Fe3O4 is applied in induction of apoptosis and death of hyphae and conidia of Fusarium graminearum.

[0010] The application of nano Fe3O4 in the preparation of a preparation for inhibiting plant pathogenic fungi Fusarium graminearum, wherein the nano Fe3O4 has a particle size of 4-10 nm, preferably 4-5 nm.

[0011] A method for inhibiting Fusarium graminearum, wherein the nano Fe3O4 is applied to the Fusarium graminearum.

[0012] As a preferred embodiment of the present application, the nano Fe3O4 has a particle size of 4-10 nm.

[0013] As a preferred embodiment of the present application, the nano Fe3O4 has a particle size of 4-5 nm, preferably 5 nm.

[0014] Advantages:

[0015] The present application discloses that 5 nm nano molecular material Fe3O4 has significant antibacterial activity on various plant pathogenic fungi Fusarium graminearum at a concentration of 10 mg / L, and the 5 nm nano molecular material Fe3O4 has a great industrialization prospect as a new green plant elicitor.

[0016] The present application specifically describes the molecular mechanism of 5 nm nano molecular material Fe3O4 in inhibiting plant pathogenic fungi (Fusarium graminearum). The experimental results show that the 5 nm nano molecular material Fe3O4 can not only inhibit the sporulation and conidial germination of Fusarium graminearum, but also can efficiently induce the abnormality of hyphae and conidia of Fusarium graminearum, thereby inhibiting the pathogenicity of Fusarium graminearum. In addition, the experimental results also show that the treatment of hyphae and conidia of Fusarium graminearum with the 5 nm nano molecular material Fe3O4 can induce the accumulation of ROS in the hyphae and conidia, and further induce the apoptosis and death of the cells of the hyphae and conidia. Further, it is proved that the inhibition of the pathogenicity of Fusarium graminearum by the 5 nm nano molecular material Fe3O4 is realized by inducing the accumulation of ROS, apoptosis and cell death of the conidia and hyphae. The finding shows that it has great application prospect in the prevention and control of plant diseases in agricultural production. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 .5nm nano molecular material Fe3O4 significantly inhibits the sporulation and conidial germination of Fusarium graminearum

[0018] Figure 2 .5nm nano molecular material Fe3O4 significantly induces the abnormality of hyphae and conidia of Fusarium graminearum

[0019] Figure 3 .5 nm nanomaterial Fe3O4 significantly induced ROS accumulation in hyphae of F. graminearum

[0020] Figure 4 .5 nm nanomaterial Fe3O4 significantly induced ROS accumulation in conidia of F. graminearum

[0021] Figure 5 .5 nm nanomaterial Fe3O4 significantly induced apoptosis in hyphae of F. graminearum

[0022] Figure 6 .5 nm nanomaterial Fe3O4 induced apoptosis in conidia of F. graminearum

[0023] Figure 7 .5 nm nanomaterial Fe3O4 significantly induced death in hyphae of F. graminearum

[0024] Figure 8 .5 nm nanomaterial Fe3O4 significantly induced death in conidia of F. graminearum DETAILED DESCRIPTION

[0025] Example 1 Nanomaterial Fe3O4 inhibited sporulation and conidia germination of F. graminearum PH-1 Experimental procedure: PH-1 strain was activated on PDA solid medium at 25°C for 3 days. CMC and YPDA liquid mixed culture medium of 10 mg / L 5 nm and 10 nm nanomaterial Fe3O4 were set up, and the activated PH-1 strain was placed in the CMC and YPDA liquid mixed culture medium, respectively, and placed in a shaker at 200 rpm, 25°C in the dark for 3 days, and the experiment was designed with three replicates.

[0026] Experimental results: In order to study whether 5nm and 10nm nanomaterials Fe3O4 can inhibit the sporulation and spore germination of F. graminearum, we set 10mg / L of 5nm and 10nm nanomaterials Fe3O4 CMC and YPDA liquid mixed culture medium, and cultured F. graminearum in CMC and YPDA liquid mixed culture medium, and FeCl3 was used as positive control. After 3d, the mycelium and spores were collected and the sporulation and spore germination of F. graminearum were counted under microscope. The experimental results showed that 10mg / L of 5nm and 10nm nanomaterials Fe3O4 not only had significant inhibitory effect on the sporulation of F. graminearum, but also had strong inhibitory effect on the spore germination of F. graminearum. The experimental data proved that 10mg / L of 5nm and 10nm nanomaterials Fe3O4 significantly inhibited the sporulation and spore germination of F. graminearum Figure 1 A and B).

[0027] Example 2 Nanomaterials Fe3O4 induced the mycelium and conidial production of F. graminearum F. graminearum Experimental steps: PDA solid medium was used to culture PH-1 strain at 25℃ for 3 days. 10mg / L of 5nm and 10nm nanomaterials Fe3O4 CMC and YPDA liquid mixed culture medium were set, and the activated PH-1 strain was placed in CMC and YPDA liquid mixed culture medium, respectively, and cultured in a shaking bed at 200rpm, 25℃ in the dark for 3d, and three replicates were designed.

[0028] Experimental results: In order to study whether 5nm and 10nm nanomaterials Fe3O4 can affect the morphology of F. graminearum mycelium and spores, we set 10mg / L of 5nm and 10nm nanomaterials Fe3O4 CMC and YPDA liquid mixed culture medium, and cultured F. graminearum in CMC and YPDA liquid mixed culture medium, and FeCl3 was used as positive control. After 3d, the changes of F. graminearum mycelium and spore structure were observed under laser confocal microscope. The experimental results showed that after treating F. graminearum with 10mg / L of 5nm and 10nm nanomaterials Fe3O4, compared with the negative control, nanomaterials Fe3O4 not only induced the mycelium of F. graminearum to produce deformity, but also efficiently induced the conidial of F. graminearum to produce deformity. Compared with the effect of 10nm nanomaterials, 5nm nanomaterials Fe3O4 more significantly induced the mycelium and conidial of F. graminearum to produce deformity Figure 2 A and 2B). By counting the spore deformity of F. graminearum treated with nanomaterials Fe3O4, it was also proved that nanomaterials Fe3O4 can efficiently induce the spore of F. graminearum to produce deformity, and further inhibit the pathogenicity of F. graminearum Figure 2 C).

[0029] Example 3: Nanomaterial Fe3O4 induces ROS accumulation in F. graminearum mycelium Experimental procedure: The PH-1 strain was activated on PDA solid medium at 25°C for 3 days. The YPDA liquid medium was set with 10 mg / L of 5 nm and 10 nm nanomaterial Fe3O4. The activated F. graminearum was placed in the YPDA liquid medium and incubated in a shaker at 200 rpm, 25°C in the dark for 3 days. The mycelium was collected and treated with DCFH-DA (dichlorofluorescein diacetate). The experiment was designed with three replicates.

[0030] Experimental results: ROS (reactive oxygen species) is a series of active oxygen clusters produced by aerobic cells during metabolism, including O-, HO and HO·, ·OH, etc. High concentration of ROS content can cause cell damage and even cell death. DCFH-DA (dichlorofluorescein diacetate) is a special ROS staining agent that emits green fluorescence at the appropriate wavelength. In order to study whether 5 nm and 10 nm nanomaterial Fe3O4 induces ROS accumulation in F. graminearum mycelium, thereby inhibiting its pathogenicity. FeCl3 was used as a positive control. After treating F. graminearum with 10 mg / L of 5 nm and 10 nm nanomaterial Fe3O4 for 3 days, the mycelium was treated with DCFH-DA (dichlorofluorescein diacetate) fluorescent dye, and the fluorescence in the mycelium was observed under a laser confocal microscope. The experimental results show that the fluorescence of F. graminearum mycelium treated with 5 nm and 10 nm nanomaterial Fe3O4 is much stronger than that of the negative control (Figures 2A and 2B), and the fluorescence intensity per unit area of the mycelium also proves this result (Figure 2C). Therefore, the experiment proves that nanomaterial Fe3O4 can inhibit the pathogenicity of F. graminearum by inducing ROS accumulation in the mycelium. Figure 3 A and 3B), and the fluorescence intensity per unit area of the mycelium also proves this result (Figure 2C). Therefore, the experiment proves that nanomaterial Fe3O4 can inhibit the pathogenicity of F. graminearum by inducing ROS accumulation in the mycelium. Figure 3 C).

[0031] Example 4: Nanomaterial Fe3O4 induces ROS accumulation in F. graminearum conidia Experimental procedure: The F. graminearum strain was activated on PDA solid medium at 25°C for 3 days. The CMC liquid medium was set with 10 mg / L of 5 nm and 10 nm nanomaterial Fe3O4. The activated F. graminearum was placed in the CMC liquid medium and incubated in a shaker at 200 rpm, 25°C in the dark for 3 days. The conidia were collected and treated with DCFH-DA (dichlorofluorescein diacetate). The experiment was designed with three replicates.

[0032] Experimental results: In order to study whether the nanomolecular material Fe3O4 induces the massive accumulation of conidial ROS of Fusarium graminearum, thereby inhibiting its pathogenicity. FeCl3 is used as a positive control. We use 10 mg / L of 5 nm and 10 nm nanomolecular material Fe3O4 to treat Fusarium graminearum for 3 days, and then use DCFH-DA (dichlorofluorescein diacetate) as a fluorescent dye to treat the collected fungal conidia, and then observe the fluorescence in the mycelium under a laser confocal microscope. The experimental results show that, compared with the negative control, the fluorescence intensity of Fusarium graminearum spores treated with 5 nm nanomolecular material Fe3O4 is very strong (Figs. 3A and 3B). By statistically analyzing the fluorescence intensity per unit area of the spores, it is proved that the treatment of Fusarium graminearum with 5 nm nanomolecular material Fe3O4 can enhance the fluorescence intensity per unit area of the conidia (Fig. 3C). Therefore, the experiment proves that 5 nm nanomolecular material Fe3O4 can inhibit the pathogenicity of Fusarium graminearum by inducing the massive accumulation of ROS in the conidial body. Figure 4 A and 4B). By statistically analyzing the fluorescence intensity per unit area of the spores, it is proved that the treatment of Fusarium graminearum with 5 nm nanomolecular material Fe3O4 can enhance the fluorescence intensity per unit area of the conidia ( Figure 4 C). Therefore, the experiment proves that 5 nm nanomolecular material Fe3O4 can inhibit the pathogenicity of Fusarium graminearum by inducing the massive accumulation of ROS in the conidial body.

[0033] Example 5: Experiment of nanomolecular material Fe3O4 inducing mycelial apoptosis of Fusarium graminearum Experimental steps: Activate the PH-1 strain on PDA solid medium at 25°C for 3 days. Set 10 mg / L of 5 nm and 10 nm nanomolecular material Fe3O4 in YPDA liquid mixed culture medium, place the activated Fusarium graminearum culture dish in the YPDA liquid mixed culture medium, and place it in a shaker at 200 rpm, 25°C in the dark for 3 days. Collect the mycelium and treat it with the cell apoptosis fluorescent Hoechst 33342 / PI double staining kit. Three replicates are designed.

[0034] Experimental results: In order to study whether 5 nm and 10 nm nanomolecular material Fe3O4 induces apoptosis of mycelial cells of Fusarium graminearum, thereby inhibiting its pathogenicity. FeCl3 is used as a positive control. We use 10 mg / L of 5 nm and 10 nm nanomolecular material Fe3O4 to treat Fusarium graminearum for 3 days, and then use the cell apoptosis fluorescent Hoechst 33342 / PI double staining kit to treat the mycelium, and then observe the fluorescence in the mycelium under a laser confocal microscope. The experimental results show that, compared with the negative control, the fluorescence of Fusarium graminearum mycelium treated with 5 nm and 10 nm nanomolecular material Fe3O4 is very strong (Figs. 5A and 5B), and this result is also proved by statistically analyzing the fluorescence intensity per unit area of the mycelium (Fig. 5C). Figure 5 A and 5B), by statistically analyzing the fluorescence intensity per unit area of the mycelium ( Figure 5C). Therefore, the experiment proves that the nanomolecular material Fe3O4 can inhibit the pathogenicity of F. graminearum by inducing a large number of apoptosis of the conidial cells of F. graminearum.

[0035] Example 6: Experiment of nanomolecular material Fe3O4 inducing apoptosis of conidial cells of F. graminearum

[0036] Experimental results: In order to study whether the nanomolecular material Fe3O4 of 5nm and 10nm induces apoptosis of the conidial cells of F. graminearum, thereby inhibiting its pathogenicity. The experiment takes FeCl3 as a positive control, and we use 10mg / L of the nanomolecular material Fe3O4 of 5nm and 10nm to treat F. graminearum for 3d, and then use the cell apoptosis fluorescence Hoechst 33342 / PI double staining kit to treat the conidial cells, and then observe the fluorescence in the hyphae under the laser confocal microscope. The experimental results show that, compared with the negative control, the fluorescence of the conidial cells of F. graminearum treated with the nanomolecular material Fe3O4 of 5nm and 10nm is very strong (Figs. 6A and 6B), and this result is also proved by the statistics of the fluorescence intensity per unit area of the conidial cells (Fig. 6C). Therefore, the experiment proves that the nanomolecular material Fe3O4 can inhibit the pathogenicity of F. graminearum by inducing a large number of apoptosis of the conidial cells of F. graminearum. Figure 6 A and 6B), through the statistics of the fluorescence intensity per unit area of the conidial cells ( Figure 6 C). Therefore, the experiment proves that the nanomolecular material Fe3O4 can inhibit the pathogenicity of F. graminearum by inducing a large number of apoptosis of the conidial cells of F. graminearum.

[0037] Example 7: Experiment of nanomolecular material Fe3O4 inducing a large number of death of hyphal cells of F. graminearum

[0038] Experimental Results: To investigate whether 5nm and 10nm Fe3O4 nanomaterials induced cell death in the hyphae of *Fusarium graminearum*, thereby inhibiting its pathogenicity, FeCl3 was used as a positive control. *Fusarium graminearum* was treated with 10 mg / L of 5nm and 10nm Fe3O4 nanomaterials for 3 days. After treatment with the hyphae using the Hoechst 33342 / PI double staining kit for apoptosis fluorescence, the fluorescence within the hyphae was observed under a laser confocal microscope. The results showed that, compared to the negative control, the fluorescence of *F. graminearum* hyphae treated with 5nm and 10nm Fe3O4 nanomaterials was significantly stronger. Figure 7 This result was also confirmed by statistical analysis of the fluorescence intensity per unit area of ​​the hyphae (A and 7B). Figure 7 C). Therefore, the experiment proved that the nanomaterial Fe3O4 can inhibit the pathogenicity of Fusarium graminearum by inducing the death of a large number of mycelial cells.

[0039] Example 8: Experimental Procedure for Direct Induction of Conidial Cell Death in *F. graminearum* by Fe3O4 Nanoparticles. The PH-1 strain was activated by culturing on PDA solid medium at 25°C for 3 days. A CMC liquid mixture of 10 mg / L 5nm and 10nm Fe3O4 nanoparticles was prepared. Activated *F. graminearum* bacterial culture dishes were placed on the CMC liquid mixture and cultured in the dark at 25°C for 3 days using a shaker at 200 rpm. Conidia were collected and treated with a Hoechst 33342 / PI double staining kit for apoptosis fluorescence. The experiment was designed with three replicates. Results: This study investigated whether 5nm and 10nm Fe3O4 nanoparticles induced apoptosis and cell death in *F. graminearum* conidial cells, thereby inhibiting its pathogenicity. We treated *Fusarium graminearum* with 10 mg / L of 5 nm and 10 nm Fe3O4 nanoparticles for 3 days. Using FeCl3 as a positive control, we treated its conidia with a Hoechst 33342 / PI double staining kit for apoptosis fluorescence. The fluorescence of the conidia was then observed under a laser confocal microscope. The results showed that, compared to the negative control, the fluorescence of red cell death in the conidia of *F. graminearum* treated with 5 nm and 10 nm Fe3O4 nanoparticles was significantly stronger. Figure 8 Figures A and 8B) also confirm this result by statistically analyzing the fluorescence intensity per unit area of ​​conidia (Figure 8C). Therefore, the experiment demonstrates that the nanomaterial Fe3O4 can directly bypass the induction mechanism to induce apoptosis in the conidial cells of Fusarium graminearum, thereby inducing massive cell death and inhibiting its pathogenicity.

Claims

1. Use of nano-Fe3O4 with a particle size of 4-10 nm in inhibiting the plant pathogenic fungus Fusarium graminearum.

2. Use according to claim 1, characterized in that The nano-Fe3O4 has a particle size of 4-5 nm.

3. Use according to claim 1, characterized in that The nano-Fe3O4 has a particle size of 5 nm.

4. Use according to any one of claims 1 to 3, characterized in that Use of nano-Fe3O4 in inhibiting the sporulation and conidial germination of Fusarium graminearum.

5. The use according to any one of claims 1 to 3, characterized in that Use of nano-Fe3O4 in inducing the abnormality of hyphae and spores of Fusarium graminearum, thereby inhibiting the pathogenicity of Fusarium graminearum.

6. Use according to any one of claims 1 to 3, characterized in that Use of nano-Fe3O4 in inducing the apoptosis and / or death of hyphae and conidia of Fusarium graminearum.

7. Use of nano-Fe3O4 with a particle size of 4-10 nm in preparing a preparation for inhibiting the plant pathogenic fungus Fusarium graminearum.

8. Use according to claim 7, characterized in that, The nano-Fe3O4 has a particle size of 4-5 nm.

9. A method of inhibiting Fusarium graminearum comprising The nano-Fe3O4 of any one of claims 2 or 3 is applied to Fusarium graminearum.

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