Preparation method and application of taurine carbon dots for controlling rice pathogenic fungal diseases

Abstract

The present invention relates to the field of bioengineering, specifically a preparation method and application of taurine carbon dot materials for preventing and controlling rice pathogenic fungal diseases. High-purity platinum wires cleaned with ultrapure water and alcohol are used as the cathode and anode and placed in a 50% taurine solution, and continuous electrolysis is carried out to obtain a taurine nano-dispersion. The taurine nano-dispersion is filtered with qualitative filter paper, centrifuged at high speed, and dialyzed with ultrapure water to obtain the taurine carbon dot materials. The particle size of the nanoparticles in the taurine carbon dot materials is 2-5 nm, there is a disordered crystal structure, and the surface has oxygen-containing and sulfur-containing functional groups. The taurine carbon dot materials can effectively inhibit the mycelial growth and spore germination of Magnaporthe oryzae and Bipolaris oryzae. Therefore, using the taurine carbon dot materials to pretreat rice can effectively reduce the occurrence of rice blast and brown spot disease.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and particularly to a method for preparing and applying taurine carbon dots for controlling pathogenic fungal diseases of rice. Background Art

[0002] Rice (Oryza sativa) is one of the important food crops, feeding more than half of the world's population. However, the occurrence of various diseases seriously affects the yield and quality of rice. Rice blast caused by Magnaporthe oryzae has always been the most serious fungal disease threatening rice production. In China, the direct loss of rice yield caused by rice blast reaches up to 3 billion kilograms annually. When the disease occurs severely, it can lead to a 40%-50% reduction in rice yield, or even a complete harvest failure. Rice brown spot is a fungal disease caused by Cochliobolus miyabeanus. When the disease occurs, elliptical brown spots appear on the leaves, with a yellow halo at the edge. When the spots merge severely, the leaves will wither and die. When planted in barren fields or drylands, the brown spot disease occurs rapidly. If not controlled in time, it will cause a large reduction in rice yield. Currently, the control techniques for rice blast and brown spot in production include selecting disease-resistant varieties, but years of use will lead to single resistance, which is easily broken through by pathogenic bacteria, resulting in a decline in disease resistance; using fungicides, such as triazoles (e.g., tricyclazole), methoxyacrylates (e.g., azoxystrobin), benzimidazoles (e.g., carbendazim), and organophosphates (e.g., isoprothiolane). The use of fungicides belongs to chemical control. Excessive use is likely to produce drug resistance, pollute the environment, and endanger health; using antagonistic microorganisms, such as Bacillus subtilis and Trichoderma, etc. The control by antagonistic microorganisms belongs to the type of biological control. The effect of such techniques is slow and greatly affected by the environment; seed treatment, such as hot water soaking of seeds, medicament dressing of seeds, etc. Seed treatment belongs to physical control. The operation in the physical control process is cumbersome and the effect is limited.

[0003] Taurine (chemical formula: C2H7NO3S) is an amino acid converted from sulfur-containing amino acids and exists in the form of free amino acids in humans and animals. As an organic osmotic regulatory substance, taurine not only participates in regulating cell volume, but also provides a basis for the formation of bile salts, and also plays an important role in modulating the intracellular free calcium concentration. In addition, taurine is an active substance that regulates the normal physiological activities of the body, and has a wide range of biological functions such as anti-inflammatory, analgesic, maintaining the osmotic balance of the body, regulating cell calcium balance, lowering blood sugar, regulating nerve conduction, participating in endocrine activities, regulating lipid digestion and absorption, and protecting myocardial cells. Taurine can be industrially produced and is a stable compound in vitro.

[0004] Although traditional chemical pesticides can effectively prevent and control the occurrence and prevalence of crop diseases, due to their biological toxicity, improper use will seriously affect food safety. With the improvement of people's requirements for food safety and the gradual enhancement of their awareness of ecological environment protection, the development of new green pesticides with low toxicity and high efficiency has become the mainstream in the field of food disease prevention and control in China and even the world.

[0005] With the rapid development of materials science, the research on nanomaterials has entered a stage of rapid development and has become increasingly prominent in agriculture. At present, nanotechnology can nanometerize some substances to obtain physical and chemical properties different from the raw materials, which provides new ideas for the development of new green pesticides. So far, there have been reports on the preparation of nano heavy metal particle bactericides by nanometerizing different metal ions. Although such nano antibacterial materials have a certain disease prevention and control effect, the accumulation of heavy metal ions in the agricultural ecological environment will pollute the environment and endanger human health. Therefore, when developing nano bactericides, attention should be paid not only to the antibacterial effect but also to the safety of the environment and the human body. Using non-toxic small molecule organic substances of biological origin as raw materials to prepare antibacterial materials through nanotechnology is a new idea for the development of new green biological pesticides. Summary of the Invention

[0006] The purpose of the present invention is to provide a preparation method of taurine carbon dots material that can effectively inhibit the growth and spore germination of Magnaporthe oryzae and Bipolaris oryzae, and the taurine carbon dots material can preventively control the occurrence of rice blast and brown spot disease.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is: The present invention provides a taurine carbon dots material, which can be used for the prevention and control of rice pathogens Magnaporthe oryzae or / and Bipolaris oryzae. The taurine carbon dots material can inhibit the mycelial growth and spore germination of Magnaporthe oryzae, and foliar spraying on rice can reduce the occurrence of rice blast; it can inhibit the mycelial growth and spore germination of Bipolaris oryzae, and foliar spraying on rice can reduce the occurrence of brown spot disease.

[0008] The present invention also provides a preparation method of taurine carbon dots material for preventing and controlling rice pathogenic fungal diseases, and the steps include:

[0009] 1) Take two platinum wires with a purity of 99.9%, a diameter of 0.5 mm, wash them with ultrapure water for 5 min, place them in 70% alcohol for ultrasonic cleaning for 10 min, and then dry the surface moisture with absorbent paper;

[0010] 2) Take 1000 ml of an aqueous solution of taurine with a mass fraction of 50% in a beaker as the electrolyte. Place the cleaned platinum wire as the cathode and anode in the electrolyte, and apply a constant voltage DC power supply of 10 - 20 V for continuous electrolysis for 25 - 30 days to obtain a taurine nano-dispersion;

[0011] 3) Centrifuge the taurine nano-dispersion at 22000 rcf for 45 min, discard the precipitate, and collect the supernatant;

[0012] 4) Pour the supernatant into a dialysis bag with a molecular weight cut-off of 500 - 1000 D and dialyze it in ultrapure water for 24 h to obtain the taurine carbon dot material that can effectively inhibit rice pathogenic fungi.

[0013] Further, in step 1), the ultrasonic power is 300 W and the frequency is 40 KHz.

[0014] Further, in step 2), during electrolysis, the distance between the two electrodes is maintained at 3 - 10 cm.

[0015] The beneficial technical effects of the present invention are as follows: In the present invention, high-purity platinum wires cleaned with ultrapure water and alcohol are used as the cathode and anode and placed in a 50% taurine solution for continuous electrolysis to obtain a taurine nano-dispersion. The taurine nano-dispersion is filtered with qualitative filter paper, centrifuged at high speed, and dialyzed with ultrapure water to obtain the taurine carbon dot material. The particle size of the nanoparticles in the taurine carbon dot material is 2 - 5 nm, there is a disordered crystal structure, and the surface has oxygen-containing and sulfur-containing functional groups. The taurine carbon dot material can effectively inhibit the mycelial growth and spore germination of Magnaporthe oryzae and Bipolaris oryzae. Therefore, using this taurine carbon dot material to pretreat rice can effectively reduce the occurrence of rice blast and brown spot disease. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 It is a transmission electron microscope image of taurine carbon dots (scale bar is 5 nm).

[0018] Figure 2 It is the particle size distribution diagram of taurine carbon dots in an aqueous solution.

[0019] Figure 3 It is the ultraviolet absorption spectrum diagram of taurine carbon dots.

[0020] Figure 4 It is the X-ray photoelectron spectroscopy (XPS) diagram of taurine carbon dots.

[0021] Figure 5 It shows the inhibition of the vegetative growth of Magnaporthe oryzae by taurine carbon dot material (scale bar is 1 cm).

[0022] Figure 6 It shows the morphological abnormality of the vegetative hyphae of Magnaporthe oryzae caused by the treatment with taurine carbon dot material (scale bar is 25 μm).

[0023] Figure 7 It shows the inhibition of the conidial germination of Magnaporthe oryzae by taurine carbon dot material (scale bar is 25 μm).

[0024] Figure 8 It shows the inhibition of the occurrence of rice blast by spraying taurine carbon dot material on rice leaves 4 hours before inoculation.

[0025] Figure 9 It shows the inhibition of the vegetative growth of Bipolaris oryzae by taurine carbon dot material (scale bar is 1 cm).

[0026] Figure 10 It shows the morphological abnormality of the vegetative hyphae of Bipolaris oryzae caused by the treatment with taurine carbon dot material (scale bar is 75 μm).

[0027] Figure 11 It shows the inhibition of the conidial germination of Bipolaris oryzae by taurine carbon dot material.

[0028] Figure 12 It shows the inhibition of the occurrence of brown spot of rice by spraying taurine carbon dot material on rice leaves 4 hours before inoculation. Detailed implementation manners

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] The experimental materials are as follows:

[0031] High-purity platinum wire: platinum content is above 99.9%, diameter is 0.5 mm. [[ID=%]]

[0032] Ultra-pure water: resistivity is 18.4 MΩ•cm -1 .

[0033] Taurine solution: volume percentage is 50%.

[0034] Example 1

[0035] Preparation of Taurine Carbon Dots Material

[0036] Step 1: Take two platinum wires with a purity of 99.9%, a diameter of 0.5 mm, wash them with ultrapure water for 5 min, place them in 70% alcohol and ultrasonically clean them for 10 min, with an ultrasonic power of 300 W and an ultrasonic frequency of 40 KHz, and then dry the surface moisture with absorbent paper;

[0037] Step 2: Take 1000 ml of a 50% aqueous solution of taurine in a beaker as the electrolyte, place the cleaned high-purity platinum wires as the cathode and anode in the electrolyte, keep the distance between the two electrodes at 3 - 10 cm, and continuously electrolyze for 25 - 30 days under an external constant voltage DC power supply of 30 V to obtain a taurine nano-dispersion;

[0038] Step 3: Centrifuge the taurine nano-dispersion at 22000 rcf for 45 min, discard the precipitate, and collect the supernatant;

[0039] Step 4: Pour the supernatant into a dialysis bag with a molecular weight cut-off of 500 - 1000 D and dialyze it in ultrapure water for 24 h to obtain the taurine carbon dots material that can effectively inhibit rice pathogenic fungi.

[0040] Example 2

[0041] Transmission Electron Microscopy Analysis of Taurine Carbon Dots Material

[0042] Perform transmission electron microscopy imaging observation on the obtained taurine nano-dispersion, and the results are as Figure 1 shown. The effective substances in the taurine nano-dispersion are in the shape of dots and have a typical graphite sheet-like structure, indicating that the nano-dispersion is a carbon dots solution. Further statistical analysis of the particle size distribution of taurine carbon dots is carried out, and the results are as Figure 2 shown, and the particle size of the carbon dots is mainly distributed between 2.5 - 4.5 nm.

[0043] Example 3

[0044] Characterization Analysis of Taurine Carbon Dots Material

[0045] Ultraviolet spectroscopy analysis shows that taurine carbon dots have a significant absorption peak at 280 nm, as Figure 3 shown, suggesting that the carbon dots have fluorescence characteristics and can be used for real-time monitoring of the interaction with microorganisms. Further, X-ray photoelectron spectroscopy (XPS) technology is used to analyze the surface functional groups of taurine carbon dots, and the results are as Figure 4As shown, in addition to rich C-O and C=O bonds on the surface of taurine carbon dots, elements such as S and N are also contained, reflecting its active surface activity.

[0046] Example 4

[0047] Inhibitory effect of taurine carbon dot material on Magnaporthe oryzae

[0048] To test the inhibitory effect of taurine carbon dot material on the growth of vegetative hyphae of Magnaporthe oryzae, in this invention, a mycelial cake of Magnaporthe oryzae with a diameter of 5 mm was transferred onto a CMⅡ medium supplemented with 400 μg / mL of taurine carbon dot material or taurine. After culturing at 28 °C for 7 days, the CMⅡ medium without any treatment was used as a control to calculate the growth inhibition rates of taurine carbon dot material and taurine on Magnaporthe oryzae. The results are as Figure 5 shown. Compared with the original substance taurine, the treatment with taurine carbon dot material can effectively inhibit the growth of vegetative hyphae of Magnaporthe oryzae. Statistical analysis of the inhibition rates of the two shows that the difference is extremely significant (**, P < 0.01, Student’s t-test).

[0049] Solid culture blocks of Magnaporthe oryzae were respectively placed into CM liquid medium containing 400 μg / mL of taurine carbon dot material or taurine, and cultured at 28 °C for 48 h at a rotation speed of 80 revolutions per minute. After that, the morphology of vegetative hyphae was observed through a microscope. It was found that the treatment with taurine carbon dot material led to abnormal vesicular morphology in the vegetative hyphal cells of Magnaporthe oryzae ( Figure 6 , indicated by the white arrow), while no obvious difference was observed in the hyphae of Magnaporthe oryzae treated with taurine compared with the control.

[0050] Conidia produced by Magnaporthe oryzae were added to 0.02% Tween-20 aqueous solution containing 400 μg / mL of taurine carbon dot material or taurine. After dropping onto a hydrophobic glass slide and placing it under moisturizing and dark conditions at 28 °C for 6 h, the spore germination rate was counted. Three groups were counted for each treatment, and each group contained more than 50 conidia. It can be seen from Figure 7 this that taurine carbon dot material has a significant inhibitory effect on the germination of conidia of Magnaporthe oryzae. ** represents that the difference is extremely significant (P < 0.01, Student’s t-test).

[0051] Example 5

[0052] Control effect of taurine carbon dot material on rice blast

[0053] To further explore the control effect of taurine carbon dots on rice blast, 4 h before spraying the conidia of Magnaporthe oryzae on rice seedlings, the rice seedlings were evenly sprayed with 400 μg / mL of taurine carbon dots at 0.5 mL / plant, and then inoculated with Magnaporthe oryzae. The disease incidence was observed 6 days later. As Figure 8 shown, compared with the control treatment, taurine carbon dots can effectively reduce the diseased area of rice blast, indicating that 400 μg / mL of taurine carbon dots has a good preventive effect on the occurrence of rice leaf blast.

[0054] Example 6

[0055] Inhibitory effect of taurine carbon dots on Bipolaris oryzae

[0056] To test the inhibitory effect of taurine carbon dots on other pathogenic fungi of rice, the present invention selected Bipolaris oryzae for further treatment. A mycelial cake of Bipolaris oryzae with a diameter of about 5 mm was transferred onto CMⅡ medium supplemented with 400 μg / mL of taurine carbon dots or taurine. After culturing at 28 °C for 5 days, the CMⅡ medium without treatment was used as the control to calculate the growth inhibition rate. The results showed that ( Figure 9 ), compared with the original substance taurine, the treatment with taurine carbon dots can effectively inhibit the growth of the vegetative mycelium of Bipolaris oryzae. The statistical analysis showed that the difference in the inhibition rates between the two was extremely significant (**, P<0.01, Student’s t-test).

[0057] The solid culture blocks of Bipolaris oryzae were respectively placed into CM liquid medium containing 400 μg / mL of taurine carbon dots or taurine, and cultured at 28 °C for 72 h at a rotation speed of 80 r / min. The growth of the vegetative mycelium was observed by fluorescence microscopy. The results showed that the treatment with taurine carbon dots could cause the vegetative mycelium cells of Bipolaris oryzae to show abnormal vesicular morphology ( Figure 10 , indicated by the white arrow), while there was no obvious difference in the mycelial morphology between the taurine treatment and the control.

[0058] The conidia produced by Bipolaris oryzae were added to 0.02% Tween-20 aqueous solution containing 400 μg / mL of taurine carbon dots or taurine. After dropping onto a hydrophobic glass slide and placing it at 28 °C under humid and dark conditions for 2 h, the spore germination rate was counted. Three groups were counted for each treatment, and each group had more than 100 conidia. As Figure 11 shown, taurine carbon dots have a significant inhibitory effect on the germination of the conidia of Bipolaris oryzae. ** represents extremely significant difference (P<0.01, Student’s t-test).

[0059] Example 7

[0060] Control Effect of Taurine Carbon Dots Material on Brown Spot of Rice

[0061] In order to further explore the control effect of taurine carbon dots material on brown spot of rice, 4 hours before spraying the conidia of brown spot pathogen on rice seedlings, the rice seedlings were evenly sprayed with 400 μg / mL taurine carbon dots material at 0.5 mL / plant, and the disease incidence was observed 3 days after inoculation. As Figure 12 shown, compared with the control treatment, the taurine carbon dots material can effectively reduce the disease area of brown spot of rice, indicating that 400 μg / mL taurine carbon dots material also has a good preventive effect on the occurrence of brown spot of rice.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: the present invention can still be modified or equivalently replaced, and any modification or partial replacement without departing from the spirit and scope of the present invention shall be covered by the scope of the claims of the present invention.

Claims

1. Application of taurine carbon dot material in preventing and controlling rice pathogenic fungal diseases, characterized in that, The rice pathogenic fungi are Magnaporthe oryzae and / or Bipolaris oryzae, and the preparation method of the taurine carbon dot material comprises the following steps: 1) Take two platinum wires with a purity of 99.9% and a diameter of 0.5 mm, wash them with ultrapure water for 5 min, place them in 70% alcohol for ultrasonic cleaning for 10 min, and then dry the surface moisture with absorbent paper; 2) Take 1000 ml of a 50% aqueous taurine solution in a beaker as the electrolyte, place the cleaned platinum wires as the cathode and anode in the electrolyte, and continuously electrolyze for 25 - 30 days under an externally applied constant voltage DC power supply of 10 - 20 V to obtain a taurine nano-dispersion; 3) Centrifuge the taurine nano-dispersion at 22000 rcf for 45 min, discard the precipitate, and collect the supernatant; 4) Pour the supernatant into a dialysis bag with a molecular weight cut-off of 500 - 1000 D and dialyze it in ultrapure water for 24 h to obtain a taurine carbon dot material that can effectively inhibit rice pathogenic fungi.

2. The application according to claim 1, wherein The taurine carbon dot material inhibits the mycelial growth and spore germination of Magnaporthe oryzae, and foliar spraying treatment on rice can reduce the occurrence of rice blast.

3. The application according to claim 1, wherein The taurine carbon dot material inhibits the mycelial growth and spore germination of Bipolaris oryzae, and foliar spraying treatment on rice can reduce the occurrence of brown spot of rice.

4. The application according to claim 1, wherein In step 1), the ultrasonic power is 300 W and the frequency is 40 KHz.

5. The application according to claim 1, wherein In step 2), during electrolysis, the distance between the two electrodes is maintained at 3 - 10 cm.

Images