Fungus nano-particles as well as synthesis method and application thereof

Abstract

The invention discloses a fungal nano-particle, a synthesis method and application of the fungal nano-particle. Wherein the fungal nano-particles are synthesized through reaction of a helminthosporium bulbigerum cell filtrate and silver nitrate, the obtained fungal nano-particles are spherical, the diameter ranges from 30 nanometers to 50 nanometers, and the uniformity is good. The fungus nanoparticles have a good weed control effect, and especially have a remarkable control effect on moleplant seeds.

Description

Technical Field

[0001] The invention relates to nano materials, in particular to fungal nano particles, and also to a synthesis method and application of the fungal nano particles. Background Art

[0002] Nanotechnology (NT) is a process that involves the study of -9 Nanomaterials are technologies that operate at scales of 100 to 200 meters (m) to create smart systems, gadgets, and materials. NTs have wide applications in several fields, including agriculture, due to their atomic-level precision. In agriculture, common nanoparticles such as gold (Au), silver (Ag), copper (Cu), zinc (Zn), titanium (Ti), aluminum (Al), silicon dioxide (SiO2), and chitin have been studied for a variety of applications such as herbicides, fungicides, pesticides, and biofertilizers. These nanomaterials help improve crop yields and quality, and are essential for the sustainable development of humans and ecosystems.

[0003] Nanomaterials have a wide range of applications in various multidisciplinary fields. With the development of nanotechnology, the wide application of metal nanoparticles such as gold, silver, platinum, copper, magnesium and zinc in the agricultural field has become increasingly important. Among them, Ag NPs are the latest developed metal nanoparticles, which have attracted much attention due to their superior properties such as strong antimicrobial activity, high catalytic efficiency, strong conductivity and strong chemical stability (Mészáros et al, 2021; Jayarambabu et al, 2023). The potential agricultural applications of bio-silver nanoparticles as nanofertilizers (Landa, 2021), nanoscale growth regulators (Manickavasagam et al, 2019), nanopesticides for seed germination (Parveen and Rao, 2015; Mahakham et al, 2017) and nanoinsecticides have been well demonstrated. Silver nanoparticles synthesized by Z. nitidum have potential medicinal value, can be used for a variety of purposes and cultivation, and have potential herbicidal activity (Jiang et al, 2023).

[0004] Plants exposed to nanopesticides showed changes in protein content, lipid peroxidation, enzymatic / non-enzymatic antioxidant activity, and chlorophyll content (Zhao et al, 2016; Zhang et al, 2019; Pontes et al, 2021; Wu et al, 2021; Oliveira-Pinto et al, 2022). Previous reports have shown that one of the cellular internalization mechanisms adopted by plants to improve the stress damage caused by nanoparticles is to sort nanomaterials into vacuoles (Panariti et al, 2012). In addition, nanoherbicides may enter host plant organelles and cause changes in their functions (Pérez-de-Luque, 2017; Mittal et al, 2020). For example, the formation of reactive oxygen species (ROS) in plant cells mainly exists in the mitochondrial electron transport chain (ETC), followed by the chloroplast electron transport chain (ETC). According to several previous studies, both enzymatic and non-enzymatic activities are involved in the antioxidant mechanisms to cope with ROS formation (Oliveira et al., 2015; Pontes et al., 2021; Wang et al., 2022). The main factors for the generation of ROS by nanoherbicides include the peroxidase-active (POD) groups attached to the surface of the nanoherbicides, the active reduction and oxidation mechanisms of the enzymes, especially in the hybrid synthesis process caused by the modification of these nanomaterials and the interaction with the host plants (Manke et al., 2013). However, more in-depth studies are needed to better assess the fate of nanoherbicides in mitochondria, which generally focus on the tricarboxylic acid (TCA) cycle (Wang et al., 2021). Similarly, in chloroplasts, various nanoherbicides lead to the ferredoxin-NADP + Oxidoreductase (NADPH / NADP + ratio), quantum yield, and total CO2 accumulation rate decreased (Oliveira et al., 2015; Forini et al., 2020; Pontes et al., 2021; Takeshita et al., 2021). Therefore, nanoagriculture requires more attention to improve the understanding of the gene-level toxicity of nanoherbicides to target and non-target plant species. Summary of the invention

[0005] One of the objects of the present invention is to provide a fungal nanoparticle.

[0006] The technical solution provided by the present invention is as follows: a fungal nanoparticle is prepared by reacting the cell filtrate of Helicoverpa orientalis with silver nitrate.

[0007] The fungal nanoparticles are spherical and have a diameter between 30 and 50 nanometers.

[0008] The cell filtrate of Helicoverpa orientalis needs to be free of hyphae.

[0009] The second object of the present invention is to provide a method for synthesizing the above-mentioned fungal nanoparticles.

[0010] The technical scheme provided by the present invention is as follows: a method for synthesizing fungal nanoparticles, wherein the cell filtrate of Helicoverpa orientalis is reacted with silver nitrate to prepare the fungal nanoparticles.

[0011] In one embodiment, 50 ml of Helicoverpa orientalis cell filtrate was mixed with 50 ml of 5 mM AgNO 3 solution for reaction.

[0012] In the present invention, the reaction temperature is 40-50° C., the reaction time is 3-4 hours, and a dark brown reaction product is obtained.

[0013] The third object of the present invention relates to the application of the above-mentioned fungal nanoparticles.

[0014] Specifically, it relates to the application of the fungal nanoparticles as nano-herbicides; it relates to the application of the fungal nanoparticles as nano-herbicides for controlling grass weeds; it relates to the application of the fungal nanoparticles as nano-herbicides for controlling Leptochloa chinensis.

[0015] A fourth object of the present invention relates to nano herbicides.

[0016] Specifically, the nano herbicide, the main component of which comprises fungal nanoparticles obtained by reacting the cell filtrate of Helicoverpa orientalis with silver nitrate.

[0017] In the present invention, the concentration of fungal nanoparticles in the nanoherbicide is 70-100 mg / L.

[0018] Advantages of the present invention:

[0019] 1. The fungal nanoparticles provided by the present invention are prepared by reacting the cell filtrate of Helicoverpa orientalis with silver nitrate. The synthesized NPs are spherical, with a diameter of 30-50 nanometers and good uniformity.

[0020] 2. Fungal nanoparticles have good weed control effects, especially on Leptochloa chinensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a visual representation of the M-Ag NPs synthesized from the cell-free filtrate of Helicoverpa orientalis.

[0022] Figure 2 is the UV-visible absorption spectrum of M-Ag NPs.

[0023] Figure 3is the simulated XRD pattern recorded from the air-dried M-Ag NPs powder.

[0024] The high peaks correspond to the characteristic diffraction peaks of elemental silver.

[0025] Figure 4 is the particle size distribution analysis and stability of M-Ag NPs.

[0026] Note: DLS of M-Ag NPs (A) and Zeta potential of M-Ag NPs (B).

[0027] Figure 5 is the Fourier transform infrared spectrum of M-Ag NPs.

[0028] Figure 6 is a scanning electron micrograph of Helicoverpa orientalis-mediated silver nanoparticles (Ag NPs).

[0029] Note: Micrographs were obtained at different magnifications of 50.0 μm.

[0030] Figure 7 This is the EDX spectrum and elemental analysis of synthetic silver nanoparticles. The highlighted area is the screening area of ​​the EDX spectrum of synthetic silver nanoparticles.

[0031] Note: (A) Elemental analysis of spectrum 1 (B), (C) Elemental analysis of spectrum 2 (D).

[0032] Figure 8 It is EDX element percentage analysis.

[0033] Spectrum 1 (A), Spectrum 2 (B).

[0034] Fig. 9 TEM micrographs of silver nanoparticles (Ag NPs) produced by Helicoverpa orientalis show a relatively spherical shape and corresponding particle size distribution.

[0035] Fig.10 This is a screening experiment to determine the differences in the control effects of different concentrations of nanoparticles and Helminthosporium orientalis on Leptochloa chinensis.

[0036] Note: All treatments showed control effects at the 4-leaf stage. All values ​​indicated significant differences at P<0.01 and P<0.001, respectively.

[0037] Fig.11 is the nanoherbicidal potential of M-Ag NPs of all formulations against the three-leaf stage; all values ​​indicate significant differences at P < 0.01 and P < 0.001, respectively.

[0038] Fig.12The effect of M-Ag NPs nanoherbicidal potential of all agents on the 4-leaf stage.

[0039] Note: All values ​​indicate significant differences at P<0.01 and P<0.001, respectively.

[0040] Fig.13 is the effect of M-AgNPs on oxidative stress parameters in bulbs, and fungi served as positive control.

[0041] Note: A) Malondialdehyde (MDA); B) Hydrogen peroxide (H2O2); C) Electrolyte leakage percentage (EL).

[0042] Fig.14 is the effect of M-Ag NPs on the antioxidant parameters of Leptochloa chinensis.

[0043] Note: All values ​​indicate significant differences at P<0.01 and P<0.001, respectively.

[0044] Fig.15 This is a flow chart of the determination of PG, PMG, CX, and βG by the DNS method.

[0045] Fig.16 is the effect of M-AgNPs on the activity of cell wall-degrading enzymes of Leptochloa chinensis.

[0046] Note: (A) PG, polygalacturonase; (B) PMG, polymethylgalacturonase; (C) βG, β-glucuronidase; (C) CX, cellulase. All values ​​indicate significant differences at P < 0.01 and P < 0.001, respectively.

[0047] Fig.17 It is to observe the effects of nanomaterials synthesized by mold on Leptochloa chinensis from a cytological perspective.

[0048] Note: (A) control; (B) leaves treated with Helicoverpa orientalis; (C) Helicoverpa orientalis-mediated silver nanoparticles (90 mg / mL); CW; cell wall; M; mitochondria; V; vacuole; Ch; chloroplast; PG; AS; stomata.

[0049] Fig.18 The effects of different concentrations of M-Ag NPs on rice crops at the 4-leaf stage, and fungi were used as a positive control.

[0050] Note: (A), Effects of different treatments on plant height (B). Effects of different treatments on SPAD values. All values ​​indicate significant differences at P<0.05 and t P<0.01 levels.

[0051] Fig.19Schematic diagram of the safety of nanomaterials to rice crops. Effects of different treatments on fresh weight of shoots.

[0052] Note: (A) and root fresh weight (B), shoot dry weight (C) and root dry weight (D), root length (E), shoot length (F). All values ​​are significantly different at P<0.05 and P<0.001 levels, respectively. DETAILED DESCRIPTION

[0053] The present invention will be further described below in conjunction with specific embodiments.

[0054] 1. Synthesis and characterization of mold-synthesized silver nanoparticles (M-Ag NPs), i.e., fungal nanoparticles of the present invention

[0055] Silver nitrate (AgNO3) and sodium hypochlorite (NaClO) were purchased from Shanghai Chemical Reagent Co., Ltd., China. Y9511 was isolated from infected Leptochloa chinensis (L.) Nees and preserved in Guangdong Microbiological Culture Collection Center, Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, with the preservation number GDMCC60535. It has been disclosed in Chinese patent application CN201910128106.2 "A strain of Y9511 and its application in controlling paddy field weeds Leptochloa chinensis". All other reagents used in this experiment were of analytical grade, and distilled water was used in all experiments.

[0056] Fungal culture maintenance

[0057] The Helminthosporium orifice was subcultured on PDA medium and stored in a biochemical incubator (SPX-250) at 28°C in the dark for 7 days (Bernhardt, 1983). The Helminthosporium orifice culture pieces were cut from the active growth area at the edge of the 7-day culture plate with a corkscrew and inoculated into a conical flask containing Richard's medium.

[0058] Preparation of filtrate from wet fungal biomass

[0059] The flasks of Richard's medium containing spores of Helicoverpa orientalis were inoculated in the dark at 28°C and 160 rpm for seven days to allow the fungal biomass to grow. Then, 100 ml of Helicoverpa orientalis was washed with distilled water and the flasks were placed on a rotary shaker at 28°C and 160 rpm for 3-5 days. The cell filtrate (CFWFC) free of fungal cells was then separated and filtered with No. 1 filter paper.

[0060] Fungal synthesis of silver nanoparticles mediated by fungal filtrate

[0061] method:

[0062] Silver nanoparticles were produced by reducing silver nitrate (AgNO3) by fungal culture. When synthesizing M-Ag NPs, 50 ml of CFWFC was first mixed with 50 ml of 5 mM AgNO3 solution, and then silver nitrate (5 mM) was placed in a flask and stirred at 40-50°C at 160 rpm on a magnetic stirrer. The flask containing the AgNO3 solution served as a negative control, while the filtrate containing the mouth-protruding umbilical cord spore was a positive control. It was noted that the color of the reaction mixture changed from brown to dark brown. The final solution was centrifuged at 13,000 rpm for 15 minutes using a centrifuge to collect Ag NPs in the form of particles and discard the supernatant. The final particles were washed three times with pure water. The particles were air-dried at 40°C. Finally, the obtained M-Ag NPs powder was collected, calcined and stored in Eppendorf tubes.

[0063] result:

[0064] The synthesis of M-Ag NPs was observed by the color change of the filtrate. The synthesis of Ag NPs was carried out by combining the cell filtrate of Helicoverpa orientalis with AgNO3 solution. Initially, the filtrate of Helicoverpa orientalis was colorless. After the addition of AgNO3 solution, the colorless Helicoverpa orientalis mycelium-free cell filtrate immediately became slightly white turbid. This may be due to the presence of certain residual anions in the cell filtrate, which can easily react with Ag. + The ions form a soluble water salt. At a temperature of 40-50°C, after 3-4 hours, the turbid light yellow gradually turns into light brown. Finally, as the reaction time is extended to 3-4 hours, the dark brown gradually stabilizes, which is the characteristic color of the Ag nanoparticle solution.

[0065] Characterization of fungal silver nanoparticles

[0066] (1) Study on the production of silver nanoparticles by fungi using UV-visible spectroscopy, dynamic light scattering and zeta potential.

[0067] method:

[0068] The preliminary characterization of fungal synthesized M-Ag nanoparticles used a UV-Vis spectrometer with a wavelength range between 200 and 800 nm. For UV-Vis spectroscopic characterization, 10 mg / ml of M-Ag NPs were suspended in distilled water as this method is based on surface plasmon resonance and its optical properties to characterize the nanoparticles. Dynamic light scattering (DLS) and zeta potential were used to measure the average particle size and surface charge of the silver nanoparticles, respectively. To determine the particle size and zeta potential, an instrument, namely, Malvern Zeta sizer, was used. The dried powder of M-Ag NPs was suspended in distilled water and sonicated for 2 hours. All measurements were repeated three times at 25°C with an angle of 90°C and the temperature was allowed to equilibrate for 1 minute.

[0069] result:

[0070] The synthesis of Ag NPs was evaluated by observing the absorption peak at 450 nm using UV-visible spectroscopy. Figure 2 When M-Ag NPs powder (10 mg / ml) is suspended in water, an ultraviolet absorption peak appears at a wavelength of 450 nanometers and a visible absorption peak appears at a wavelength of 300-400 nanometers.

[0071] (2) X-ray diffraction analysis and Fourier transform infrared spectroscopy analysis

[0072] method:

[0073] X-ray diffraction (Bruker D8 Advance, Germany) was used to determine the crystal structure of the nanoparticles with a scanning angle of 2θ between 30° and 90° and a scanning speed of 0.05° / s. Fourier transform infrared spectroscopy (Nicolet IS10, Nicolet, USA) was used to identify the functional groups present in the AgNPs. The 400–4000 cm -1 The infrared spectrum was recorded in the middle wavelength range with a resolution of 0.01 cm -1 .

[0074] result:

[0075] X-ray diffraction spectroscopy was used in this experiment to determine the crystal properties of the synthesized Ag NPs. XRD can determine the crystalline phase of the nanoparticles. The diffraction peaks in the figure show that the Ag NPs synthesized by Helicoverpa orientalis have strong absorption peaks at angles of 31.8°, 32.7°, 43.6°, 55.0°, and 57.0°. The absence of other sharp peaks proves the purity of the sample. The unclear background noise is caused by the protein shell surrounding the nanoparticles.

[0076] The average particle size and surface charge of the nanoparticles were measured using DLS and zeta potential, respectively. The DLS results are based on the hydrodynamic diameter of the synthesized NPs, where the NPs are dissolved in the dispersant, forming non-covalent interactions, resulting in a larger particle size than that obtained using SEM and TEM techniques. DLS showed that the average particle size of the silver NPs was 128.58 nm. Figure 4 As shown in A, the stability of silver NPs was determined by measuring the zeta potential. The zeta potential of silver NPs was -24 mV ( Figure 4 B). Zeta analysis shows that charges are recorded on the surface of Ag NPs. These results indicate that the current fungal synthesis method can produce silver nanoparticles with good uniformity and a size of only a few nanometers.

[0077] Fourier transform infrared spectroscopy analysis predicted the presence of functional groups responsible for the reduction and capping of the synthesized nanoparticles based on the vibrations of different types of bonds present, which absorb different frequencies of light when infrared light is passed through the sample. -1 Various absorption peaks corresponding to different functional groups were detected at Figure 5 ). Hydroxyl at 544.60cm -1 The carbon-carbon stretching (C=C) of alkanes has an absorption peak at 994.78 cm -1 、1928cm -1 , carboxyl (OH), choline extension, strong amide (NH), strong amide extension (1777.48), aromatic compound (CH) extension (1650cm -1 ) and carbonyl stretch (1650.15cm -1 ) The absorption peak appears at . Based on the above analysis, it is concluded that the proteins and enzymes released by fungi can act as capping agents to stabilize the nanosilver particles.

[0078] (3) Scanning electron microscopy and high-resolution transmission electron microscopy (EDX)

[0079] method:

[0080] The structural and elemental analysis of Ag NPs was performed by scanning electron microscopy (SEM) with a voltage of 3-5 kV, a resolution of 50 nm, and a magnification of ×10K, using an energy dispersive X-ray (EXD) detector. SEM and EDX analysis were performed in a Hitachi Horbia EMAX mics2 (No. SU8010). During the analysis, a fine layer of sample was prepared on a carbon-coated grid, and the size and particle size distribution of silver oxides were analyzed using TEM (FEl Tecnai G2 F30, FEI Amercia) on the instrument Hitachi Horbia EMAX mics2. A drop of M-Ag NPs was dropped on a copper grid and the water was evaporated before observation. The sample was then observed under TEM.

[0081] result:

[0082] The surface topology of the synthesized Ag NPs was investigated using scanning electron microscopy. Figure 6 The micrographs shown indicate that the synthesized NPs are spherical in shape with diameters ranging from 30 to 50 nm and also show agglomeration.

[0083] EDX analysis can determine the elemental position of the sample. Elemental composition analysis by EDX shows that the signal of silver (Ag) is the strongest at 3keV, with an area of ​​100%, and the signals of C, N, O, P, S and Cl atoms are weaker, with atomic percentages of 22.38%, 4.71%, 56.01%, 5.60%, 0.17% and 0.53% respectively ( Figure 7 ). Optical absorption bands were recorded in the range of 1 to 9 kilovolts. However, M-Ag NP showed a peak at 3keV, indicating the surface plasmon resonance of the silver crystal. This also indicated its pure crystalline nature consisting entirely of silver ( Figure 8 ). EDX analysis of Ag NPs synthesized using fungi revealed the presence of other elements, including chlorine, phosphorus, oxygen, and sulfur. Various organic compounds present in the fungal cells are responsible for the production of these elements, which are used to convert the silver salts into nanoparticles.

[0084] The particle size distribution of M-Ag NPs was measured by TEM. The results showed that M-Ag NPs were monodisperse with a particle size range of 37 nm ( Fig. 9). This image shows individual silver nanoparticles as well as some aggregates. TEM results are based on electron scattering when the sample is irradiated. It can be seen that the silver nanoparticles are almost spherical. The TEM results show the core size of the NPs, and furthermore, the particle size distribution estimated from the TEM images shows that most of the particles have a size range of 30.50nm. In comparison to the DLS results, most of the particles have a particle size of 120.58nm. Therefore, the particle size recorded in the DLS is relatively large compared to the actual core size of the particles observed in the TEM images, which is due to the hydrodynamic diameter of the particles.

[0085] 2. Herbicidal activity of fungal-synthesized silver nanoparticles (M-Ag NPs)

[0086] 1. Screening test to evaluate the bioherbicidal potential of mold-synthesized silver nanoparticles (M-Ag NPs) under controlled conditions in a greenhouse

[0087] method:

[0088] In this experiment, silver nanoparticles mediated by Helminthosporium orientalis were used to control Leptochloa chinensis. The seeds of Leptochloa chinensis were collected from the Ningxi teaching practice base of South China Agricultural University in Zengcheng District, Guangzhou City, Guangdong Province. The seeds were surface disinfected with 2% sodium hypochlorite solution, then dried with a paper press and stored in a refrigerator. The indoor cultivation conditions of Leptochloa chinensis were 26±1℃, relative humidity 50-75%, and photoperiod 12L﹕12D (day and night temperature difference 35 / 25±5℃, photoperiod 12 hours).

[0089] In the experiment, Helminthosporium orientalis-mediated silver nanoparticles (M-Ag NPs) used the following concentrations of M-AgNP1; 50ppm, M-AgNP2; 60ppm, M-AgNP3; 70ppm, M-AgNP4; 80ppm, M-AgNP5; 90ppm to fight against Leptochloa chinensis. Positive and blank controls were also performed. The same concentration of fungal spore suspension without Ag NPs was used as a positive control, and the blank control treatment was sprayed with 0.5% (v / v) Tween 80 solution; each treatment was repeated three times.

[0090] result:

[0091] In order to study the nano-herbicidal effect of Helminthosporium orientalis, a preliminary screening experiment was conducted under greenhouse conditions ( Fig.10 After 14 days of treatment, the effects of M-Ag NPs on fresh weight control ( Fig.10 B) Fresh weight control efficiency ( Fig.10 C) and plant control efficiency ( Fig.10D) herbicidal activity. The results showed that M-AgNP4 had the highest plant control efficiency at a concentration of 80ppm, followed by M-AgNP5 at a concentration of 90ppm, compared with the blank control and the positive control. Similarly, there was a significant difference in the fresh weight inhibition effect of the positive control (81.99%) with M-AgNp4 (85.07%) and M-AgNP5 (92.72%), while the blank control had the highest biological herbicidal effect on Leptospermum chinense. These results indicate that nanoparticles mediated by Helicoverpa orientalis can be used to prepare biological herbicides, and the use of fungal synthesized nanoparticles can further improve the efficacy of Helicoverpa orientalis. According to the above results, these nanoparticle concentrations M-AgNP5 and M-AgNP4 mediated by Helicoverpa orientalis improved the biological herbicidal effect of Helicoverpa orientalis, and had the greatest biological herbicidal potential based on the toxicity control and the blank control, and can be used for further eco-friendly experiments.

[0092] 2. In vitro herbicidal activity analysis of M-AgNPs

[0093] method:

[0094] An experiment was designed in a greenhouse to determine the potential herbicidal activity of Ag NPs synthesized by molds against grass weeds. Pots filled with sterile sandy loam soil and weighing 10 kg were used. Four weeds were planted in each pot, each 10 / 15 cm long with drainage holes at the bottom. To determine the herbicidal activity, seven live Leptothrix plants were foliarly sprayed with different concentrations of Ag NPs, M-AgNP1: 70 mg / L, M-AgNP2: 80 mg / L, and M-AgNP3: 100 mg / L, from the three-leaf stage to the four-leaf stage with the help of an atomizer. After spraying, the pots were transferred to a dew chamber at 25 °C for 48 h and then to a greenhouse under controlled conditions for 14 days, with the controlled conditions being: 28 °C and a photoperiod of 12 L﹕12 D. The fresh weight of each plant was determined 14 days after inoculation. Positive and blank controls were also performed. The same concentration of fungal spore suspension without Ag NPs was used as a positive control, and the blank control was sprayed with 0.5% (v / v) Tween 80 solution; each treatment was repeated three times.

[0095] 1) Preparation of fungal conidia suspension or inoculum

[0096] After 14 days of culture, conidia of Helicoverpa orientalis were harvested from the PDA plate, and the hyphae were scraped into a 0.5% (v / v) Tween 80 solution in a test tube to prepare a conidia suspension with a concentration of 100 mg / L.

[0097] 2) Determination of fresh weight and plant control efficiency

[0098] After 14 days, the plant control efficiency and fresh weight control efficiency were investigated. At harvest, the yield parameters fresh weight and number of surviving plants were observed and recorded. Based on the survey data, the fresh weight control efficiency (%) and plant control efficiency (%) of each treatment were calculated according to the following formulas (1) and (2), respectively:

[0099] Ew = (W0-W1) / W0 (1)

[0100] En = (N0-N1) / N0 (2)

[0101] Among them, Ew and En represent the fresh weight control effect and plant control effect respectively; W0 represents the fresh weight of weeds in the control treatment; W1 represents the fresh weight of weeds in the treatment; N0 represents the number of weeds in the control; N1 represents the number of weeds in the treatment.

[0102] result:

[0103] Based on the results of the in vitro control screening experiment, the herbicidal effect of M-Ag NPs nanoherbicides was further evaluated. Fig.11 A) to 4-leaf stage ( Fig.12 A) were used for further experiments. At the three-leaf stage, M-AgNP3 had the highest plant control effect, followed by M-AgNP2. The fresh weight control effect showed the greatest effect at both the three-leaf and four-leaf stages compared with the blank control and the positive control. Similarly, in terms of fresh weight control effect, the difference between the positive control and the blank control was statistically significant, with both M-AgNP2 (97.20%) and M-AgNP3 (92.72%) showing higher bioherbicidal effects, while the fresh weight control effect of the positive control (96.64%) was based on the blank control as a reference. The control effect of M-AgNP1 was relatively low compared with the positive control. Therefore, the results showed statistical differences compared with the blank control. In addition, under the treatment of M-AgNP3, the leaf diameter measured by detached leaves showed that the leaves wilted sharply ( Fig.12 B).

[0104] 3. Determination of Reactive Oxygen Species (ROS) Levels in Weed Leaves

[0105] method:

[0106] 1) Determination of malondialdehyde (MDA) and hydrogen peroxide (H2O2) activity

[0107] Malondialdehyde activity was determined by using the method outlined by (Ghouri et al., 2023). 0.1 g of plant sample was ground in liquid nitrogen and the supernatant was prepared with 0.05 M phosphate buffer at pH 6.5. The supernatant was prepared with TCA (0.1%) and TBA (0.5%) and the absorbance was measured at 532 nm by spectrophotometer (Riaz et al., 2022). Hydrogen peroxide activity was determined using the protocol of (Ghouri et al., 2023). Leaves (0.25 g) were ground with 5 ml of TCA solution and the absorbance of the supernatant was measured at 390 nm by spectrophotometer (Jana and Choudhuri, 1982).

[0108] 2) Relative electrolyte leakage rate (REL%)

[0109] The relative electrolyte leakage (REL) of the selected weed plants was determined by following the protocol of Kaur et al. (2010). (Kaur et al., 2010; Ahmad et al., 2020). 200 mg of leaf tissue was added to sterile distilled water and soaked in a test tube at 25 °C for 2 hours, and then the electrical conductivity (EC) was measured. After the test tube containing the leaf material was boiled for 30 minutes, the conductivity (E2) was measured again. The relative electrolyte leakage (%) was calculated using the following formula:

[0110] REL(%)= E1 / E2 × 100 (3)

[0111] result:

[0112] The accumulation of reactive oxygen species was significantly increased in the leaves of Leptochloa chinensis treated with the silver nanoparticles mediated by Helicoverpa orientalis and the cell filtrate of Helicoverpa orientalis. This was evident from the increased levels of malondialdehyde (MDA), hydrogen peroxide (H2O2) and extravasation (EL%) in the weeds ( Fig.13 ). Compared with the control group, M-AgNP3 had the highest MDA accumulation of 85%, followed by M-AgNP2; 74%, M-AgNP1; 66%, and similarly, the MDA accumulation of the fungal culture filtrate treatment was 63%. All these treatments significantly increased the accumulation level of MDA content in weed plants, resulting in a significant decrease in the growth of weed plants ( Fig.13A). Similarly, a sharp increase in REL% was observed on weed leaves, with the REL% on weed leaves sprayed with fungal silver nanoparticles and fungal culture filtrate being 65%, followed by M-AgNP2; 56%, M-AgNP1; 55%, and fungal effect being 55%. M-AgNP3 showed the highest REL value in weed plants at 65%, followed by M-AgNP2; 56%, M-AgNP1; 55%, and fungal effect being 55%, compared to the control group. All these treatments showed a significant increase in electrolytic leakage of weeds ( Fig.13 C). To determine the ROS species in the leaves, O2 and H2O2 in Leptochloa chinensis were analyzed. The accumulation of the selected ROS species increased significantly in all treatments. Silver nanoparticles significantly increased the hydrogen peroxide levels in the weeds compared to the control. Compared to the control, the highest hydrogen peroxide activity level was calculated for M-AgNP3; 236μg FW-1, followed by the fungal filtrate at 200μg FW-1, and the calculated hydrogen peroxide activity levels for M-AgNP2 and M-AgNP1 were 183μg FW-1 and 180μg FW-1, respectively. Fig.13 B). All these treatments significantly increased the activity of hydrogen peroxide compared to the control. The growth inhibition efficiency was highest in weed seedlings, leading to systemic ROS accumulation in the leaves of weed seedlings. This suggests that all treatments had significant systemic effects on the physiological functions of Leptochloa chinensis.

[0113] 4. Determination of Antioxidant Enzyme Activities in Leptochloa crus-galli Leaves

[0114] method:

[0115] Preparation of enzyme solution: Weigh 0.5 g of weed leaves into a 10 mL centrifuge tube, add 5 mL of pre-cooled phosphate buffer and a small amount of steel beads, grind them into a homogenate using a -35 ° C freezer grinder, then add buffer to 10 mL to make up the volume, centrifuge at 6000 rpm for 15 minutes, and take the supernatant as the crude enzyme solution for various enzyme activities.

[0116] Prepare 0.05 mol / L phosphate buffered saline (PBS, pH 7.8): Take 228.75 mL of solution A and 21.25 mL of solution B, and dilute to 1 L with pure water. Add 10 g of polyvinyl pyrrolidone (PVP) and 0.029 g of ethylenediaminetetraacetic acid disodium salt (EDTA-Na2). This buffer is used for subsequent experiments. Solution A): 0.2 mol / L sodium sulfate dibasic solution; Na2HPO4·12H2O (molecular weight 358.14) 71.7 g; Solution B): 0.2 mol / L sodium dihydrogen sulfate solution. Take 31.2 g of NaH2PO4·2H2O (molecular weight 156.01), and dilute to 1 L with pure water for later use.

[0117] 1) Superoxide dismutase (SOD) activity assay

[0118] Reagent preparation:

[0119] 130mmol / L methionine (Met) solution: weigh 1.399g Met and dilute to 100mL with phosphate buffer.

[0120] 100μmol / L EDTA-Na2 solution: Weigh 0.0372g EDTA-Na2 and dilute to 1L with phosphate buffer.

[0121] 750 μmol / L nitroblue tetrazolium (NBT) solution: weigh 0.06133 g NBT and dilute to 100 mL with phosphate buffer.

[0122] 20μmol / L riboflavin solution: Take 0.0075g riboflavin and dilute to 1L with pure water.

[0123] Color reaction:

[0124] Take 5mL centrifuge tubes, 3 of which are sample tubes, 1 is a control tube, and the other is a blank tube, and quickly add each reagent according to the table. After mixing, place the blank tube in a dark place, and react the other tubes in the morning sunlight on a sunny day for 20 minutes, changing the direction from time to time to ensure that each tube receives the same light.

[0125] Table 1 Reagents and concentrations

[0126]

[0127] After the reaction is completed, the UV spectrophotometer is zeroed with a lightless blank tube, and the absorbance values ​​of the sample tube and the control tube at a wavelength of 560 nanometers are measured.

[0128] 2) Peroxidase (POD) activity assay (guaiacol method)

[0129] Quantification of peroxidase activity was performed by monitoring the dehydrogenation of guaiacol (Malik and Singh, 1980; Bukhari et al., 2021). For the preparation of crude enzyme extracts, 100 mg of frozen leaf powder was placed in 1 mL of phosphate buffer solution (PBS, pH 7.0). The mixture was centrifuged at 8,064 x g for 5 min at 5 °C. Enzyme extraction was performed in 3 mL of 100 mM PBS (pH 7.0) and stirred at 5 °C and 8,064 x g for 15 min. 3 mL of 100 mM PBS, 0.05 mL of 20 mM guaiacol, and 0.03 mL of H2O2 (12.3 mM or 0.04%) were added to 0.1 mL of plant extract and then stirred vigorously. The optical density was monitored at 436 nm and the absorbance changes were recorded over time.

[0130] 3) Catalase (CAT) activity assay

[0131] The crude enzyme extract was prepared by adding 0.5 g of frozen leaf sample to 25 mM potassium phosphate buffer (pH 7.8) composed of 1 mM ascorbic acid, 0.4 mM EDTA and 2% (w / v) polyvinyl pyrrolidone. The mixture was shaken at 15,000 rpm for 1 min to homogenize its contents, and the precipitate was removed by centrifugation at 15,000 g for 20 min. The supernatant was filtered through a syringe filter to clear the crude enzyme extract. 50 mM potassium phosphate buffer and 10 mM H2O2 were added to the crude enzyme extract. The determination of catalase enzymatic activity was performed by monitoring the decomposition rate of H2O2 at 240 nm (Aebi, 1984; Asim et al., 2022).

[0132] 5. Determination of Physiological Characteristics of Leptochloa chinensis Leaves

[0133] The chlorophyll content of leaves was measured using a SPAD-502plus (Konica Minolta Sensing, Japan). The SPAD value was determined using the fully expanded uppermost leaf. The average SPAD value was calculated from 10-12 readings (including 5 or 6 readings on each side of the midrib). These values ​​were then converted to milligrams of chlorophyll per square centimeter.

[0134] result:

[0135] The activity of antioxidant enzymes was investigated in this study to understand the mechanism of weed control by fungal-mediated bioherbicides. Catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) are antioxidant enzymes, and inhibition of antioxidant enzymes significantly increases the stress level of plants, leading to a significant decrease in plant defense mechanisms and plant growth. Different antioxidant tests were performed when treating Leptochloa chinensis with different concentrations of nanofungal particles, while fungal extracts were used as positive controls to evaluate the nature of oxidative stress in seedlings. The activities of catalase, superoxide dismutase, and peroxidase were significantly reduced in the presence of nanoherbicides compared to the control ( Fig.14 The peroxidase activity of all treatments decreased significantly, with M-AgNP3 showing the largest decrease (0.29 unit / g), followed by M-AgNP2 (0.39 unit / g) and M-AgNP1 (0.48 unit / g). Similarly, the peroxidase activity of the fungal filtrate was significantly reduced (1.4 unit / g) compared with the control group ( Fig.14 B). M-AgNP3 showed the largest decrease in catalase activity (1.5 μM / g(FW)), followed by M-AgNP2 (2.2 μM / g(FW)) and M-AgNP1 (2.6 μM / g(FW)). Similarly, fungal filtrate also showed a significant decrease in catalase activity to (2.5 μM / g(FW)) compared to the control group (5.6 μM / g(FW)) ( Fig.14 C). Similarly, all treatments showed a significant decrease in SOD activity. Compared with the control group (34 μg-1FW), M-AgNP3 (10 μg-1FW) showed the greatest decrease, followed by M-AgNP2 (13 μg-1FW) and M-AgNP1 (15 μg-1FW). Similarly, fungal filtrate also had a significant decrease effect (18 μg-1FW) ( Fig.14 A). The inhibition of SOD and CAT prevented the formation of OH from H2O2 and O2. The inhibition rate of chlorophyll content of M-AgNP3 was the highest, 88.1%, followed by M-AgNP2; 84.6%, M-AgNP1; 83.8%, while the inhibition rate of fungal culture filtrate was 81.1% ( Fig.14 D) ROS production is locally enhanced, thereby trapping invading pathogens. Likewise, chlorophyll was significantly reduced in all treatments compared to the control.

[0136] 6. Determination of Cell Wall Degrading Enzyme Activity in Leptochloa crus-galli Leaves

[0137] method:

[0138] About 6-7 cubes of Helminthosporium orificeum were cut from the edge of the PDA plate with vigorous growth and grown in 100 mL Richard liquid medium, cultured in the dark at 30°C and 160 rpm for 48 hours, then filtered and centrifuged, and the supernatant was stored in a refrigerator at -20°C for later use. The dry weight of mycelium was calculated once for each sample and repeated three times. Galactose acid standard curve and glucose standard curve were drawn.

[0139] Substrate preparation: After the substrate is prepared, store it in a refrigerator at 4°C, and prepare the buffer and substrate solution from scratch. The substrate preparation method is as follows. (1) Polygalacturonase (PG) substrate: Prepare a polygalacturonase solution with a concentration of 10 g / L and 1.00 g of polygalacturonic acid, dissolve it in 50 mol / L, pH 5.5 sodium acetate buffer, and reduce the volume to 100 mL. (2) Pectin methylgalacturonase (PMG) substrate: Prepare a pectin solution with a concentration of 10 g / L, weigh 1.0 g of pectin, dissolve it in 50 mol / L, pH 5.5 sodium acetate buffer, and make up to 100 mL. (3) Cellulase (Cx) substrate: Prepare a 10 g / L carboxymethyl cellulose solution, weigh 1.00 g carboxymethyl cellulose solution and dissolve it in 50 mol / L citric acid-sodium citrate buffer at pH 5.0, heat until dissolved, and dilute to 100 mL. (4) β-glucosidase (βG) substrate: 10 g / L salicylic acid solution 1.00 g salicylic acid (CMC) is dissolved in 50 mol / L citric acid-sodium citrate buffer at pH 4.5, and dilute to 100 mL.

[0140] Determination of polygalacturonase (PG) and pectin methylgalacturonase (PMG) activities

[0141] The 3,5-dinitrosalicylic acid method (DNS) was used to determine PMG, PG, CX and βG (PMG substrate is pectin, PG substrate is polygalacturonic acid). (βG substrate is salicylic acid, CX substrate is carboxymethyl cellulose). The above treatment was repeated 3 times ( Fig.15 ). The in vitro activities of PG, PMG, βG and CX can be calculated as follows:

[0142] Enzyme activity = (m×5.14×V') / (V×t×m')(4)

[0143] m: mass of galacturonic acid in the standard curve (unit: mg);

[0144] 5.14: micromoles of galacturonic acid in 1 mg (1000 / 194.14);

[0145] V': total volume of enzyme solution after mycelium filtration (unit: ml);

[0146] V: total volume of sample taken during measurement (unit: mL);

[0147] t: enzyme reaction time (unit: minutes); m': mass of dry mycelium (unit: mg).

[0148] result:

[0149] Based on the transcriptome sequence of Exserohilum rostratum, cell wall degrading enzymes play a key role in improving its pathogenicity against L. chinensis. The main cell wall degrading enzymes belonging to the Cazymes family are PG, CMC, CX, and βG, which are significantly involved in the pathogenicity of Exserohilum rostratum to L. chinensis. Therefore, this experiment also determined the effect of Exserohilum rostratum-mediated silver nanoparticle cell wall degrading enzymes on L. chinensis, paving the way to determine the virulence mechanism of M-AgNPs on L. chinensis. This experiment also evaluated the recovery of fungal properties during the synthesis of silver nanoparticles. The results showed that the activity of cell wall degrading enzymes was significantly increased after the treatment of fungi and M-Ag NPs. After application of M-Ag NPs, polygalacturonase (PG) activity increased significantly, with the same effect on fungus-stressed weed plants, while M-AgNP2 showed a decrease in PG activity (0.42) and positive plants (E. rostratum) (0.36). PMG did not show a significant increase in polymethylgalacturonase activity ( Fig.16 B). Application of AgNP3 slightly increased CX activity, while low concentrations significantly decreased CX activity of the fungus. Application of M-AgNP3 slightly increased polygalacturonase (CX) activity, with the same effect on fungus-stressed weed plants, while M-AgNP2 showed a decrease (0.235) and a decrease (0.267) in positive plants (E. rostratum) ( Fig.16 C).

[0150] 7. Cytological Observation of Rhizobia and Effects of M-Ag NPs on Leptochloa chinensis

[0151] method:

[0152] The transmission electron microscope was JEM1200; the resin was an embedding kit from SPI; the anhydrous ethanol and acetone reagents were analytical grade AR produced by Sinopharm Chemical Reagent Co., Ltd.; glutaraldehyde was from Solarbio; and the infiltrating acid, uranyl acetate, and lead citrate were analytical grade AR produced by Ruixin Laboratory Supplies Co., Ltd.

[0153] After fixing the leaves of Leptochloa spp. with 2.5% glutaraldehyde solution at 4°C overnight, they were processed as follows. First, the fixative was poured off and the samples were rinsed three times with 0.1M, pH 7.0 phosphate buffer for 15 minutes each time. The samples were then fixed with 1% heavy acid solution for 1-2 hours. The heavy acid waste was carefully removed and the samples were rinsed three times with 0.1M, pH 7.0 phosphate buffer for 15 minutes each time using five concentration gradients, including 30%, 50%, 70%, 80%, 90% and 100%. The samples were dehydrated with different concentrations of ethanol solutions, each for 15 minutes, and finally transferred to pure acetone for 20 minutes. The samples were then treated with a mixture of acetone and resin (3:1) for 2 hours, and then dehydrated with a mixture of acetone and resin (1:1): 1) for 3 hours, acetone and resin (1:3) for 3 hours, and finally treated overnight in pure resin; the infiltrated samples were placed in a mold and heated in a gradient: 35℃-60℃-80℃, 5 hours per step, to finally obtain embedded samples. After rough trimming, 70-90nm sections were cut using a LEICA EMUC7 ultramicrotome. The sections were stained with dioxyacetic acid (15 minutes) and lead citrate (5 minutes), then dried and photographed.

[0154] result:

[0155] Transmission microscopy confirmed cytological damage in leaves of Leptochloa spp. Abnormalities such as cell deformation, greater degree of demorphogenic vacuolation, plasma membrane disruption and intercellular matrix dispersion, microvesicle and endoplasmic reticulum abnormalities, chloroplast disruption and deformation were observed in both chlorophyll-stressed and nano-stressed plants. Fig.17 In addition, cell and protoplast damage is more severe than that of fungi ( Fig.17 B). Both treatments significantly damaged the cell walls and organelles of the leaves of Leptochloa chinensis. The lipid bodies in the cells were heavily stained, which is due to the lack of peroxisomes caused by lipid metabolism disorders ( Fig.17 C).

[0156] 8. Evaluating the safe administration of nanoparticles to rice crops

[0157] method:

[0158] The safety of nanomaterials to plants requires determining the effects of nanomaterials on plant biological capabilities.

[0159] To evaluate the safety of nanotechnology on crops, rice was chosen. The rice seeds were first surface sterilized with 2% sodium hypochlorite and then washed three times with distilled water. The study used the petri dish filter paper method. The rice seedlings were then moved into soil-filled pots and hydroponic chambers for further growth under controlled conditions in a greenhouse.

[0160] 1) Determine growth parameters

[0161] To determine the safety of the nanomaterials to rice plants, in vitro pot experiments were conducted, while hydroponic experiments were also conducted to evaluate the root characteristics. At the harvest stage, the rice plants were removed from the potting soil and hydroponic chambers and various growth parameters such as plant height, root and shoot length, plant fresh weight, dry weight, root fresh weight, and root dry weight were evaluated using standard measurement techniques.

[0162] 2) Physiological property determination

[0163] Leaf chlorophyll content was estimated using a SPAD-502plus (Konica Minolta Sensing, Japan). The fully expanded uppermost leaf was used to estimate the SPAD value. The average SPAD value was calculated from ten readings (including five readings on each side of the midrib).

[0164] result:

[0165] Under the biotic stress of Helicoverpa orientalis, the height, root length, stem length, plant height, root fresh weight and dry weight of rice plants were not significantly reduced compared with healthy plants ( Fig.18 ). However, after foliar application of silver nanoparticles, morphological parameters were significantly enhanced at a concentration of 70 mg / L. The greatest positive effects were observed at a concentration of 70 mg / L. At this concentration, plant height increased significantly: 33.5 cm at 70 mg / L and 80 mg / L, and 30 cm at 100 mg / L ( Fig.18 A, B), the stem length reached the maximum at 70 mg / L: 23.5 cm ( Fig.18 B), and 21.3 cm at 100 mg / L, while the fresh and dry weights of the roots decreased slightly at 80 mg / L and 100 mg / L concentrations ( Fig.19 A). Meanwhile, the effect of Helminthosporium orientalis as a positive control showed similar effects on plant morphological attributes, the effects were the same as those of healthy plants.

[0166] The results of this experiment showed that the nanoparticle treatment significantly increased the SPAD value of weeds. The effect was most obvious at a concentration of 70 mg / L. The SPAD value of the leaves of Leptochloa chinensis treated with M-AgNP1 increased significantly, while the SPAD value of the leaves treated with M-AgNP3 decreased slightly. The effect of fungal filtrate and M-AgNP2 was the same as that of healthy plants ( Fig.18 B).

Claims

1. A fungal nanoparticle, characterized in that: It is prepared by reacting the cell filtrate of Helicoverpa orientalis with silver nitrate.

2. The fungal nanoparticles according to claim 1, characterized in that: The fungal nanoparticles are spherical and have a diameter between 30 and 50 nanometers.

3. The fungal nanoparticles according to claim 1, characterized in that: The cell filtrate of Helicoverpa orientalis needs to be free of hyphae.

4. A method for synthesizing fungal nanoparticles, characterized in that: The cell filtrate of Helicoverpa orientalis was reacted with silver nitrate to prepare fungal nanoparticles.

5. The method for synthesizing fungal nanoparticles according to claim 4, characterized in that: Take 50 ml of the cell filtrate of Helicoverpa orientalis and mix it with 50 ml of 5 mM AgNO3 solution for reaction.

6. The method for synthesizing fungal nanoparticles according to claim 4, characterized in that: The reaction temperature is 40-50°C, the reaction time is 3-4 hours, and a dark brown reaction product is obtained.

7. Use of the fungal nanoparticles according to claim 1, 2 or 3 as nanoherbicides.

8. Use of the fungal nanoparticles according to claim 1, 2 or 3 as nanoherbicides for controlling grass weeds.

9. Use of the fungal nanoparticles according to claim 1, 2 or 3 as nanoherbicides for controlling Leptochloa chinensis.

10. Nano herbicide, characterized in that: The main ingredient contains fungal nanoparticles obtained by reacting the cell filtrate of Helicoverpa orientalis with silver nitrate.

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