Nano silver, nano copper oxide treat blast fungus in rice and brown spots on citrus

The biosynthesis of nanoparticles (NPs) from green fungi is a promising environment-friendly method for large-scale production. In this study, silver (Ag), copper oxide (CuO) and zinc oxide (ZnO) nanoparticles were biosynthesized using the cytological filtrate of a Trichoderma harzianum strain as reducing and stabilizing agent. . The structure, morphology and physical and chemical properties of NPs are characterized through transmission electron microscopy, dynamic light scattering, wide-angle X-ray scattering and gravimetric thermal analysis. Since nanotechnology can provide promising applications in the agricultural sector, we evaluated the ability of nanoparticles to reduce the growth of important plant pathogens such as Alternaria Alternata. (causes brown spot disease in citrus), Pyricularia oryzae (causes rice blast disease) and Sclerotinia sclerotiorum (causes rot disease). Nano silver and nano CuO  significantly reduced A. Alternata and P. oryzae mycelium growth depending on the dose. This is the first report of multi-nanoparticle extracellular biosynthesis from T. harzianum and the first time to obtain CuO and ZnO nanoparticles from this fungus. In addition, we emphasize the potential of nano silver and CuO to control plant pathogenic fungi. On the other hand, these three types of nano can be easily and sustainably produced on a large scale with the opportunity to have many applications in biotechnology processes.

Nano silver treat blast disease in rice

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1. Introduce

Nanoparticles (NPs) are microscopic materials with dimensions less than 100 nm, of great interest because they can be used in a number of processes involving materials science, agriculture, real industry. cosmetics, cosmetics, medical and diagnostic applications 1.
Inorganic nanomaterials can be synthesized from pure metals such as aluminum, silver, gold, cadmium or from metal oxides such as CuSO 4, TiO 2 and ZnO 2. Among them, the silver nanoparticles (AgNPs) were marked for their antiviral and antibacterial properties 3. Some NPs, such as metal oxides, play a very important role in the manufacture of sensors, 4 surface coating catalysis processes, medical area or antibacterial, as well as 5.
Conventional physical and chemical methods for the synthesis of metallic NPs, high in cost, low yield, and requiring the use of hazardous solvents that produce hazardous products for the environment 6. Biological methods have been and are emerging as an economically viable option to be produced in large quantities at the same scale at a reasonable cost for their simplicity and environmentally friendly methods. 7.
Absence of organic solvents and biosynthesis in the aqueous phase under room temperature and pressure are the main advantages 8. Bacteria, algae, plants, diatoms and fungi are considered NPs production plants because of their properties as reducing agents and stabilizers 9. Fungi are of particular interest due to the rapid growth of mycelium. , increase surface area, easy to handle biomass and large-scale culture. In addition, their ability to secrete a significant amount of protein amplified the 10 nanoparticle synthesis yield.
The genus Trichoderma (Ascomycetes, Hyprocreales) contains species of great economic importance because they produce industrial enzymes, antibiotics and bioactive metabolites 11. In particular, Trichoderma harzianum is the main mycoparasite used to combat pathogens in plants. A literature review has shown that several species of Trichoderma, including T. harzianum, that can synthesize AgNPs, have great potential as antimicrobial agents 12, 13, 14. However, their ability to synthesize metal oxide-based nanomaterials, which may have many applications, has yet to be explored 15, 16.
In agriculture, the use of metal nanoparticles has great potential to contribute to the improvement of current and future crops 17. They may be involved in plant pest inhibition, monitoring or detection of plant diseases and are a good alternative to reducing the dosage of chemical products as a pesticide. Importantly, the nanoparticles can improve drug delivery or slow down the release of the active ingredient to increase their effectiveness 18.
Nano silver have antibacterial activity against bacteria, fungi and viruses 3. In agricultural production, the use of bulk copper compounds has been successful in minimizing plant diseases caused by fungi and bacteria. They are cost-effective and low-risk in creating resistance to the pathogen, due to their multi-site mode of operation 19. In particular, copper NPs induces changes in the expression of proteins as a key to inhibiting microbial growth 20. Furthermore, the ZnO NPs have been characterized to be effective against microorganisms, and mainly due to their antimicrobial properties based on photo-oxidation and photocatalytic effects and are considered biosafety. 21.
Fungi are the main cause of about 70% of plant diseases22, causing significant damage and negative economic effects in certain crops affecting agricultural systems worldwide 23, 24. Therefore, some representative fungi species massively infect different crops are: Sclerotinia sclerotiorum, affecting production of soybeans, tomatoes, lettuce, beans and sunflower 25, Pyricularia oryzae most important pathogen on cereals such as rice and wheat 26 and Alternaria Alternata, are a broad host range of pathogenic, opportunistic and pathogenic fungi that causes leaf spots and blight on many parts of the plant.

In this context, although considerable work has been done to examine the effects of NPs on plant pathogens, a few of which have been done with biosynthetic NPs 28. Therefore, the aim of this study is to attempt to evaluate the cytoplasmic extraction potential from the biocontrol bacterium T. harzianum as a reducing agent and stabilizer for the synthesis of NP Ag, CuO and ZnO. . To the best of our knowledge, very few reports of using only T. harzianum in the synthesis of AgNPs 13, 14, 29 and no reports from this fungus on the synthesis of NP CuO and ZnO from metal sulfate salts. type. Our results demonstrated the successful formation of three metallic NPs from the same strain of T. harzianum using a simple green process. Biological NPs are characterized to validate synthesis and structure, and to define parameters including size distribution, multiple percentile index, zeta potential, and morphology. Furthermore, their antifungal activity against plant microbial strains A. Alternata, P. oryzae and S. sclerotiorum was demonstrated.

2. Materials and methods to synthesize nano silver, nano CuO, and nano ZnO

2.1. Fungal strains and biomass production

A strain of Trichoderma harzianum from INBIOTEC Culture Collection IB-363 was renewed in a 9 cm diameter Petri dish containing potato-dextrose agar (Britania) at 24 ° C for 7 days. To create fungal biomass for the synthesis of nanoparticles, two agar plugs of young mycelium were harvested from the plate and transferred to 500 ml flasks containing the liquid medium as follows: KH 2 PO 4 (7 g L −1); K 2 HPO 4 (2 g L −1); MgSO 4 · 7H 2 O (0.1 g L −1); (NH 2) SO 4 (1 g L −1); yeast extract (0.6 g L −1); glucose (10 g L −1). The flasks were incubated in an orbital shaker at 24 ± 2 ° C and stirred at 150 rpm for 72 hours under dark conditions. After growth, approximately 10 g of mycelium is harvested through a plastic sieve, washed with twice sterile distilled water to remove the culture media components from the biomass. The fungal biomass was exposed to 150 mL -1 of twice sterile distilled water for 48 hours at 22 ° C in a 500 mL -1 flask. After incubation, the fungal biomass was separated from a water-free culture filtrate (CFCF) by passing it through a Whatman no filter paper. first.

2.2. Synthesis of metal nanoparticles

Nano silver, copper oxide and zinc oxide are synthesized using 50 ml of aqueous CFCF in flasks by stirring their respective metal saline solution. Accordingly, the amount of AgNO3, CuSO4.5H2O and ZnSO4.7H2O is added to CFCF to create a solution with a final concentration of 1–2 mM. The reaction is carried out under dark conditions at 45 ° C with strong agitation. The effect of pH on the synthesis of nanoparticles has been studied through experiments on the range from 6 to 12.
Formation of nanoparticles was observed by their color change and further confirmed using the various techniques described below. Also, the positive control of CFCF without silver nitrate, copper or zinc sulfate, and their respective negative control, 1–2 mM per metal salt on deionized water, were also examined for comparison. Nanoparticles were separated by centrifugation, (8,000 rpm for 10 minutes) were washed twice with distilled water twice and dried by freeze-drying for 24 hours. Then, NP was stored in polypropylene pipes at room temperature and dark conditions.
2.3. Physical and chemical properties of metal nanoparticles
Suspensions of silver, CuO and synthetic ZnO nanoparticles were diluted in ultra-pure water, ultrasound for 20 min at room temperature and their size distribution measured by dynamic scattering of the scattering intensity. lighting (DLS) using the Zetasizer Nano ZS, (Malvern Instruments Ltd., UK). The measurements provide the mean hydrodynamic diameters of the particles, the peak values ​​in the hydrodynamic diameter distribution, and the polygonal dispersion index (PdI) describing the width of the particle size distribution.
The PdI scale ranges from 0 to 1 (where 0 is monodisperse and 1 is polydisperse) 30. All measurements are performed three times with a temperature equilibrium time of 1 min at 25 ° C with an angle of 90 °. C. The data processing mode is set to high multimodal resolution. Thermal gravimetric analysis (TGA) was performed to determine the thermal degradation of NPs using the TGAQ500 V20.13 Build 39 (TA Instruments Co., USA) with approx. 10 mg of sample, under N2 medium and at the the heating temperature is 5 ° C from room temperature to 900 ° C under the airflow.
Wide angle X-ray scattering measurements (WAXS) of powdered silver nanoparticles, CuO and ZnO were recorded using XEUSS 2.0 XENOCS. The samples were registered with an X-ray detector with a 100 k 2D Pilatus photon count pixel (DECTRIS, Swizerland) positioned near the sample at an angle of 36 °. The scattering intensity, I (2θ), is recorded as a function of the scattering angle 2θ where λ = 0.15419 nm is the weighted average of the X-ray wavelength of Cu-K α12 emission lines. Due to the small beam size in the sample (1 mm × 1 mm), the blurring effect is not taken into account. The samples were kept under vacuum in two kapton windows. Measurements are performed in transmission mode. Each sample was collected in two different samples to detect distances for 10 minutes, to cover a range of 15 ° to 60 ° 2θ.
Morphology of transmitted electron microscopy (TEM) nanoparticles was performed to prepare a drop of sample of NPs Ag, CuO or ZnO in water included in a carbon-coated copper mesh, dried and kept. in a vacuum before uploading the sample holder. The image obtained by the device JEOL, JEM-2100 (Japan) operates at an accelerated voltage of 200 kV with an energy dispersion spectrometer (EDS). Microscopic images obtained at 150,000 × magnification and particle size distribution of NPs were evaluated using ImageJ 1.45 software.
2.4. Biological activity of synthetic NP
The effects of nano silver, CuO and biosynthetic ZnO on mycelium growth on plant pathogenic fungi A. Alternata, P. oryzae and S. sclerotiorum from the INBIOTEC culture collection were evaluated. . Therefore, a poisson test was performed placing a 5 mm mycelium node of each phytopathogen in the center of a Petri dish containing potato-dextrose agar supplemented with 5, 10 and 20 ppm of each nanoparticle and Keep for 7 days at 24 ± 2 ° C under 12 h L / D optical cycle. Negative control cultures of fungal strains grew only in potato-dextrose agar. After that time, the diameter of the mycelium was measured. The growth effects of individual NPs synthesized from T. harzianums stress were evaluated based on their own development. All tests were performed in three times.
2.5. Statistical analysis
In toxicity tests, mycelium growth was compared with their respective negative control, and experimental data were statistically analyzed by one-way analysis of variance (ANOVA). Mean values ​​and standard deviations were calculated and checked by the Tukey test at p <0.05. Analysis was performed using GraphPad Prism v software. 6.0.

3. Results and discussion of nano silver, nano CuO, and nano ZnO

3.1. Biosynthesis and characterization of nanoparticles
From the water-based CFCF of T. harzianum, silver, copper and zinc oxide NPs, have been successfully synthesized in separate experiments after adding 1–2 mM silver nitrate, copper sulfate or zinc sulfate. The formation of metallic NPs was demonstrated by changing the CFCF color suspension after adding their respective salts under continuous stirring at 45 ° C under dark conditions. Although pH responses of 6 to 12 were evaluated, rapid precipitation of the NPs was observed within 10 minutes, when the initial CFCF at pH 6 was alkaline with NaOH to pH 12.
Immediately notice a decrease of Ag + to Ag 0 by CFCF. after adding AgNO 3, it changes from light brown to colloidal dark brown due to the formation of AgNPs 30, 31. Meanwhile, the formation of NP CuOs occurred after the reduction of sulfate ions from Cu +2 to Cu 0, which was observed by the immediate change of the blue CFCF solution to a light brown yellow color. The color turns dark brown colloidal after continuous stirring 32. In contrast, colorless to pale white is observed in CFCF after addition of ZnSO 4, where Zn +2 is reduced to Zn 0 to form a solution containing ZnO NP demonstrated by a fine white powder precipitate 33 , after the same condition as mentioned above. Therefore, this is the first work in which a single T. harzianum CFCF can synthesize three metallic NPs under the same conditions.
3.2. Physical and chemical properties of biological nanoparticles
The size distribution of biological AgNPs silver nanoparticles from T. harzianum determined by DLS showed mean hydrodynamic diameter of 582.4 nm ± 112.78 nm with PDI value 0.36 ± 0, 07 and the mean diameter Z is 657.93 nm ± 127.62 nm. The distribution profile is single (Figure 1 a) shows low size change and good physical and chemical stability. In the case of CuO NPs, two peaks detected (Figure 2a) oscillating the mean size peak1 of 276.43 nm ± 76.77 nm and peak 2, 58.87 nm ± 10.00 nm with the The PDI was 0.49 ± 0.06 and the mean diameter Z was 249.30 nm ± 61.24 nm. For ZnO NPNs, only one peak of size 517.8 nm ± 50.1 nm and mean diameter Z 792.4 nm ± 60.5 nm with PDI value 0.563 ± 0.05 and fractional Narrow distribution of NPN ZnO size is shown in turn.

Figure 1 Size and morphological distribution of silver nanoparticles

Figure 1 Size and morphology distribution of bio-synthesized silver nanoparticles by Trichoderma harzianum strain IB-363. (A) dynamic light scattering (DLS, n = 3), (b) nanoparticle tracking analysis (n = 3), and (c) transmission electron microscope (TEM) combined with analysis Energy dispersive X-ray spectroscopy (EDS) (d) WAXS pattern, White stars represent Ag 2 O diffraction peaks (COD: 00-101-0486).

Figure 2 Size distribution of CuO nanoparticles

Figure 2 Size distribution of CuO nanoparticles biosynthesized by Trichoderma harzianum fungus strain IB-363 (a) dynamic light scattering (DLS, n = 3), (b) nanoparticle tracking analysis (n = 3), and (c) transmission electron microscope (TEM) together with energy dispersion X-ray spectroscopy (EDS) (d) WAXS sample for NP CuOs. The stars correspond to diffraction CuO 1 (00-721-2242) and red arrows for Cu (OH) 2 (00-900-9157).

Figure 3 Size distribution of ZnO nanoparticles

Figure 3 Size distribution of biosynthetic ZnO nanoparticles with Trichoderma harzianum fungus strain IB-363. (A) dynamic light scattering [DLS, n = 3), (b) nanoparticle tracking analysis (n = 3), (c)] transmission electron microscope (TEM) combined with analysis energy dispersion X-ray spectroscopy (EDS) (d) WAXS form for the NPs of ZnO .

For example, the weight loss of CuO NP is about 7%, which shows the thermal stability of the compound more than 90% at 400 ° C 35. For both NPs, the composition change is completed at 400 ° C , in which the steady weight reduction is up to 900 ° C. For example, in the case of NP ZnOs, a high rate of mass loss continuously occurs in the range 300 to 900 ° C in the 15% range, due to water evaporation is absorbed on the surface, and may be at higher temperatures due to the decomposition of zinc sulfate (Figure 3 b). This is the first report of bio-synthesized CuO or ZnO NPs, through the TGA method using CFCF from Trichoderma in combination with sulfate salts. According to the document, only for ZnO, a similar type of decline, as observed in our study, is consistent with Moharran et al. 36 where NPs is prepared by a physical and chemical method based on the hydrolysis and condensation of zinc acetate dihydrate by potassium hydroxide in an alcohol medium at low temperature.
XRD analysis confirmed the crystal nature of the NPs with peaks corresponding to nano silver, Cu and Zn. The XRD pattern for AgNPs (Fig. 1 d) revealed the nano noise at diffraction peaks at 38,11, 46,18, 63,44, 77,21 and 2.11 were assigned. The WAXS model shows that the vertices coincide with the calculated form of Ag 2 O block. As shown in Figure 2 d for CuO NPs, four strong absorption peaks can be observed at angles 29, 37, 44 and 62. , according to Khatami et al. . 5. The WAXS form, which presents the vertices coinciding with the calculated form of simple CuO, The complementary vertices have been identified as Cu (OH) 2, with a rhombohedral crystal structure. For the NPs of ZnO, as seen in Figure 3d, the WAXS form shows all the vertices that match the computed form of the hexagonal ZnO. The difference in width shows the formation of anisotropic structures, with preferred development in the 002 direction.
Transmission electron microscopy images confirmed that the three types of metal-synthesized NPs are in the nanoscale range. The shape of the nano silver is almost spherical with few aggregates and a distribution of sizes from 5 to 18 nm (Figure 1 c). Two explanations of this observation can be proposed: first, agglutination may occur due to a possible accumulation of proteins and enzymes, which may be secreted during biosynthesis, and second Second, the filtration process can prevent the passage of various protein molecules produced during biosynthesis. The same nanoparticles, obtained from our T. harzianum strain, are similar in shape and size to those observed by Guilguer et al. 13 and Elamawi et al.30 from T. harzianum and T. longibrachiatum respectively.
Dispersed and elongated fibers were shaped for the CuO NPs that were observed (Fig. 2 c) and in size ranged from 38 to 77 nm in width and 135–320 nm in length. Therefore, the morphology of bioavailable CuO NPs synthesized in our study from T. harzianum is the first to date reported. In the literature, the most commonly biosynthetic CuO NPs are mainly from different plant species, in which spherical or cubic forms have been observed 37. There are very few reports of CuO NPs synthesized from Trichoderma species. The most relevant of our research is intracellular synthesis from T. koningiopsis, in which NPs are obtained from dead biomass, where a few spherical aggregates with an average diameter of 87, 5 nm.38.
More recently, the CuO NPs were synthesized from the CFCF of T. asperellum in a dense and spherical agglutination like shape 12. Zhu et al. Observed a similar morphology of CuO NPs. 39 people prepared CuO NPs by adding solid NaOH to an aqueous solution of copper acetate after heating the solution up to 100 ° C from room temperature. These conditions formed elliptical particles of diameter. 100 nm is similar in our work. This process regulates nucleation and growth of CuO particles resulting in the aggregation of CuO crystals and the production of highly dispersed ellipsoite particles. Furthermore, Alishah et al. 40, the extended CuO NPs observed under hydrothermal synthesis conditions had an average diameter of 328.27 nm.
The morphology of the biological ZnO NPs resembles a fan-shaped structure and a bouquet as shown in Figure 3 c. And the dimensions are between 27–40 nm wide and 134–200 nm. As proposed by Ludi and Niederberger 41, this type of NPs structure is observed only in inorganic processes of crystal formation using benzyl alcohol, in which the nanocrystals are primary and the bonding. has orientation and their resurfacing occurs inside agglomerates. In addition, Pacholsky et al. 42 reported that in solutions containing alkaline water, the adsorption of OH or H 2O as well as antionic, should differ. Therefore, different surface charges may interfere or permit orientation attachment in different planes, as it occurs during crystal growth during biological mineralization. It has been determined that the pH plays an important role in modeling the morphology and size of the NPs as well as the pH increases, the number of nucleation centers also increases by 43. Therefore, our study is the first report on the abnormal geometry of bio-synthesized ZnO NPs using T. harzianum’s CFCF determined by an alkaline medium.
As expected, the difference between the dimensions determined by the DLS and the TEM is due to the characterization technique. Although the DLS showed the hydrodynamic radii of the nanoparticles, the TEM showed their actual size. Therefore, DLS is larger than the actual size of the nanoparticles.
It is known that the effects of the reaction conditions can have different effects on the morphology of the synthetic nanocrystals. The conditions of our work are simple and affordable to meet, due to the temperature around 45 ° C and the alkaline conditions after NaOH addition up to pH 12; all of these induce rapid precipitation of the three types of NPs.
3.2. Potential of nano silver, nano CuO, and nano ZnO to control plant fungi
The potential of silver, CuO and ZnO nanoparticles to control A. Alternata, P. oryzae and S. sclerotiorum was tested when plating fungal culture media supplemented with 5, 10 and 20 ppm of each of the NPs and sugars. Glass of mycelium growth is measured after 7 days. Both NP Ag and CuO significantly impaired mycelium growth of A. alterta and P. oryzae at dose-dependent concentrations. As shown in Figure 4 a under Ag treatment, the mycelium diameters of A. alterta decreased by 18% at 5 ppm, 42% at 10 ppm and 52% at 20 ppm. On the other hand, mycelium growth of P. oryzae was reduced by 22%, 46% and 68% for each NP dose respectively.
Furthermore, when CuO was treated, a similar inhibitory effect compared with nano-silver was observed (Fig. 4 b). Both the Ag and CuO treatment, although no effect on the mycelium diameter on S. sclerotiorum was observed, a small mycelium and small sclerotia with distribution at the edge of the plate were observed ( not shown). On the other hand, when treated with ZnO NPs, although there was a tendency to reduce mycelium growth of plant pathogens, these results were not statistically significant (data not shown).

Figure 4 Effects of inhibiting mycelium growth

Figure 4 Inhibiting effect of mycelium Alternaria Alternata and Pyricularia oryzae on potato-dextrose agar containing (a) AgNP or (b) CuO NP for 7 days.
Interestingly, the three biosynthetic NPs did not show adverse effects on T. harzianum growth at all tested concentrations, compared with their negative control. As has been reported for fungi such as T. harzianum and T. asperellum are able to metabolize various heavy metals 44, so it is not surprising if T. harzianum can be applied in combination with particles. nano, but the development will not be affected.
Although the effects of AgNPs toxicity have been demonstrated against 14 different plant pathogens, it is the first time that A. Alternata, P. oryzae and S. sclerotiorum have been tested concurrently with three types of NPs. synthesized from a single T. harzianum strain. We propose, due to the same toxic effect between silver and copper nanoparticles compared with these phytopathogens.
Since the ZnO NPs are not significantly toxic to the fungal pathogens tested in this study, other microorganisms may be considered targets for further trials. Furthermore, these NPs have been reported to be very effective antimicrobial agents against a wide range of bacterial species, and because of their high surface relative to their volume ratio they have toxic, physical and chemical properties. unique 45. In addition, other studies have demonstrated the antifungal potential of ZnO NPs against other fungal pathogens including Fusarium sp., Botrytis cinerea, Penicillium expansum, Aspergillus niger and Rhizopus stolonifer 18, 46, 47.
In later studies, possibly another possibility to enhance ZnO’s effects against phytopathogens could be possible fusion of NPs to produce bimetallic nanoparticles as demonstrated by Paszkiewicz. et al. 48, in which study they found that the combination of Cu and Ag increased their toxicity strength as an antibacterial and antifungal compound.

4. Conclusion efficiency nano silver, CuO, ZnO

In this work, the successful synthesis of three types of nano silver, CuO and ZnO from T. harzianum was carried out in a green, affordable and non-toxic method. This is the first report in which a non-aqueous cell culture filtrate (CFCF) from a single strain of fungi, can synthesize different metallic NPs using a one-step, condition-based process. alkaline. These promising results provide new insights for generating different NPs that take advantage of the simplicity of a single strain’s method of maximizing the potential.
The physical and chemical properties of each metal NPs show narrow size distribution, thermal stability and crystal XRD spectrum, and especially to date, the morphology observed for CuO and ZnO NPs is very rare. The antibacterial effects of nano silver and CuO show similar inhibitory effects against radial growth of A. Alternata, P. oryzae and the sclerotia formation of S. sclerotiorum in a dependent manner. dosage, highlighting their potential for agricultural applications. Although it does not have antifungal properties, the NPs of ZnO can be tested against other target microorganisms. Notably, the NPs obtained in this study may have strong applications in other fields such as the pharmaceutical, cosmetics, clothing and food industries. In conclusion, the use of non-pathogenic T. harzianum fungus could be a good tool for large-scale production processing.

Reference source: Mycosinthetized Ag, CuO and ZnO nanoparticles from a promising Trichoderma harzianum strain and their antifungal potential against important phytopathogens