Preparation of Ag/SiO₂ nanocomposite and assessment of its antifungal effect on soybean plant (a Vietnamese species DT-26)

Procedure for preparation of Ag/SiO₂ nanocomposite was presented in figure 1. To prepare AFSPs, 100 g of silica powder (particle size 20–30 nm, Sigma-Aldrich) was mixed with 150 ml of 4% APTES (Sigma-Aldrich) solution in a 500 ml beaker. Due to the fact that the surface of silica particles as a rule contains a number of silanol groups (≡Si−OH), the functionalization process on their surface takes place. APTES is hydrolyzed in an acidic water solution to form 3-aminopropyltrihydroxylsilane which then reacts with active OH groups on the surface of the silica substrate [15]:

$$\begin{eqnarray*}&&{({{\rm{C}}}_{2}{{\rm{H}}}_{5}{\rm{O}})}_{3}{{\rm{SiC}}}_{3}{{\rm{H}}}_{6}{{\rm{NH}}}_{2}\,+\,{{\rm{H}}}_{2}{\rm{O}}\,+\,{{\rm{H}}}^{+}\\ &&\,\to {({\rm{HO}})}_{3}{{\rm{SiC}}}_{3}{{\rm{H}}}_{6}{{\rm{NH}}}_{2}\,+\,3{{\rm{C}}}_{2}{{\rm{H}}}_{5}{\rm{OH}}\end{eqnarray*}$$

$$\begin{eqnarray*}&&{({{\rm{SiO}}}_{2})}_{{\rm{n}}}\phantom{\rule{-0.10}{0ex}}\equiv \phantom{\rule{-0.10}{0ex}}{\rm{Si}}\phantom{\rule{-0.16}{0ex}}-\phantom{\rule{-0.16}{0ex}}{\rm{OH}}\,+\,{({\rm{HO}})}_{3}{{\rm{SiC}}}_{3}{{\rm{H}}}_{6}{{\rm{NH}}}_{2}\\ &&\,\to {({{\rm{SiO}}}_{2})}_{{\rm{n}}}\,\equiv \,{\rm{Si}}-{\rm{O}}-{\rm{Si}}{({\rm{OH}})}_{2}{{\rm{C}}}_{3}{{\rm{H}}}_{6}{{\rm{NH}}}_{2}\,+\,{{\rm{H}}}_{2}{\rm{O}}\end{eqnarray*}$$

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This process finally tailors amino-functional groups on the surface of silica particles. The reaction mixture was aged at 80 °C for 3 h and then removed APTES residue by filtration. Finally, the AFSPs were dried at 80 °C for 20 h to obtain the product in a powder form.

To synthesize Ag/SiO₂ nanocomposite (figure 1), 100 g of AFSPs were added to 200 ml of 0.5% AgNO₃ (Merck) solution in a 500 ml beaker and stirred for 4 h followed by filtering and washing with distilled water to remove free silver ions adsorbed on the silica surface. Then the silica particles with attached Ag⁺ ions were dispersed into 500 ml distilled water in a 1000 ml beaker and stirred 7000 rpm for 15 min Then, without disrupting stirring, 0.05 M NaBH₄ (Cica) solution was added by drop to the mixture and the stirring was continued for another 15 min The color of the solution slowly became yellow, indicating the formation of silver NPs immobilized on the surface of the silica particles. The APTES-functionalized Ag/SiO₂ nanocomposite was washed with distilled water in order to remove free APTES and other components. Then sample was dried overnight at 50 °C to obtain Ag/SiO₂ nanocomposite in a powder form.

FTIR spectra were recorded on Perkin Elmer (model 783) IR spectrometer in KBr medium at room temperature in the region 4000–500 cm⁻¹. Ultraviolet visible (UV-Vis) spectroscopy method (UV-VIS 2450 Shimazu) was applied to prove the existence of AgNPs decorated the surface of the silica particles. The wavelength scanning interval ranged from 300 nm to 800 nm. Particle size was determined by transmission electron microscopy (TEM, JEM1010 JEOL).

Soybean seeds of a Vietnamese species DT-26 were procured from Legumes Research and Development Center of the Field Crop Research Institute, Vietnam Academy of Agricultural Sciences. For coating soybean seeds a rotating drum was used and the following raw materials were needed: soybean seed of 25 g, bentonite clay with montmorillonite content of ≥50% (1.4 g), carboxymethyl cellulose (CMC) 2% solution of 2 ml and APTES-functionalyzed Ag/SiO₂ nanocomposite of 2.6 g.

Bentonite clay was taken from Tam Bo deposit of Lam Dong province, processed and purified in Institute of Environmental Technology, Vietnam Academy of Science and Technology (VAST).

To prepare soybean seed pellets, 25 g of soybean seeds were put into a drum. As drum rotated, 2 ml of 2% CMC solution was sprayed onto the seeds followed by adding bentonite and Ag/SiO₂ nanocomposite. The pellets gradually formed during drum rotation and slightly increased in size with each turn of the drum. At the end of the pelleting process, the pelleted seeds were dried 1 h at 40 °C [1].

2.3.1. Fungal inhibition test on the Ag/SiO₂ nanocomposite

Fungal species Fusarium oxysporium and Rhizoctonia solani were obtained from the Research Institute of Plant Protection. The fungi were maintained on potato dextrose agar (PDA) medium and incubated for 7 days at 28 °C and served as the testing fungi for antifungal activity assay.

An adequate quantity of Ag/SiO₂ nanocomposite (nanosilver concentration 10–200 ppm) was introduced to the molten PDA by mixing and 30 ml of the mixture was poured onto a petri dish. After solidification, one fungus-containing agar bit (0.5 cm) was placed in the center of the petri dish. Incubation period of 48–168 h at 28 °C was maintained for determination of antifungal activity. The activity was evaluated by measuring the zone of fungal growth. The inhibition percentage of fungal growth was calculated by the following formula [18]:

$$\begin{eqnarray*}&&{\rm{Inhibition}}( \% )=\displaystyle \frac{{{\rm{f}}}_{{\rm{c}}}-{{\rm{f}}}_{{\rm{t}}}}{{{\rm{f}}}_{{\rm{c}}}}\times 100,\end{eqnarray*}$$

where f_c is diameter of fungal growth zone on the control sample and f_t is diameter of fungal growth zone on the tested sample.

2.3.2. Fungal inhibition test on the soybean seed coating

2.3.3. Field experiment

Field experiment included soybean seed coated with Ag/SiO₂ nanocomposite and those without coating, which were sown in the spring using Ag/SiO₂ nanocomposite. The experiment was conducted on an experimental plot at the Center for Legumes Research and Development of the Field Crop Research Institute in randomized complete block design mode and replicated three times. The plot with a size of 51 m² was divided into 6 compartments (8.5 m²/compartment, 5.0 × 1.7 m²), containing 4 rows with 25 cm apart each other and a plant spacing of 35 cm. In total, 320 soybean seeds were shown on the plot. The fungal infection was estimated as follows:

$$\begin{eqnarray*}&&{\rm{Disease}}\,{\rm{incidence}}\,( \% )=\displaystyle \frac{{\rm{Number}}\,{\rm{of}}\,{\rm{infected}}\,{\rm{plants}}}{{\rm{Total}}\,{\rm{number}}\,{\rm{of}}\,{\rm{plants}}\,{\rm{tested}}}\times 100.\end{eqnarray*}$$

FTIR spectra of silica and APTES-attached silica particles were shown in figure 2. The absorption band at about 951.03 cm⁻¹ proves the existence of Si−OH groups (curve a) which are essential for immobilization of APTES on the silica surface. But after immobilization of APTES (curve b) this band dramatically decreased, witnessing the formation of the linkage between APTES and silanol groups on the silica surface. The absorption band at 1182.15 cm⁻¹ is caused by the stretching of Si−O−Si. These results suggested that APTES was successfully anchored on the silica surface [16, 17].

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UV-Vis spectrum of Ag/SiO₂ nanocomposite presented in figure 3 showed that the broad peak at about 400 nm is attributed to the plasmon resonance absorption of silver nanoparticles. Appearance of this peak affirmed the presence of AgNPs in the nanocomposite [16].

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Considering TEM image of Ag/SiO₂ nanocomposite presented in figure 4 one can distinguish clearly two phases of the nanocomposite: uncoated (white color) and silver-coated (black color) silica nanoparticles with an average size of 6–12 nm. This is in good agreement with the results from the study [14, 16, 17].

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Effects of Ag/SiO₂ nanocomposites on the fungal germination growth were shown in table 1 and demonstrated in figure 5. Briefly, freshly obtained sclerotia of Rhizoctonia solani were placed at the center of petri dish. At highest nanosilver concentration of the Ag/SiO₂ nanocomposite (200 ppm) sclerotial germination of Rhizoctonia solani was almost inhibited. After 2 days colony diameter was smallest (0.63 cm) compared to 8.77 cm of the control, equivalent to 92.82% of inhibition. Meanwhile at lower nanosilver concentration (10−50 ppm) the fungal inhibition still remained considerable (53%–79%). For the case of Fusarium oxysporium, after 7 days at nanosilver concentration 200 ppm the inhibition effect on sclerotial germination attained 75.26%, while at the lowest nanosilver concentration (10 ppm) the inhibition reached only 14.82%. These results demonstrated that inhibition effect of Ag/SiO₂ nanocomposite on Rhizoctonia solani is stronger than on Fusarium oxysporium, and depends on nanosilver concentration.

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Effects of the seed coating mode on the soybean germination were presented in table 2 and figures 6 and 7. The results indicated that coating soybean seed with Ag/SiO₂ nanocomposite increased its ability against the fungal infection. In this experiment, 100% of soybean seed without coating and contacted with fungi Rhizoctonia solani (R. solani) and Fusarium oxysporium (F. oxysporium) were infected depending on the exposition time. Using Ag/SiO₂ nanocomposite for coating soybean seed positively stimulated the seedling growth of soybean plant even in the presence of pathogenic fungi.

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Results of the field experiment presented in table 3 and figure 8 indicated that coating soybean seed with nanocomposite affected the disease incidence on soybean plant at different growth stages. The experimental data showed that almost all the growth stages were affected by fungal diseases at different level, but in the presence of Ag/SiO₂ nanocomposite this impact considerably decreased.

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