Rice (
Nanotechnology is an emerging area that could provide potential solutions to problems encountered in agriculture (Iavicoli et al., 2017). Nano-formulations of currently used chemical and biological products are in vogue to enhance their efficacies. Silver ion (Ag+) and its compounds are highly toxic to microorganisms, exhibiting strong biocidal effects on many species of bacteria and fungi (Park et al., 2006; Kim et al., 2007; Lara et al., 2011). Recently, Ag nanoparticles (AgNP) have shown evidence of being a potentially effective nematicide (Cromwell et al., 2014; Abdellatif et al., 2016; Hassan et al., 2016; Nassar, 2016; Taha, 2016).
The first report on the bio-efficacy of AgNP on a plant-parasitic nematode was provided by Cromwell et al. (2014) against root-knot nematode on bermuda grass with mixed results, the lab assays revealed promising results but field experiments were not conclusive. Subsequent studies have explicitly revealed promising results of AgNP (Abdellatif et al., 2016; Hassan et al., 2016) and FeNP (Sharma et al., 2017) against
The objectives of this project were to (i) characterize the physical properties of a citrate reduction synthesized AgNP formulation compared to a commercially available AgNP (Silvox 500®), (ii) determine the nematicidal concentration of lab synthesized AgNP against
AgNP were prepared by chemical route using a citrate reduction method (Mulfinger et al., 2007). Briefly, 50 ml aqueous solution of 1 mM AgNO3 was heated to boiling temperature. After that, 5 ml of 10 mM trisodium citrate aqueous solution was added dropwise under vigorous stirring until the color of the solution changed to pale yellow. The stirring was continued for another 15 min, and the sample was allowed to cool to room temperature.
UV–Visible (UV–Vis) spectroscopy was used to optically analyze the AgNP and commercially available Silvox 500® formulation using UV–Vis-Near Infra-red (NIR) spectrophotometer (CARY 5000 series), Agilent technologies. The absorbance was recorded in the range of 200 to 600 nm. The X-ray diffraction (XRD) characterization was carried out using Rigaku Miniflex 600 diffractometer operated at 30 KV with CuKα radiation (
A pure culture of rice root-knot nematode,
A laboratory trial was conducted using oven sterilized (100°C) sand contained in 5-cm diameter glass petri dishes. The final water saturation level was 6 ml per 25 g sand. About 500 J2 of
This and subsequent experiments were conducted in a glasshouse (30±2°C) at National Phytotron Facility of ICAR-IARI, New Delhi. Rice seedlings were raised in a soilless system in 10×5 cm plastic trays (Kumar et al., 2017). Double strength solutions of the lab synthesized AgNP product was poured into the trays at 10 ml per tray and 1000 J2 of
Clay loam soil collected from a rice field of Genetics Division at ICAR-IARI farm was sterilized in an autoclave at 1.5 kg per sq. cm. (121.6°C) for 2 hr, cooled and filled in 10-cm plastic pots. Lab synthesized AgNP was applied as seed soaking or soil drench at 1, 2, and 3 µg/ml. For seed soaking, rice seeds were kept overnight in respective concentrations of AgNP solutions or water (control). Nematode inoculum (1000 J2 per pot) was released uniformly over the soil surface in 50 ml water suspension. For soil drenching, nematode inoculation was followed by application of equal amount of respective double strength AgNP solutions. In both cases, 100 rice seeds (cultivar Pusa Basmati 1121) were placed over the soil surface that was covered with small quantity of the same soil. The plants were maintained in the glasshouse and watered regularly. Root gall counts and fresh weight of 10 randomly selected seedlings were recorded at the time of termination (25 days from inoculation) of experiment. Four replicates were maintained for each treatment, and all the pots were arranged in a complete randomized design.
Field soil naturally infested with
All the experiments in laboratory and glasshouse were conducted in a completely randomized design. All the experiments were conducted twice using the same methods. The data were subjected to analysis of variance (ANOVA) using statistical package of ICAR–Indian Agricultural Statistical Research Institute, New Delhi, India available online (webfmc.iasri.res.in). Least significant differences (LSD)/critical difference (CD) were calculated for each trial. Means were separated at
UV–Vis spectra of the AgNP revealed a well-defined surface plasmon resonance absorption peak centered at 417 nm (Fig. 1A), which is the characteristic peak related to spherical AgNP. The broadness of the absorption peak indicated the formation of poly-dispersed nanoparticles. The crystalline structure of synthesized nanoparticles was investigated using X-rays diffractometer having CuKα source. The diffraction peak at a 2
Silver nanoparticles (AgNP) characterization (A) ultra violet-visible (UV–Vis) absorption spectra exhibiting 417 nm absorbance related to surface plasmon resonance of AgNP, (B) X-ray diffractogram showing the different reflections from crystalline planes of AgNP, indicating the formation of face-centered cubic (FCC) structure of AgNP, (C) transmission electron microscopy (TEM) image showing the formation of poly-dispersed spherical AgNP with average size of 20 nm, and (D) high resolution TEM image of single AgNP of 25 nm showing characteristic inter-planar spacing of silver (Ag).
Silvox 500® only contained silver ions without the presence of silver nanoparticles as confirmed by lack of 417 nm peak on Silver 500®. Similarly, bare AgNO3 also did not form a peak at 417 nm in the US-Vis spectra (Fig. 2).
Ultra violet-visible (UV–Vis) spectra of silver nanoparticles (AgNP), AgNO3, H2O2, and commercial Silvox 500® showing the distinctive features of AgNP which is absent in Silvox 500®.
A benchmark of 100% nematode mortality was targeted to identify the lowest concentration of AgNP. J2 were normal up to 0.08 µg/ml concentration. Immobility of J2 was evident within 1 hr from 0.09 µg/ml concentration onwards and it increased to 100% from 3 hr onwards at 0.10 µg/ml and higher concentrations. The lowest concentration of AgNP for 100% mortality was identified as 0.1 µg/ml by 12 hr. The J2 did not recover after rinsing with water. Trisodium citrate alone had no effect on nematode immobility/mortality; therefore, the nematode mortality is attributed to AgNP exclusively.
Unlike water screening, 0.1 µg/ml of AgNP proved ineffective even up to 72 hr (Table 1). Juvenile mortality started at 0.2 µg/ml, and it increased with increasing concentration of AgNP and time (
Percentage mortality of
Percent juvenile mortality after | |||
---|---|---|---|
AgNP conc. (µg/ml) | 24 hr | 48 hr | 72 hr |
0a | 0 | 0 | 0 |
0.1 | 0 | 0 | 0 |
0.2 | 13.3 | 6.7 | 16.3 |
0.3 | 36.7 | 30 | 55 |
0.4 | 43.3 | 46.7 | 68.3 |
0.5 | 60 | 83.3 | 80 |
1.0 | 73.3 | 96.7 | 93.3 |
2.0 | 100 | 100 | 100 |
3.0 | 100 | 100 | 100 |
Notes: aControl (no AgNP, water alone). Means are averages of three replications. Data were subjected to 9 × 3 (AgNP concentration × incubation time) factorial analysis of variance (ANOVA), critical difference (CD) for incubation time is 0.027, CD for AgNP concentrations is 0.468, and that for time × concentrations is 0.81.
The AgNP treatments at 1, 2, and 3 µg/ml concentrations in soilless system were equally effective (
Effect of silver nanoparticles (AgNP) on root galling by
(A) Rice seedlings grown in soilless medium, (B) root mats of different treatments showing galls, and (C-F) magnified view of root mats showing galls.
(A) Scanning electron microscope (SEM) micrographs of rice roots without silver nanoparticles (AgNP) treatment, (B) energy-dispersive X-ray spectroscopic elemental analysis of rice roots without AgNP, (C) SEM micrographs of rice roots with AgNP, and (D) energy-dispersive X-ray spectroscopic elemental analysis of rice roots with AgNP.
No adverse effect was observed with seed soaking in AgNP solutions for 12 hr or soil drenching with AgNP on seed germination and plant growth (Table 2). Root galling was not suppressed with AgNP at 1 µg/ml when applied to the seed or as a soil drench. AgNP at 2 and 3 µg/ml, whether applied as seed soak or soil drench were equally effective in root gall suppression compared to untreated check (
Effect of silver nanoparticles (AgNP) as seed soak or soil drench on rice seedling growth and root galling by
AgNP conc. | Fresh weight of 10 seedlings (g) | No. of galls/seedling |
---|---|---|
|
||
1 µg/ml | 1.411a | 4.96ab |
2 µg/ml | 1.649a | 3.33b |
3 µg/ml | 1.291a | 2.33c |
|
||
1 µg/ml | 1.334a | 6.03a |
2 µg/ml | 1.402a | 3.70bc |
3 µg/ml | 1.457a | 2.23c |
|
1.277a | 7.20a |
Notes: The experiment was repeated, the Trial × Treatment interaction was not significant (p > 0.05). Data are means of two trials. All treatments had four replications randomized completely. Values with same letters in a column denote non-significant differences according to test of significance (p ≤ 0.05) in a completely randomized design.
AgNP, at all the doses reduced root galling significantly compared to untreated control. Although, all the doses of AgNP were statistically similar, a gradual reduction in galling was recorded with increasing dose. Silvox 500® was not effective in terms of gall reduction and was similar to untreated control. Fresh weight of seedlings was not significantly different among all treatments (Table 3).
Effect of silver nanoparticles (AgNP) as soil drench on rice seedling growth and root galling by
AgNP conc. | Fresh weight of 10 seedlings (g) | No. of galls/seedling |
---|---|---|
3 µg/ml | 1.637a | 4.9bc |
6 µg/ml | 1.365a | 4.3bc |
9 µg/ml | 1.679a | 3.9bc |
12 µg/ml | 1.318a | 2.8c |
Silvox® solution | 1.567a | 5.4ab |
Untreated control | 1.600a | 7.5a |
Notes: Drenching at 100 ml per pot. Initial nematode population = 1 J2 per g soil. The experiment was repeated, the Trial × Treatment interaction was not significant (p > 0.05). Data are means of two trials. All treatments had four replications randomized completely. Values with same letters in a column denote non-significant differences according to test of significance (p ≤ 0.05) in a completely randomized design.
In this study, laboratory assays involving direct exposure in water unequivocally established strong nematicidal properties of synthesized AgNP at a low concentration of 0.1 µg/ml killing 100% J2 of
The initial studies on the toxicity of AgNP involved free-living species
In sand screening tests, the lethal dose was 20 times higher (2 µg/ml) compared to water screening. This could be due to the adsorption of AgNP by soil particles which is expected to hinder the contact of AgNP with nematodes (Klitzke et al., 2015). This is further established by the
Nano-products of chemical and biological pesticides have proved to be more efficacious in combating pests and diseases leading to reduced dosages and are therefore, more economical. Some commercial products like Silvox 500® are being marketed as nano-formulation with a blend of hydrogen peroxide. However, the current studies examining the particle size using UV–Vis NIR spectrophotometer did not support Silvox® to be a true nano-product. The efficacy of Silvox 500® (when used at equivalent silver nitrate basis in the product) was negligible in terms of gall reduction in comparative study with AgNP synthesized in this study.
It is, therefore, concluded that AgNP synthesized by citrate reduction and tested in this study may prove to be very promising at a dosage of 3 µg/ml, and further field studies are warranted. Though application of AgNP by seed dip and soil drench in nursery were equally effective, both have their own merits. While seed dipping can save the quantities of chemical used, soil drenching can ward off infection by soil-borne pathogens as well. This study has opened up new vistas in the application of AgNP for nematode management in crop plants. However, its safety to the environment needs further study. Ultimately, AgNP may provide an alternative to currently used chemical nematicides and resolve nematode problems on other crops.