Skip to main content
Beni-Suef University Journal of Basic and Applied Sciences
Download PDF

Green production of silver nanoparticles, evaluation of their nematicidal activity against Meloidogyne javanica and their impact on growth of faba bean

Beni-Suef University Journal of Basic and Applied Sciences volume 8, Article number: 9 (2019) Cite this article

Abstract

Background

Cyanobacterium-based silver nanoparticles are considered not only as an efficient nano-nematicide but also as a bio-stimulant material for plant growth. They could be employed as a part of an integrated program for controlling some plant diseases.

Results

In this study, silver nanoparticles (Ag-NPs) were biosynthesized from aqueous extract of the cyanobacterium, Nostoc sp. PCC7524. Full characterization of the biosynthesized Ag-NPs was monitored by UV-vis spectroscopy, transmission electron microscopy, X-ray diffraction pattern, Zeta sizer, and Fourier transform infrared spectroscopy. In vitro assay against the root-knot nematode Meloidogyne javanica showed that Ag-NPs significantly decreased egg hatching of M. javanica at different applied concentrations (3, 6, 12, 25, and 50%, v/v). Fifty percent of Ag-NPs induced the highest reduction percent (94.66%). Moreover, Ag-NPs and AgNO3 significantly increased the percentages of larval mortality of the second-stage juveniles (J2) with concentration and time-dependent responses. Ag-NPs or AgNO3 at 2.4 ml/l, 24 h, completely inhibited the growth of J2 compared to 23% inhibition using aqueous cyanobacterial extract (ACE). In vivo effect of Ag-NPs on faba bean-infected plant under greenhouse conditions was achieved by treating soil with three different concentrations of 1, 2, and 3 ml/kg soil over two consecutive seasons. Ag-NPs significantly reduced root galling from 39.6 to 78.7% and J2 population in the soils from 32.2% to 86.7% in the 2018 season and from 21.9 to 78.1% and 40.0 to 81.0% in the 2019 season, respectively. Moreover, 3 ml/kg soil of Ag-NP treatment showed statistically comparable effects to that of vydate nematicide but with remarkable enhancement of faba bean growth parameters as compared to those of vydate or AgNO3 treatments in the two seasons.

Conclusions

The considerable in vitro and in vivo nematicidal potential of the cyanobacterium-based Ag-NPs, besides their bio-stimulant effect on plant growth, makes them feasible for the biological control of M. javanica.

Background

The plant-parasitic nematodes especially the root-knot nematode Meloidogyne spp. pose a potential threat to most of the agricultural crops in Egypt [1]. Faba bean (Vicia faba L.) is a major grain legume widely cultivated in many countries for food and feed purposes [2], and it is one of the oldest legume crops grown in Egypt. The root-knot nematode species attack the roots of the crops and considerably reduce the yield of legumes [3]. Chemical treatment using different types of nematicides has been proposed for the management of root-knot nematodes. However, nematicides do not provide long-term suppression of root-knot nematodes. Furthermore, their environmental and human health problems increased restrictions on their uses [4]. Thus, a considerable number of recent studies extensively focused on the feasibility of biological synthesis of environmentally friendly, non-toxic nanocomposites [5,6,7]. The synthesis of metallic nanoparticles using various biological materials is a green chemistry approach, it is also known as “green biosynthesis.” This approach was proposed as an alternative to chemical and physical methods due to few chemicals and less energy input needed for their production.

Indeed, a number of microorganisms including microalgae, fungi, yeast, bacteria [8,9,10,11], and plant extract [12] have been used in green biosynthesis of silver nanoparticles (Ag-NPs). Meanwhile, microalgae are considered as a promising tool for nanoparticle production due to their high growth rates and high biomass production. Cyanobacteria, such as Gloeocapsa sp., Lyngbya majuscula, Spirulina platensis, and Anabaena variabilis, have been reported for the biosynthesis of intracellular silver, gold, palladium, and platinum nanoparticles (NPs) [7, 13,14,15,16]. Qualities of nanoparticles are strongly correlated to their size where the high surface-to-volume ratio of nanoparticles resulted in tremendous increase in their physical, chemical, and biological properties [17].

Several studies reported the use of nanoparticles like Ag-NPs in biological control of different plant-parasitic nematodes [18,19,20]. However, employing Ag-NPs in the soil is controversial due to their impact on biosystem, e.g., changing the soil bacterial diversity [21], induction of species-specific phytotoxicity in higher plants [22]. Therefore, it is necessary to investigate the mode of interaction of Ag-NPs with plant and their effects on plant growth and development. Previous studies indicated that Ag-NPs induced the variable mode of action on crop plants [23]. In this study, exposure to plant-mediated Ag-NPs resulted in a significant reduction in plant elongation, fresh weight of shoot and root, and total chlorophyll content of seedling of Lupinus termis L. Moreover, 0.5 mg/l of Ag-NPs decreased carbohydrates and protein contents and increased accumulation of proline in seedlings [24]. On the contrary, 50 and 75 mg/l of biologically synthesized Ag-NPs protected wheat plants against heat stress and improved plant growth and biomass as compared to control [25]. A similar inductive effect on growth parameters and chemically valuable phytochemical production has been reported for Fenugreek (Trigonella foenum-graecum L.) by soaking the seedling in a concentration of 1 μg/ml of Ag-NPs for 5 days [26]. Effectiveness of Ag-NPs is best associated with their chemical composition, size, surface covering, reactivity, and most importantly the applied concentration at which they are effective [27]. The optimum growth promotion and increased root nodulation were observed at 50 mg/l treatment in cowpea (Vigna sinensis, var. Pusa Komal), while improved shoot parameters were recorded at 75 mg/l in Brassica (Brassica juncea, var. Pusa Jai Kisan) [21]. Therefore, looking for a cost-effective, safe, and efficient nematicide is a serious requirement to ensure quality of life for future generations. The main objectives of the present study were to prepare Ag-NPs using filamentous cyanobacterium Nostoc sp. PCC7524 as a green route and to evaluate the nematicidal activity of Ag-NPs against the root-knot nematode Meloidogyne javanica infecting faba bean plant in vivo and investigate their impact on plant growth parameters in two consecutive seasons of cultivation.

Methods

Cyanobacterial species and culture condition

The cyanobacterium, Nostoc sp. PCC7524 was isolated from the Egyptian agricultural lands and was identified based on the morphotaxonomic and molecular standpoints [28]. The cyanobacterium strain, Nostoc sp. PCC7524 was cultivated and propagated for 21 days in 500 ml BG11 medium [29] in 1-L round bottles at 30 °C and under continuous light illumination provided by white fluorescent lamps with light intensity of 30 photon/m2/s1. The cultures were aerated with a bubbling air at a regular pressure (200 ml/min and 50 Hz frequency) and sealed with a rubber plug having a narrow glass tube as an air outlet.

Preparation of cyanobacterial extract and biosynthesis of Ag-NPs

The batch cultures were cultivated until reaching the late exponential growth phase, and the biomass was collected by centrifugation at 5000 rpm for 20 min using a Hettich centrifuge (D-78532 Tuttlingen, Germany). The supernatant was discarded and the pellets were then washed well using deionized water to remove trace salts of the applied growth medium. The fresh biomass was air-dried in clean glass plates and then crushed by a porcelain mortar. The aqueous extract was prepared by soaking 1 g of the cyanobacterial powder in 100 ml of sterilized and deionized water in a 250-ml conical flask. The mixture was shaken thoroughly using a shaker incubator at 200 rpm for 12 h at room temperature. The aqueous cyanobacterial extract (ACE) was obtained by filtration and used as a stock for biosynthesis of Ag-NPs. The biosynthesis was conducted according to the method of Morsy et al. [30] with a little modification (crude ACE was used instead of water-soluble extracellular polysaccharides/matrix; low concentration of silver nitrate (AgNO3) solution was used for biosynthesis). A sample of 50 ml from ACE was mixed thoroughly with 50 ml of 1 mM sterilized AgNO3 in a 250-ml conical flask. Flasks of reaction mixtures were plugged by a cotton stopper and autoclaved for 5 min at 15 psi and 121 °C. The positive control was prepared by mixing 50 ml of ACE with 50 ml of deionized water while solution of 1 mM sterilized AgNO3 was used as a negative control.

Characterizations of cyanobacterium-based Ag-NPs

Indication of Ag-NP biosynthesis by aqueous extract of Nostoc sp. was evidenced by monitoring the UV-visible spectrum using a UV-vis spectrophotometer (UV-2600 Schimadzu, Germany). Samples were measured between 200- and 500-nm wavelengths, and the maximum range was recorded. All measurements were carried out at room temperature. Transmission electron microscopy (TEM) micrographs were obtained by FEI, Netherlands, Tecnai G20 Super twin, double tilt TEM, operating at an accelerating voltage of 200 kV. The sample suspension was sonicated for 5 min to separate the agglomerated particles and to get a homogeneous solution. A drop from the suspension was placed on a film of support grid. The samples were examined after the solvent has completely evaporated. The crystallization degree and elemental composition of the developed Ag-NPs were assessed and identified by an X-ray powder diffractometer (202964 Panalytical Empyrean) with CuKá1 radiation; the voltage and the current of the X-ray source were 40 kV and 30 mA, respectively. The sample was drop-coated onto a silica plate by applying many layers of small amounts of the sample on the plate with intermittent drying, this leads to a thick coat of the sample. Surface charge of cyanobacterium-based Ag-NPs was measured using Malvern Zeta sizer instruments (MAL1121994). Fourier transform infrared spectroscopy (FTIR) spectra is commonly used for identifying biomolecules responsible for the bioreduction of Ag ions and capping of the biosynthesized Ag-NPs. The samples of the freeze-dried synthesized nanoparticles were ground with potassium bromide (KBr), and the spectrum was recorded on FTIR spectroscopy Vertex 70.

Preparation of Meloidogyne javanica culture

The inoculum of M. javanica was prepared by collecting individual egg masses of distinct root-knot nematode from diseased tomato plants by a special needle, and then they were immersed in sterilized water for 7–10 days. The obtained new healthy second-stage juveniles (J2) were reared on tomato seedling planted cv. Super strain B. The tomato seedlings were maintained in a greenhouse at the Department of Plant Pathology, Sakha Agricultural Research Station for more than 45 days to prepare eggs and larval suspensions from the infected roots with galls. Eggs of M. javanica were extracted from infected tomato using sodium hypochlorite solution according to Hussey and Barker [31]. The re-extracted nematodes from diseased tomato plants were identified by the examination of Perineal patterns of adult females according to Barker [32]. Identification of M. javanica was confirmed by polymerase chain reaction (PCR) using OPA-01 primer according to a method described by Cenis [33].

Nematicidal bioassays

Assessment of in vitro nematicidal activity

The inhibitory effect of cyanobacterium-based Ag-NPs, ACE, and AgNO3 solutions against egg hatching of M. javanica was performed using five different concentrations (3%, 6%, 12%, 25%, and 50%, v/v). Solutions were prepared by mixing original stocks with sterilized distilled water as diluents. Nine milliliters from each concentration was mixed with 1 ml of egg suspension containing 2000–2500 eggs of M. javanica in 25-ml vials. The vials were incubated at 27 °C for 14 days. The percent of egg hatching was recorded using the research microscope. The nematicidal activity of cyanobacterium-based Ag-NPs, ACE, and AgNO3 solutions against the second larval instars (J2) of M. javanica was also managed in vitro by testing five different concentrations (0.15, 0.3, 0.6, 1.2, and 2.4 ml/l, v/v) from the original stocks as aforementioned. Similarly, 9 ml from each concentration was pipetted in 25-ml vials containing 1 ml of juvenile (J2) suspension (200–300 juveniles). The vials were incubated at 27 °C for 24, 48, and 72 h. The number of active and inactive J2 larvae was counted, and the percentages of inactive larvae were calculated to evaluate the net percentage (%) of larval mortality at each incubation period. The initial % of inactive J2 at control conditions was 2.3% after 24 h of incubation, this value was considered in the calculation. Distilled water was used as a negative control. All treatments were conducted in three biological replicates.

Assessment of in vivo nematicidal activity

The nematicidal efficacy of cyanobacterium-based Ag-NPs, ACE, and AgNO3 against M. javanica infecting faba bean was performed under greenhouse experiments in two consecutive cultivation seasons (2018 and 2019). Faba bean seeds (Sakha 2 cultivar) were sown in 30-cm-diameter pots filled with a mixture of clay and sand soil in a ratio of 2:1. Two weeks after sowing, the plants were thinned into two seedlings per pot. Each pot was inoculated with approximately 5000 eggs and newly hatching J2 of M. javanica per plant. The inoculums were added inside three holes in the soil, around the base of stems, then pots were irrigated. The inhibitory activity of cyanobacterium-based Ag-NPs, ACE, and AgNO3 was investigated at three different concentrations from each solution (1, 2, and 3 ml/kg soil). Vydate® SL. (24%) was used as the standard chemical nematicide in a dose of 0.01 ml per pot. Treatments were added to pots after infesting the pots with nematode. Infested pots with only M. javanica were used as control1 while untreated pots were used as control2. All pots were arranged in the greenhouse in a randomized block design and were kept at 20 ± 5 °C. After 60 days, faba bean plants were uprooted, and the roots were gently washed with a stream of distilled water. The roots with nematode galls were rated on a 1–9 scale of gall index (GI) according to Sharma et al. [34] as follows: 1 = no galls, 2 = 1 to 5 galls, 3 = 6 to 10 galls, 4 = 11 to 20 galls, 5 = 21 to 30 galls, 6 = 31 to 50 galls, 7 = 51 to 70 galls, 8 = 71 to 100 galls, and 9 = > 100 galls per root system. The number of galls developed on roots, final nematodal population (J2) in 250 cm3 of soil, fresh and dry weights of shoots, length of plants, and number of flowers per plant was determined. All treatments were conducted in three biological replicates.

Statistical analysis

Analysis of variance (ANOVA) and regression coefficient analyses were performed by WASP-Web Agri Stat Package statistical analysis software. Treatment means were separated using Duncan’s multiple range test [35]. All analyses were conducted at the significance value of p ≤ 0.05.

Result

Characterization of cyanobacterium-based Ag-NPs

A distinct shifting in color from light-blue (Fig. 1c) to brown (Fig. 1d) was observed in reaction mixture flask of aqueous cyanobacterial extract (ACE) with 1 mM AgNO3 after 5 min of heating. The obtained UV-vis spectroscopy spectrum showed a single surface plasmon resonance (SPR) absorption band at 424 nm (Fig. 1). TEM micrographic analysis revealed the formation of predominantly spherical structures with an average size which ranged between 6.4 and 16 nm (Fig. 2a). The crystallinity of the cyanobacterium-based Ag-NPs was also confirmed by the X-ray diffraction pattern (XRD) (Fig. 2b). The diffraction pattern revealed the existence of 4 distinct peaks at 2θ values of 38.0°, 44.2°, 64.5°, and 76.9° which were assigned to 111, 200, 220, and 311 crystal planes, respectively, of fcc Ag0 (JCPDS, card file no: 04-003-7118), and other 3 peaks at 2θ values of 27.6°, 32.1°, and 46.1° were indexed as 111, 200, and 220 crystal planes, respectively, of fcc AgCl (JCPDS, card file no: 04-007-3906). The intensity of the peaks denotes to intensity of crystallinity of the produced Ag and AgCl-NPs. The obtained cyanobacterium-based Ag-NPs were also demonstrated through their characteristic peak of the surface charge observed in Zeta size image, where the bioformed nanoparticles showed an average potential value with − 18.0 mv (Fig. 2c). FTIR spectroscopy analysis was used to identify the possible biomolecules which existed in the aqueous extract of Nostoc sp. (Fig. 3). The obtained pattern showed 10 distinct peaks at 3275, 3083, 2861, 1630, 1528, 1448, 1393, 1237, 1052, and 556 cm−1.

Fig. 1
figure 1

UV-vis spectroscopy analysis of cyanobacterium-based Ag-NPs using the aqueous extract of Nostoc sp. Direct visualization of cyanobacterium-based Ag-NP. (A) Negative control (1 mM AgNO3). (B) Positive control aqueous cyanobacterial extract (ACE). (C) and (D) are ACE with 1 mM AgNO3 before and after heating, respectively

Full size image
Fig. 2
figure 2

Characterization of cyanobacterium-based Ag-NPs by reduction of 1 mM Ag+ ions by the aqueous extract of Nostoc sp. a Transmission electron microscopy (TEM) micrograph. b X-ray diffraction pattern of purified Ag-NPs. c Zeta potential analysis of Ag-NPs

Full size image
Fig. 3
figure 3

FTIR spectrum of cyanobacterium-based Ag-NPs bioformed by reduction of 1 mM Ag+ ions by the aqueous extract of Nostoc sp

Full size image

In vitro nematicidal activity of cyanobacterium-based Ag-NPs

Data listed in Table 1 indicate that cyanobacterium-based Ag-NPs, ACE, and AgNO3 remarkably decreased the percentages (%) of egg hatching of M. javanica in a concentration-dependent manner. Among tested treatments, cyanobacterium-based Ag-NPs showed a significant reduction in % of egg hatching by 72.6–84.6% (p ≤ 0.05) at the concentrations 3–12.5%. Additionally, 50% of cyanobacterium-based Ag-NPs and AgNO3 showed the highest inhibitory effect against egg hatching by 94.7% and 84.3% respectively which were statistically comparable (p ≤ 0.05). Interestingly, low concentrations of cyanobacterium-based Ag-NPs (3% and 6%) showed a significant toxic effect on egg hatching as compared to AgNO3 (p ≤ 0.05) by 4.56- and 4.58-folds, respectively. Regression analysis in Fig. 4 showed positive and significant relationships between concentrations of cyanobacterium-based Ag-NPs and ACE with reduction % of egg hatching by R2 = 0.790 and 0.871, respectively, whereas a positive insignificant relationship was found between the concentration of AgNO3 and the reductions % of egg hatching by R2 = 0.755 (p ≤ 0.05).

Table 1 Effect of the different concentrations of cyanobacterium-based Ag-NP, ACE, AgNO3 solutions on the percentage of egg hatching of Meloidogyne javanica
Full size table
Fig. 4
figure 4

Relationship between different concentration of a cyanobacterium-based Ag-NPs, b aqueous extract of Nostoc sp. (ACE), and c AgNO3 solution and reduction % of egg hatching of M. javanica

Full size image

Percentages of J2 larval mortality are shown in Table 2. Overall, Ag-NPs and AgNO3 significantly increased % of larval mortality with concentration and time-dependent responses (p ≤ 0.05). Meanwhile, the cyanobacterial-based Ag-NPs showed insignificant values of larval mortality compared to AgNO3 solution at all tested concentrations. Upon exposure to 2.4 ml/l of cyanobacterial-based Ag-NPs or AgNO3, growth of J2 was completely inhibited. On the contrary, 2.4 ml/l of ACE showed the lowest percentage of larval mortality by 23–39% at 24–72 h of incubation periods, respectively. The detected LC50 values of cyanobacterial-based Ag-NPs were 0.6 ml/l at 24 h and ˂ 0.15 ml/l at 48 h or 72 h of incubation periods, respectively. Results of regression analysis showed positive and significant relationships between different concentrations of the cyanobacterium-based Ag-NPs, ACE, and AgNO3 solutions and the percentage of larvae mortality at different incubation periods with R2 ranging from 0.745 to 0.973. For instance, cyanobacterial-based Ag-NPs and ACE increased % of larval mortality at all tested concentrations (p ≤ 0.05) (Fig. 5a–f). In contrast, AgNO3 treatment showed a significant effect only at high concentrations (˃ 1.2 ml/l) (p ≤ 0.05) (Fig. 5g–i).

Table 2 Effect of the different concentrations of the cyanobacterium-based Ag-NP, ACE, AgNO3 solutions on the percentage of larvae mortality of the J2 juveniles of M. javanica at different incubation periods
Full size table
Fig. 5
figure 5

Relationship between different concentrations of the cyanobacterium-based Ag-NPs (a–c), different concentrations of aqueous extract of Nostoc sp. (d–f), and different concentrations of AgNO3 solution (g–i) on the percentage of larvae mortality of the J2 juveniles of M. javanica at different incubation periods

Full size image

In vivo nematicidal activity of cyanobacterium-based Ag-NPs on M. javanica

In the present study, we observed that soil treated with cyanobacterium-based Ag-NPs or AgNO3 solution showed a similar inhibitory effect on reduction of the root galling and nematode population of M. javanica over the two conducted seasons (Table 3). Their inhibitory effects were dose-dependent and were significantly higher than that of ACE (p ≤ 0.05). For instance, Ag-NPs significantly reduced root galling and J2 population in the soils from 39.62 to 78.67% and 32.22 to 86.66%, respectively, in the 2018 season and from 21.87 to 78.12% and 40 to 81%, respectively, in the 2019 season as compared to control. Additionally, 3 ml/kg soil of cyanobacterium-based Ag-NPs or AgNO3 solution showed statistically comparable effects on root galling and J2 population as compared to the vydate nematicide at both seasons. In contrast, ACE showed the lowest reduction value of root galling and J2 population in the infected soil. Root morphology of faba bean with developed galls of M. javanica at the end of the experiment is shown in Fig. 6a–f.

Table 3 Effect of the different concentrations of the cyanobacterium-based Ag-NP, ACE, AgNO3 solutions and vydate nematicide on the number of galls developed on faba bean roots and J2 population of Meloidogyne javanica in soil under greenhouse conditions during 2018 and 2019 seasons
Full size table
Fig. 6
figure 6

a–f Representative faba bean root galling. a (Control1) nematode-infected soil. b (Control2) untreated soil. c Treated plants with vydate nematicide. d Treated plants with ACE. e Treated plants with AgNO3 solution. f Treated plants with cyanobacterium-based Ag-NPs

Full size image

Influence of cyanobacterium-based Ag-NPs on growth parameters of faba bean

All tested concentrations of cyanobacterium-based Ag-NPs, AgNO3, and ACE significantly improved some growth parameters of faba bean plant but with variable degrees as compared to control1 (nematode-infected soil) and control2 (untreated soils) during seasons 2018 and 2019 (Table 4). For more details, upon treating soil with 3 ml/kg soil of cyanobacterium-based Ag-NPS or ACE significantly improved the fresh weight of shoots by 71% and 72% (p ≤ 0.05), respectively, and increased the dry weight of shoots by 58% and 66% (p ≤ 0.05), respectively. In addition, both solutions also enhanced the plant heights by 83% and 80% and increased the number of flowers per plant by 206.9% and 248.8%, respectively, as compared to (control1) during the 2018 season. Comparable results have been confirmed in 2019 where cyanobacterium-based Ag-NPS and ACE at a concentration of 3 ml/kg soil noticeably increased fresh weight of shoots by 70% and 74%, respectively, and dry weight of shoots by 64% and 81%, respectively. Similarly, plant heights were induced by 59% and 58%, respectively, and the number of flowers/plant were also increased by 103% and 125%, respectively, compared to control1. Overall, 2 and 3 ml/kg soil from cyanobacterium-based Ag-NPs and ACE significantly increased the fresh weight of plant shoots (p ≤ 0.05) as compared to AgNO3, vydate, and control1,2 during the season of 2018. Although AgNO3 solution showed a significant reduction of root galling and J2 population in the soil, it had the lowest effect on improving plant growth parameters as compared to cyanobacterium-based Ag-NPS or ACE.

Table 4 Effect of the different concentrations of cyanobacterium-based Ag-NP, ACE, AgNO3 solutions and vydate nematicide on some growth parameters of faba bean plant infected by root-knot nematode, M. javanica under greenhouse conditions during seasons 2018 and 2019
Full size table

Discussion

Characterization of cyanobacterium-based Ag-NPs

Biosynthesis of cyanobacterium-based Ag-NPs was confirmed by a set of physicochemical analyses. The UV-vis spectroscopy spectrum showed a distinct absorption peak at 424 nm, this might be attributed to the excitation of surface plasmon resonance (SPR) of the synthesized Ag-NPs [36]. Many investigators indicated that Ag-NPs exhibited UV-vis absorption spectra which ranged between 410 and 440 nm which were also assigned to other different metal nanoparticles [6, 7, 37]. The negatively charged polysaccharides of the algal extract tightly bonded with Ag-NPs, and these confined the free electrons of the nanoparticles in a smaller volume, which resulted in a high free electron density and a high plasmon frequency. Therefore, a sharp peak was observed at a lower wavelength [38]. The produced brown color also indicated the green biosynthesis of Ag-NPs and reduction of Ag+ ions. We assumed that Ag+ was reduced to Ag0 NPs by some biomolecules that exist in the ACE. It is worth mentioning that chlorine content in the algal extract, previously obtained from BG-11 medium components could be elucidated as the main source for the formation of AgCl-NPs in this study. This observation is in accordance with other previous studies which used bacterial and microalgal species for AgCl-NP biosynthesis [6, 7, 39]. In the present study, Zeta sizer measurement revealed a high potential value of the bioformed Ag-NPs. This characteristic feature might be due to the high negative-negative repulsion force between the nanoparticle which supports well the long-term stability, good colloidal nature, and high dispersity of the Ag and AgCl-NPs [40, 41].

FTIR spectrum showed a broad peak at 3275 cm−1 corresponding to primary amide linkage in the protein [42], also 3083 cm−1 indicated the presence of stretching vibration modes of secondary amines [43]. The peak at 2861 cm−1 was attributed to C–H stretching vibrations of thiols [44]. Moreover, the strong absorption band at 1630 cm−1 was characteristic of amide I [45] while the peak at 1528 cm−1 indicated the presence of amide II bonds involving carbonyl and N–H stretching of the proteins [46]. The peak located at 1448 cm−1 suggested the involvement of CH2 and CH3 groups of protein [47]. Similarly, the peak observed at 1393 cm−1 was corresponding to asymmetric deformation of CH3 and CH2 in proteins [48]. The peak located at 1237 cm−1 could be assigned to –C–O– of aromatic acids [49] while the peak observed at 1052 cm−1 suggested the –C–O stretching vibrations from polysaccharides [50]. Also, the broad absorption band 556 cm−1 indicated the possible involvement of alkyl halides [42]. Overall, FTIR data suggested that the proteins and amino acid residues in the cyanobacterial extract that have strong binding ability with metals, possibly formed a layer surrounding the metal nanoparticles and acted as a capping agent to prevent agglomeration of NPs [47]. In addition, polysaccharides perhaps involved in the biosynthesis and capping of Ag-NPs produced by Nostoc sp. Our results are in agreement with previous studies of Ali et al. [49] and Ahmed et al. [43] who proposed that proteins were responsible for the reduction and stabilization of silver nanoparticles and metal nanoparticles. Rajeshkumar et al. [45] and Ferreira et al. [37] suggested that proteins and polysaccharides in the extract of Turbinaria conoides and Chlorella vulgaris act as reducing and stabilizing agents in the biosynthesis of gold and silver chloride-NPs, respectively.

Nematicidal activity of cyanobacterium-based Ag-NPs against M. javanica

Our results showed that cyanobacterium-based Ag-NPs induced a significant reduction in egg hatching % and induced a marked larval mortality of M. javanica, in vitro. The obtained results are in accordance with previous reports which used Ag/AgCl-NP solution against Meloidogyne incognita [6, 18,19,20]. The inhibitory effect of the bioformed Ag-NPs was attributed to their physical structure (e.g., size, shape, and homogeneity) which, probably played a key role in the cell wall penetration of the nematode eggs, followed by cell dysfunction [51]. Ag-NPs induced oxidative stress and upregulation of sod-3 and daf-12 genes which caused the failure of reproduction in Caenorhabditis elegans worms [52]. Toxicity of Ag-NPs is not a species-specific; therefore, it can be applied to control other plant-parasitic nematodes and plant-pathogenic fungi. Its mode of action is associated with disrupting and malfunctioning of several cellular mechanisms, such as membrane permeability, ATP synthesis, and physiological response to oxidative stress in both eukaryotic [53] and prokaryotic cells [54]. Our results under greenhouse conditions over two cultivation seasons suggest the efficacy of the cyanobacterium-based Ag-NPs on the reduction of root galling and J2 population in infected soil. A similar observation was reported by Cromwell et al. [18], Hassan et al. [19], and Nour El-Deen and El-Deeb [20]. Nevertheless, AgNO3 also showed a comparable inhibitory effect against M. javanica under both laboratory and greenhouse conditions. In this context, Jung et al. [55] attributed the toxic effect of silver ion on Staphylococcus aureus and Escherichia coli to membrane dysfunction of the bacterial cell which possibly causes cell death.

Bio-stimulant effect of cyanobacterium-based Ag-NPs on faba bean

Based on the obtained results, cyanobacterium-based Ag-NPs seem to be a good candidate for efficient biocontrol of M. javanica infecting faba bean plant as well as a bio-stimulant material for plant growth. A similar finding has been reported by Iqbal et al. [25] and Nour El-Deen and El-Deeb [20]. The bio-stimulant effect by both ACE and cyanobacterium-based Ag-NPs on faba bean growth parameters is possibly due to micronutrients and many organic compounds such as auxins, gibberellins, and precursor of ethylene and betaine produced by algal cells [56]. In this context, green synthesized Ag-NPs had no plant phototoxicity when they were used to control the nematodal infection by M. javanica [57]. Further investigations are still needed to separate and purify of cyanobacterium-based Ag-NPs from the suspension mixture with AgNO3. We assume that some unreacted Ag ions are still in solution which possibly interfere with Ag-NP action.

Conclusion

The present study provides a promising technique for green production of Ag-NPs using the aqueous extract of the heterocytous cyanobacterium Nostoc sp. PCC7524. This was likely due to its simplicity and accessibility. The study also indicated the nematicidal activity of cyanobacterium-based Ag-NPs on % egg hatching and larval mortality of the root-knot nematode Meloidogyne javanica in vitro and under greenhouse conditions which suggest their use in biological control of M. javanica. The study also highlighted the positive role of cyanobacterium-based Ag-NPs in improving some plant growth parameters cultivated in nematode-infected soil. Therefore, cyanobacterium-based Ag-NPs could be suggested as multifunctional nano-nematicide to avoid the harmful effect of chemical amendments.

Availability of data and materials

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Abbreviations

ACE:

Aqueous cyanobacterial extract

AgCl-NPs:

Silver chloride nanoparticles

Ag-NPs:

Silver nanoparticles

FTIR:

Fourier transform infrared spectroscopy

GI:

Gall index

J2 :

Second-stage juveniles

LC50 :

50% lethal concentration

NPs:

Nanoparticles

PCR:

Polymerase chain reaction

SPR:

Surface plasmon resonance

TEM:

Transmission electron microscopy

References

  1. Ibrahim IKA, Mokbel AA, Hammad SE (2014) Host suitability of some solanaceous plant cultivars to the root-knot nematodes, Meloidogyne spp. Global Adv Res J Agric Sci 3:136–140

    Google Scholar 

  2. Sillero JC, Villegas-Fernández AM, Thomas J, Emeran MM, Rojas-Molina AA, Fernández-Aparicio M, Rubiales D (2010) Faba bean breeding for disease resistance. Field Crops Res 115:297–307

    Article  Google Scholar 

  3. Montasser SA, Mahmoud NA, EL-Mesalamy AF, Abdel-Mageed MA (2017) Evaluation of six leguminous crops against the root-knot nematode, Meloidogyne javanica infection. Pakistan J Nematol 35(1):79–84

    Article  Google Scholar 

  4. Abd-Elgawad MMM (2008) The current status of phytonematode management in Egypt with special reference to applicable nematicides. Egyptian J Agronematol 6:33–46

    Google Scholar 

  5. Panyala NR, Peña-Méndez EM, Havel J (2008) Silver or silver nanoparticles: a hazardous threat to the environment and human health ? J Appl. Biomed 6:117–129

    CAS  Article  Google Scholar 

  6. Hamed SM, Mostafa AMA, Abdel-Raouf N, Ibraheem IBM (2016) Biosynthesis of silver and silver chloride nanoparticles by Parachlorella kessleri SAG 211-11 and evaluation of its nematicidal potential against the root-knot nematode; Meloidogyne incognita. Australian J Basic Appl Sci 10(18):354–364

    Google Scholar 

  7. Hamed SM, Abdel-Alim MM, Abdel-Raouf N, Ibraheem IBM (2017) Biosynthesis of silver chloride nanoparticles using the cyanobacterium Anabaena variabilis. Life Sci J 14(6):25–30

    CAS  Google Scholar 

  8. Merin DD, Prakash S, Bhimba BV (2010) Antibacterial screening of silver nanoparticles synthesized by marine micro algae. Asian Pacific J Tropical Med 3(10):797–799

    CAS  Article  Google Scholar 

  9. Singh T, Jyoti K, Patnaik A, Singh A, Chauhan R, Chandel SS (2017) Biosynthesis, characterization and antibacterial activity of silver nanoparticles using an endophytic fungal supernatant of Raphanus sativus. J Genetic Eng Biotechnol 15:31–39

    Article  Google Scholar 

  10. Niknejad F, Nabili M, Daie Ghazvini R, Moazeni M (2015) Green synthesis of silver nanoparticles: Advantages of the yeast Saccharomyces cerevisiae model. Curr Med Mycol 1(3):17–24

    CAS  Article  Google Scholar 

  11. Das VL, Thomas R, Varghese RT, Soniya EV, Mathew J, Radhakrishnan EK (2014) Extracellular synthesis of silver nanoparticles by the Bacillus strain CS 11 isolated from industrialized area. 3 Biotech 4:121–126

    Article  Google Scholar 

  12. Balashanmugam P, Kalaichelvan PT (2015) Biosynthesis characterization of silver nanoparticles using Cassia roxburghii DC. aqueous extract, and coated on cotton cloth for effective antibacterial activity. Int J Nanomedicine 10:87–97

    CAS  Article  Google Scholar 

  13. AL-Katib M, AL-Shahri Y, AL-Niemi A (2015) Biosynthesis of silver nanoparticles by cyanobacterium Gloeocapsa sp. Enhanced Res Sci Technol Eng 4(9):60–73

    Google Scholar 

  14. Bakir EM, Younis NS, Mohamed MEI, El Semary NA (2018) Cyanobacteria as nanogold factories: chemical and anti-myocardial infarction properties of gold nanoparticles synthesized by Lyngbya majuscula. Mar. Drugs 16:217. https://doi.org/10.3390/md16060217

    CAS  Article  PubMed Central  Google Scholar 

  15. Muthusamy G, Thangasamy S, Raja M, Chinnappan S, Kandasamy S (2017) Biosynthesis of silver nanoparticles from Spirulina microalgae and its antibacterial activity. Environ Sci Pollut Res 24:19459–19464

    CAS  Article  Google Scholar 

  16. Brayner R, Barberousse H, Hernadi M, Djedjat C, Yepremian C, Coradin T (2007) Cyanobacteria as bioreactors for the synthesis of Au, Ag, Pd, and Pt nanoparticles via an enzyme-mediated route. J Nanosci Nanotechnol 7:2696–2708

    CAS  Article  Google Scholar 

  17. Ditta A (2012) How helpful is nanotechnology in agriculture? Adv Nat Sci Nanosci Nanotechnol 3(3):2–33

    Article  Google Scholar 

  18. Cromwell WA, Yang J, Starr JL, Jo Y-K (2014) Nematicidal effects of silver nanoparticles on root-knot nematode in Bermudagrass. J Nematol 46:261–266

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Hassan MEM, Zawam HS, El-Nahas SEM, Desoukey AF (2016) Comparison study between silver nanoparticles and two nematicides against Meloidogyne incognita on tomato seedlings. Plant Pathol J 15:144–151

    CAS  Article  Google Scholar 

  20. Nour El-Deen AH, El-Deeb BA (2018) Effectiveness of silver nanoparticles against root-knot nematode, Meloidogyne incognita infecting tomato under greenhouse conditions. J Agric Sci 10(2):148–156

    Google Scholar 

  21. Mehta PCM, Srivastava R, Arora S, Sharma AK (2016) Impact assessment of silver nanoparticles on plant growth and soil bacterial diversity. 3 Biotech 6:254

    PubMed  PubMed Central  Google Scholar 

  22. Musante C, White JC (2012) Toxicity of silver and copper to Cucurbita pepo: differential effects of nano and bulk-size particles. Environ Toxicol 27(9):510–517

    CAS  Article  Google Scholar 

  23. Dietz KJ, Herth S (2012) Plant nanotoxicology. Trends Plant Sci 16(11):582–589

    CAS  Article  Google Scholar 

  24. Al-Huqail AA, Hatata MM, AL-Huqail AA, Ibrahim MM (2018) Preparation, characterization of silver phyto nanoparticles and their impact on growth potential of Lupinus termis L. seedlings. Saudi J Bio Sci 25:313–319

    CAS  Article  Google Scholar 

  25. Iqbal M, Raja NI, Mashwani Z-U-R, Hussain M, Ejaz M, Yasmeen F (2017) Effect of silver nanoparticles on growth of wheat under heat stress. Iran J Sci Technol Trans Sci. https://doi.org/10.1007/s40995-017-0417-4

    Article  Google Scholar 

  26. Jasim B, Thomas R, Mathew J, Radhakrishnan EK (2017) Plant growth and diosgenin enhancement effect of silver nanoparticles in Fenugreek (Trigonella foenum-graecum L). Saudi Pharm J 25:443–447

    CAS  Article  Google Scholar 

  27. Khodakovskaya MV, Silva KD, Biris AS, Dervishi E, Villagarcia H (2012) Carbon nanotubes induce growth enhancement of tobacco cells. ACS Nano 6(3):2128–2135

    CAS  Article  Google Scholar 

  28. Hamed SM, Raut MP, Jaffé SRP, Wright PC (2017) Evaluation of the effect of aerobic–anaerobic conditions on photohydrogen and chlorophyll a production by environmental Egyptian cyanobacterial and green algal species. Int J Hydrogen Energy 42(10):6567–6577

    CAS  Article  Google Scholar 

  29. Allen MM, Stanier ST (1968) Selective isolation of blue-green algae from water and soil. J Gen Microbiol 51:302

    Google Scholar 

  30. Morsy FM, Nafady NA, Abd-Alla MH, AbdElhady D (2014) Green synthesis of silver nanoparticles by water soluble fraction of the extracellular polysaccharide/matrix of the cyanobacterium Nostoc commun and its application as a potent fungal surface stabilizing agent of seed crop. Universal J microbiol Res 2:36–43

    CAS  Google Scholar 

  31. Hussey RS, Barker RK (1973) A comparison of methods of collecting inocula of Meloidogyne spp. including a new technique. Plant Dis Rep 57:1025–1028

    Google Scholar 

  32. Barker K.R. (Committee Chairman). 1978. Determining nematode population response to control agents. In: Methods for Evaluating Plant Fungicides, Nematicides, and Bactericides pp 114-125 (Edited by Zehr E.I., Chairman) American Phytopathological Society. St. Paul

  33. Cenis JL (1993) Identification of four major Meloidogyne spp. by random amplified polymorphic DNA (RAPD-PCR). Phytopathology 83:76–80

    CAS  Article  Google Scholar 

  34. Sharma SB, Mohiuddin M, Jain KC, Remanandan P (1994) Reaction of pigeonpea cultivars and germplasm accessions to the root-knot nematode, Meloidogyne javanica. J Nematol 26:644–652

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Duncan DB (1955) Multiple ranges and multiple F. test. Biometrics 11:1–42

    Article  Google Scholar 

  36. Mulvaney P (1996) Surface plasmon spectroscopy of nanosized metal particles. Langmuir. 12(3):788–800

    CAS  Article  Google Scholar 

  37. Ferreira DVS, Conz MEF, Lima LMTR, Fraśes S, Wanderley DS, Sant’Anna C (2016) Green production of microalgae-based silver chloride nanoparticles with antimicrobial activity against pathogenic bacteria. Enzyme Microb Technol. https://doi.org/10.1016/j.enzmictec.2016.10.018

    CAS  Article  Google Scholar 

  38. Dallas P, Sharma VK, Zboril R (2011) Silver polymeric nanocomposites as advanced antimicrobial agents: classification, synthetic paths, applications, and perspectives. Adv Colloid Interface Sci 166:119–135

    CAS  Article  Google Scholar 

  39. Paulkumar K, Rajeshkumar S, Gananajobitha G, Vanaja M, Malarkodi C, Annadurai G (2013) Biosynthesis of silver chloride nanoparticles using Bacillus subtilis MTCC 3053 and assessment of its Antifungal activity. ISRN Nanomaterials:1–8

    Article  Google Scholar 

  40. Dobre N, Petica A, Buda M, Anical L, Visan T (2014) Electrochemical synthesis of silver nanoparticles in aqueous electrolytes. UPB Sci Bull 76:127–136

    Google Scholar 

  41. Roychoudhury P, Gopal PK, Paul S, Pal R (2016) Cyanobacteria assisted biosynthesis of silver nanoparticles a potential antileukemic agent. J Appl Phycol. https://doi.org/10.1007/s10811-016-0852-1

    CAS  Article  Google Scholar 

  42. Rajeshkumar S, Ponnanikajamideen M, Malarkodi C, Malini M, Annadurai G (2014) Microbe-mediated synthesis of antimicrobial semiconductor nanoparticles by marine bacteria. J Nanostruct Chem. 4:96. https://doi.org/10.1007/s40097-014-0096-z

    Article  Google Scholar 

  43. Ahmed EA, Abdel-Hafez EH, Ismail AFM, Elsonbaty SM, Abbas HS, Salah El Din RA (2015) Synthesis of silver nanoparticles by Spirulina platensis & Nostoc sp. Global Adv Res J of Microbiol 4(4):036–049

    Google Scholar 

  44. Elumalai S, Santhose BI, Devika R, Revathy S (2013) Collection, isolation, identification and biosynthesis of silver nanoparticles using microalga Chlorella pyrenoidosa. Nanomechanics Sci Technol Int J 4:59–66

    Article  Google Scholar 

  45. Rajeshkumar S, Malarkodi C, Vanaja M, Gnanajobitha G, Paulkumar K, Kannan C, Annadurai G (2013) Antibacterial activity of algae mediated synthesis of gold nanoparticles from Turbinaria conoides. Der Pharma Chemica 5:224–229

    CAS  Google Scholar 

  46. Gajbhiye M, Kesharwani J, Ingle A, Gade A, Rai M (2009) Fungus-mediated synthesis of silver nanoparticles and their activity against pathogenic fungi in combination with fluconazole. Nanomed Nanotech Biol Med 5:382–386

    CAS  Article  Google Scholar 

  47. Jena J, Pradhan N, Dash BP, Sukla LB, Panda PK (2013) Biosynthesis and characterization of silver nanoparticles using microalga Chlorococcum humiccola and its antimicrobial activity. Int J Nanomater Bios 3:1): 1–1): 8

    Google Scholar 

  48. Rajeshkumar S, Kannan C, Annadurai G (2012) Synthesis and characterization of antimicrobial silver nanoparticles using marine brown seaweed Padina terastromatica. Drug invent Today 4:511–513

    CAS  Google Scholar 

  49. Ali DM, Sasikala M, Gunasekaran M, Thajuddin N (2011) Biosynthesis and characterization of silver nanoparticles using marine cyanobacterium, Oscillatoria willei NTDM01. Digest J Nanomater Bios 6(2):385–390

    Google Scholar 

  50. Giordano M, Kansiz M, Heraud P, Beardall J, Wood B, McNaughton D (2001) Fourier transformed infrared spectroscopy as a novel tool to investigate changes in intracellular macromolecular pools in the marine microalga Chaetoceros muellerii (Bacillariophyceae). J Phycol 37:271–279

    CAS  Article  Google Scholar 

  51. Sharon M, choudhary AK, Kumar R (2010) Nanotechnology in agricultural diseases and food safety. J Phytol 2:83–92

    Google Scholar 

  52. Roh J-Y, Sim SJ, Yi J, Park K, Chung KH, Ryu D-Y, Choi J (2009) Ecotoxicity of silver nanoparticles on the soil nematode Caenorhabditis elegans using functional ecotoxicogenomics. Environ Sci Technol 43:3933–3940

    CAS  Article  Google Scholar 

  53. Lim D, Roh J-Y, Eom H-J, Hyun JW, Choi J (2012) Oxidative stress-related PMK-1 P38 MAPK activation as a mechanism for toxicity of silver nanoparticles to reproduction in the nematode Caenorhabditis elegans. Environ Toxicol Chemist 31(3):585–592

    CAS  Article  Google Scholar 

  54. Choi O, Hu Z (2008) Size dependent and reactive oxygen species related nano silver toxicity to nitrifying bacteria. Env Sci Technol 42:4583–4588

    CAS  Article  Google Scholar 

  55. Jung WK, Koo HC, Kim KW, Shin S, Kim SH, Park YH (2008) Antibacterial Activity and Mechanism of Action of the Silver Ion in Staphylococcus aureus and Escherichia coli. Appl environ microbial 74(7):2171–2178

    CAS  Article  Google Scholar 

  56. Craigie JS (2011) Seaweed extracts stimuli in plant science and agriculture. J Appl Phycol 23:371–393

    CAS  Article  Google Scholar 

  57. Abdellatif KF, Hamouda RA, El-Ansary MSM (2016) Green nanoparticles engineering on root-knot nematode infecting eggplants and their effect on plant DNA modification. Iran J Biotech 14(4):e1309. https://doi.org/10.15171/ijb.1309

    Article  Google Scholar 

Download references

Acknowledgements

The authors wish to thank Soils, Water and Environment Research Institute (SWERI), Agriculture Research Center, Giza, Egypt, for the facilities provided to conduct this research work.

Funding

No funding was received from any agency.

Author information

Affiliations

  1. Department of Soil Microbiology, Soils, Water and Environment Research Institute, Agricultural Research Center, P.O. 175, El-Orman, Giza, Egypt

    Seham M. Hamed

  2. Plant Pathology Research Institute, Sakha Research Station, Agricultural Research Center (ARC), P.O. 175, El-Orman, Giza, Egypt

    Eman S. Hagag

  3. Botany and Microbiology Department, Faculty of Science, Beni-Suef University, Beni-Suef, 62521, Egypt

    Neveen Abd El-Raouf

Authors
  1. Seham M. Hamed
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eman S. Hagag
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Neveen Abd El-Raouf
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SMH and ESH designed the experimental approach, analyzed the data, and wrote the manuscript. SMH contributed to the Ag-NP biosynthesis and full physicochemical characterization of Ag-NPs. ESH carried out the in vitro and in vivo studies. SMH and ESH generated all the tables and figures in the manuscript. NA helped to draft the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Seham M. Hamed.

Ethics declarations

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hamed, S.M., Hagag, E.S. & El-Raouf, N.A. Green production of silver nanoparticles, evaluation of their nematicidal activity against Meloidogyne javanica and their impact on growth of faba bean. Beni-Suef Univ J Basic Appl Sci 8, 9 (2019). https://doi.org/10.1186/s43088-019-0010-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s43088-019-0010-3

Keywords

  • Silver nanoparticles
  • Biosynthesis
  • Nostoc sp.
  • Meloidogyne javanica
  • Nematicide
  • Faba bean