Bioactivity and Biomodification of Ag,ZnO,and CuO Nanoparticles with Relevance to Plant Performance in Agriculture

Abstract

In late 1996, the Nanotechnology Working Group was established in the US, comprised of members of various government agencies, academia, and industry to focus on advancing nanotechnology. It is estimated that the worldwide market for products with nanotechnology components will reach $1 trillion by 2015.¹ Nanoparticles (NPs), which are found in a variety of household, industrial, and medical products, have both benefits and risks related to their small size, which confers enhanced and often unique attributes compared to large-sized particles of similar chemistry. This review focuses on environmental factors that modify the biological activity, transformation, and potential relevance of silver (Ag), copper oxide (CuO), and zinc oxide (ZnO) NPs to help evaluate their risk to agriculture. We chose to study these particular NPs because they possess antimicrobial properties toward certain human pathogens, including those resistant to traditional antibiotics. However, in soil, plant growth and biogeochemical cycles rely on microbial activity that may be susceptible to intentionally or inadvertently introduced NPs. Biological activity of metal and metal oxide NPs toward microbes and plants is observed, although aggregation of the NPs occurs. The NPs act as primary sources of soluble metal so that exposed microbes and plants are faced with both particle-specific and ion-related toxicities. Bioactivity is mitigated by factors present on microbial cell surfaces, components exuded by plant roots, and materials present in soil pore water. At sub-lethal levels, the NPs change bacterial and plant metabolism to make risk prediction complex. Serendipitously, this aspect of NP interaction with bacterial cells or plants could be utilized in the production of commercially valuable metabolites. The potential for NPs to benefit plant productivity by enhancing nutrient availability and improving plant health is discussed herein. The utilization of plant and microbial metabolism for green synthesis of NPs or in remediation of NP-contaminated soils is also addressed. Maximizing these potentials demands a deeper understanding of the complex interactions and interplay between NPs, plants, and microbes relevant to the variability of different ecosystems.

Introduction

Nanoparticles (NPs) are being embraced by a variety of industries in which their unique properties, arising from sub-100-nm dimensions, are leveraged to impart enhanced function to numerous products. Consumers now frequently encounter NPs in such common items as textiles, clothing, cosmetics, and paints.^2,3 New construction materials, catalysts, electronics, and optical devices are also driving higher production of NPs. The medical field is developing NPs for drug delivery, contrast agents, tumor-specific hyperthermia therapy, analytical assays, and real-time sensing. Additionally, certain NPs have antimicrobial activity and are increasingly being investigated against microbial pathogens with acquired resistance to traditional antibiotics.⁴ For instance, zinc oxide (ZnO) NPs are bactericidal (minimal inhibitory concentration 25 mg/L) to Campylobacter jejuni, a leading cause of poultry contamination worldwide; changes in growth morphology, membrane damage, and increased expression of oxidative and general stress-related genes are induced by the treatment.⁵ However, nanotechnology brings new challenges for environmental risk, as well as rewards.^1,6
–8

Although this review focuses mainly on studies of silver (Ag) NPs and the nanometallic oxides ZnO and copper oxide (CuO), and their potential for risk and benefits in agriculture, studies of other NPs such as iron, cerium, and titanium oxides that have shown interesting outcomes also are discussed. Ag, CuO, and ZnO NPs are studied because their ability to restrict growth of human pathogenic bacteria is promoting their utilization in the medical field. However, not all microbes are pathogens; in agriculture many are important in promoting plant health and in soil carbon and nitrogen cycling. Intrinsic to the selected NPs is the release of soluble metals from NP suspensions that, depending on the environment and the target, will have toxic effects. Our discussion focuses on basic findings in the interactions of these NPs with beneficial bacteria and plants, with interpretation of the consequences to agriculture. The use of NPs in agriculture is in its infancy, with relatively few publications compared to the medical field, as discussed by Gogos et al.⁹ Two other recent reviews cover the potential applications of NPs in crop protection and nutrition, and for control of parasitic plants.^10,11 We focus mainly on the responses of pseudomonads to the NPs because these microbes are found globally in soils and are major players in plant growth and health promotion. These findings are used to demonstrate some of the technologies involved in the exploration of NP responses. Through this evaluation, the following general factors are highlighted:

• Biological activity of NPs occurs, although there is evidence of aggregation. Bulk products, however, do not mimic the results observed with the mixtures of aggregates and NPs.

• NPs act as primary sources for ion release. The large surface area of the NPs promotes ion release. The ions, depending on their chemistry and target, may be a toxic hazard.

• There are different ways in which environmental factors may change the activity of NPs. This variability makes it difficult to predict correctly the potential environmental risk associated with NPs. Some interactions could be modeled by considering the fate of bulk products or ions but others will be NP-dependent.

• The NPs modify important aspects of metabolism of microbes and plants at sub-lethal levels. These changes, some of which may be viewed as beneficial and others detrimental, add to the complexity of the microbial interactions with plants in the soil.

Each of these factors may be of significance in the technological applications of the NPs. Whether NPs provide an efficacy and cost that justifies their development for agricultural use is yet unproven.^9,10 The information in this review is presented in three sections: characterization of NPs with emphasis on modification of the structures and release of soluble metal; responses of plant-associated pseudomonads to NPs; and responses of the plant to NPs.

Characterization of NPs: Factors Affecting Size, Shape, Charge and Agglomeration

Our focus is on understanding the factors in soil that may influence the effects of Ag, ZnO, and CuO NPs in their interactions with soil-borne bacteria and plants and thus have an impact on agricultural productivity. Studies on soil-borne bacteria complement plant knowledge, because in nature plants are in constant association with microbes, some of which are pathogens, whereas others are beneficial to plant performance.

A range of instrumentation is frequently used to measure NP physical attributes: X-ray analysis reports on NP crystallinity; electron- and scanning-probe microscopies reveal nanoscale structure; X-ray absorption near-edge spectroscopy (XANES) indicates chemistry and oxidation states of NPs; inductively coupled plasma mass spectroscopy (ICP-MS) detects trace element composition; and Fourier-transform infrared (FTIR) spectroscopy reveals bond structures. NP size, shape, agglomeration, and surface charge in solutions are generally ascertained through atomic force microscopy (AFM), dynamic light-scattering (DLS), and zeta potential measurements.

Environmental Factors Causing Coating, Size, and Charge Differences

Stability of NP size and surface chemistry is crucial to activity in any environment and is influenced by common environmental factors. The charge on the surface of the NP is balanced by counter-ions to form an electrical double layer. The double-layer thickness, given as the Debye length, is defined by counter-ion concentration and valency, which may lead to repulsive or attractive forces between charged NPs. The thickness of the double layer is suppressed, leading to aggregation, by an increase in the valency of the electrolyte in the surrounding solution. For example, negatively charged NPs that remain dispersed in sodium chloride solution will aggregate, not only in the presence of higher valency cations (e.g., Ca²⁺), but also with increasing NaCl concentration.^12
–14

Considering associations between NPs and plants and bacteria, secreted and surface compounds on the latter will influence NP charge through surface coatings mediated primarily through ionic interactions that act as capping layers and thus influence the release of metal from the NPs.^{12

–19} Root exudates to a large extent determine the pH, microbial diversity, and soluble metals present in the region of soil influenced by the root, termed the rhizosphere.^20
–22 Similarly, the bacterial extracellular polymeric materials (EPS) as well as secreted products, such as metal-chelating siderophores, will interact with NPs and/or metal ions they release.

The agglomeration of particles and the association of NPs with bacterial cell surfaces are readily revealed using AFM.^23,24 Fig. 1A shows NPs of original size (<100 nm) as well as submicron- to micron-sized agglomerates in the aqueous fractions from microcosms containing wheat seedlings grown from seed for 14 days in sand with NP amendments. Defined changes in shape and size are observed when ZnO NPs are immersed in bacterial culture medium or after incubation in the aqueous fraction from sand.^16,25,26 Why this shape change is specific to ZnO NPs is not known; it is not seen with CuO NPs. Whether shape modification affects bioreactivity also is not known. These findings raise further questions on how manufacturers' coatings or doping of different particles will influence bioreactivity.

Fig. 1.

Topographical AFM images of CuO and ZnO NPs and their aggregates recovered from the aqueous fractions of sand after incubation for two weeks with growth of wheat seedlings (A). Images of NPs from CuO or ZnO NPs suspended in water or in root wash. Root wash was generated from 1-week-old wheat seedlings using previously published methods (B).¹⁹

AFM imaging further shows coating of the NPs in suspensions with root wash generated from the wheat seedling roots (Fig. 1B), with EPS from bacterial cells, or with components in pore water from soil.^{18,23,24,27,28} DLS measurements show size changes and zeta potential measurements reveal charge differences in the particles present in these mixtures compared with the NPs alone suspended in double distilled water. These changes highlight the extent to which interactions are occurring between NPs and components from plants and the soil.^18,26 The findings illustrate that the behavior of the NPs will differ widely depending on the environment in which they are used. Thus, variability between agricultural soil types is anticipated.

Release of Soluble Metals

Numerous studies reveal that the Ag, CuO, and ZnO NPs release soluble metals. Examining solubility and bioactivity in double distilled water allows evaluation of properties without speciation of metals released from the NPs with media components; speciation will alter metal chemistry, bioavailability, and toxicity. Table 1 provides the levels of soluble Ag, Cu, and Zn released from the NPs in different culture media compared to water and illustrates the effects of time and dose. Media with Cl^- would result in formation of AgCl by removing Ag ions. Geochemical modeling (GEOCHEM-PC) also predicts that no free Ag ions would be available in siderophore and indole-3-acetic acid (IAA)-inducing media (SIIM) due to complexation with histidine.²⁹ Cu and Zn ions also would complex with histidine and other amino acids in SIIM. Although these examples all use laboratory media, the findings illustrate that the soil composition and, thus, the materials in the pore water, and the chemicals in the applications used to fertilize and protect crops from pests could influence solubilization of ions from the NPs.

Table 1.

Summary of Nanoparticle Dissolution in Water or Medium and Correlation with Observed Effects on Growth and Metabolism of P. chlororaphis O6 (PcO6)^{16,23
–25,28}

NP TYPE	NP CONCENTRATION (mg/L)	MEDIA TYPE	TIME (h)	DISSOLVED METAL^a (mg/L)	pH	TOXICITY/METABOLIC EFFECT OF LEVEL OF DISSOLVED METAL ON PLANT AND BACTERIA
Ag	10	Water	1	0.28	6	Non-toxic
	10	Water	1	2.3	7	Bactericidal (100%)
	1	SIIM	0 24	0.18	6.8	Not tested, but 1 mg/L Ag ion only bacteriostatic and reduces IAA^b levels
			48	0.38
				0.08
	2	SIIM	0 24	0.22	6.8
			48	0.36
				0.32
	3	SIIM	0 24	0.29	6.8
			48	0.29
				0.31
CuO^c	10	Water	1	0.2	6	Non-toxic
	200	Water	1	1.0	6	∼18.8% toxicity
	500	Water	1	1.4	6	∼47.5% toxicity
	10	Water	1	0.3	7	Non-toxic
	200	Water	1	0.6	7	∼6.3% toxicity
	500	Water	1	1.1	7	∼20.7% toxicity
	200	SIIM	1 24	3	6.49	Bacteriostatic, no effect to increased IAA level
			48	29
				43
	200	SIIM	1	2.4	6.8	Bacteriostatic, increased PVD^b level
	500	SIIM	1	7.6	6.8	Not tested
	200	SIIM	48	21	6.8	Not toxic, quenches PVD fluorescence
ZnO	10	Water	1	2.8	6	Non-toxic
	200	Water	1	15	6
	500	Water	1	23.3	6
	10	Water	1	1.65	7
	200	Water	1	3.7	7
	500	Water	1	4.9	7
	200	SIIM	1	4.96	6.8	Bacteriostatic, then improves cell growth; stimulates early PVD and then quenches fluorescence
	500	SIIM	1	5.12	6.8
		SIIM
	500	SIIM	1 24	5	6.89	Bacteriostatic, no effect to reduced IAA level
			48	12
				20

Metals were assessed by inductively coupled plasma mass spectrometry (ICP-MS) on supernatants obtained after centrifugation to remove NPs and insoluble species. No acid digestion was used so that only soluble metal was determined.

IAA = indole-3-acetic acid; PVD = pyoverdine.

CuO NPs from the same distributor vary in antimicrobial activity between batches. Data shown are for one batch only.

The data in Table 1 also reveal that solubility of Ag NPs in water did not follow observations with bulk products, i.e., greater dissolution under more acidic pH, an observation also made with Ag NPs by Fabrega et al.¹⁹ The addition of base may have increased solubility through complexation of Ag ions with hydroxide ions.²⁴ For CuO NPs in water, solubility is not pH-dependent but increases with dose. Dissolution is more in SIIM than in water, and increases in SIIM with a higher level of tryptophan to permit IAA production.^16,25 For ZnO NPs in water, solubility shows both dose and pH dependency; Wang et al. also observed this effect.³⁰ In SIIM, soluble Zn levels are independent of dose but dependent on time.

Because solubilization is affected differently by pH, time, and dose according to the NP and suspension medium, it is difficult to predict outcomes in the agricultural field. From a biotechnology perspective, these unique properties may be leveraged and made to be advantageous. Clearly, this work is in its infancy as far as understanding NP behavior in the environment. Data are needed on how plant root or bacterial exudates or pore water components modify release of soluble material from the NPs.

In summary, the dynamic nature of NPs complicates the subject of nanotoxicology. NP stability, strength of aggregation or agglomeration, dissolution to ions, and remodeling to form novel NPs makes prediction of risk difficult.¹⁴ Is it the original NPs, generated aggregates or agglomerates, dissolved ions from the NPs, or novel NPs formed from dissolved ions that are causing the observed bioreactivity?

Responses of Plants to Beneficial, Soil-Borne Pseudomonads

Microbial biochemical and geochemical transformations are the basis for many industrial, agricultural, and environmental processes. Soil bacteria are important in element cycling, in bioremediation, and in interaction with other soil organisms, such as plants, where they may be pathogenic or beneficial.^31,32 The genus Pseudomonas has many isolates that function in these roles. Currently, there are few studies that report the effects of NPs on beneficial microbes with and without plant interactions. All plants in nature are microbially colonized. The findings with pseudomonads, however, alert us to the types of effects that NPs may have on plant growth and productivity through altering microbial traits. The responses of each of the NPs with the isolate P. chlororaphisO6 (PcO6), as an example of a beneficial plant-associated microbe, is discussed; colonization of plant roots by this bacterium promotes the plant's ability to counteract both abiotic and biotic stresses.^33

–36 We also show effects on Pseudomonas putida KT2440 as another soil and root colonizer with beneficial properties in bioremediation.³⁷ Changes in the function of these microbes by NPs thus could affect plant performance and carbon cycling in the soil.

Bacterial Sensitivity to NPs is Variable

Nanoparticulate size alone does not confer an ability to kill microbes. Toxicity of Ag, ZnO, and CuO NPs correlates, in part, with release of the ions, which themselves are toxic. The two soil pseudomonads are more tolerant than some human pathogens to Ag (10 nm), ZnO (50–70 nm), and CuO (33 nm) NPs, yet they display different inhibitory levels from each other.^23,24,37 These findings imply that there is likely to be extreme variability in the dose-response levels between different NPs and the diverse microbial populations that regulate plant performance.

Ag Np-Pseudomonad Interactions

Substantial work from many research groups supports the hypothesis that the antimicrobial activity of Ag NPs is due to superior delivery of Ag ions to the bacterial cells. A proteome analysis demonstrated that altered membrane function leading to ATP depletion of Escherichia coli cells caused by the NPs is duplicated by higher concentrations of Ag ions. McQuillan et al. showed the importance of Ag ion release from the NPs at the cell membranes.^38,39 Xiu et al. reported that restriction of Ag ion release limits antimicrobial effects of Ag NPs.⁴⁰ These findings indicate that environmental factors promoting or limiting Ag ion release from Ag NPs will regulate the antimicrobial activity of this NP.

As reviewed by Kahru et al., we, like other research groups, use biosensors to establish a major role of Ag ion release from Ag NPs in bacterial toxicity.⁴¹ One study compared the response of a wild type E. coli strain to a mutant strain defective in an Ag⁺/Cu⁺ efflux pump (strain GG44).⁴² The cultures, grown at 37°C in a rich medium—lysogeny broth (LB) lacking chloride ions—are challenged with Ag NPs or Ag ions for 6 h before determining cell density. The data shown in Fig. 2 illustrate that the mutant strain is more sensitive to killing on exposure to the Ag NP preparation than is the wild type strain. AFM analysis of this preparation confirms the presence of NPs. When Ag ions are used at the concentrations indicated, the results obtained with the NPs are replicated. This result supports the hypothesis that the Ag ions are the toxic factor for the E. coli cells.

Fig. 2.

Biosensor responses to NPs. Exposure of cells of the wild type (black bars) and mutant GG44 (lighter bars) E. coli strains to Ag NPs or to equivalent concentrations of Ag ions has the same effect on growth. Growth effects were monitored by measurement of OD_600nm of the cultures after 6 h of challenging the cells with the Ag products (A). Soluble Ag contained in the supernatant (SN) generated by centrifugation of a suspension of Ag NPs in water have the same effect as the intact NP suspension (Ag NPs) on light output (relative light units, RLU) in the biosensor strain, P. putida KT2440 (B). Mitigation by EPS of toxicity from exposure of P. putida KT2440 to Ag NPs at 0.5 mg/L. EPS was obtained from the pseudomonad cell surface by published procedures.¹⁵ Data are means of three replicate readings and are from one of three studies showing the same trends for protection of loss of light output (C). Protection by components in the wheat root wash (RW), indicated by restoration of the loss in light output (RLU) in P. putida KT2440 following challenge with CuO and ZnO NPs (D).

A second study used a different biosensor, generated in P. putida KT2440, in which genes endowing light production were present.³⁷ This biosensor shows reduced light output under toxic conditions.³⁷ Fig. 2B shows that loss in light output is equal whether the native Ag NPs preparation or only a supernatant with soluble Ag is used following centrifugation to pellet the NPs.²⁴ Release of soluble Ag from the Ag NPs is confirmed by ICP-MS analysis of the elemental composition of the supernatants generated after removal of NPs by centrifugation.²⁴ High variability is found in Ag solubility with various commercial preparations of Ag NPs that we have examined, making it difficult to compare results from studies in which researchers have used different sources. These findings also support the need to characterize the properties of each NP preparation to understand the mechanisms involved in bioresponses.

From a biotechnology perspective, the sensitivity of human pathogens to Ag NPs has medical importance in the purification of water and in formulations for antiseptics, especially when the pathogen being targeted is resistant to traditional antibiotics.⁴³ However, of concern is environmental contamination of water streams with Ag because of its extreme toxicity to non-target bacteria and other life forms. As shown, reporter microbes can be employed as “sentinel organisms” to provide relatively rapid (minutes) toxicity analysis. Positive results for toxicity would be confirmed with sensitive analytical methods; plate counts would reveal changes in the ability to culture microbes.

As illustrated above and reviewed by Kahru et al., understanding the responses of an organism to a challenge has permitted the construction of useful biosensors.⁴¹ Using the biosensor in strain KT2440, we observe that toxicity of Ag NPs is neutralized by EPS isolated from pseudomonads (Fig. 2C), at least on the time scale of these experiments. Whether the coated particles still release metal needs to be determined.

In a comparison of the antimicrobial effects of Ag NPs in soil versus sand, we find that the soil negates killing activity.¹⁸ Antimicrobial activity correlates with the levels of free Ag ion, again demonstrating the importance of the Ag NPs as a primary source for Ag ion release.¹⁸ By adding various factors back into the sand and by running geochemical models for free ion activity in the presence of pore water, the major factor limiting available free ion in the soil is the Cl^- content of the pore water.²⁹ Thus, precipitation of Ag ions as insoluble AgCl is a key factor in eliminating toxicity. Another protective factor is the coating of the NPs with materials in the pore water; here, the humic acid component of the soils coats and causes aggregates to form. These findings support earlier studies in which humic acid was shown to coat and alleviate the antibacterial effects of Ag NPs, possibly due to restrictions on release of Ag ions.¹⁹ Clearly, the fate of NPs in the agricultural environment will vary with soil and water components.

CuO/ZnO Nps-Bacterial Interactions

At similar NP concentrations, both pseudomonads display higher resistance to CuO and ZnO NPs than certain pathogens.^4,23,44 The full genomes of the PcO6 and KT2440 isolates reveal potential genes for resistance to Cu or Zn ions released from the NPs.^36,45 The functionality of certain metal-resistance genes is demonstrated by using proteomics in isolate KT2440 to identify proteins induced by Cu versus Cd ions.⁴⁶ The P. putida KT2440 biosensor shows that the cells are detecting Zn ions released from the NPs because there is activation in expression of light output at levels of Zn below the toxicity threshold.³⁷ This response is anticipated because the luxAB genes conditioning light output in the biosensor are expressed under control of a heavy metal-activated promoter.^37,46 As time progresses, the toxic effect of the ZnO NPs takes over, as seen by loss in light output.³⁷

Measurements of Cu and Zn levels show that metals are released when the CuO and ZnO NPs are suspended in fluids (Table 1).^{16,23
–25,28,41} Cell culturability assays with the pseudomonads show that EPS protects against loss in culturability of PcO6 as does the presence of a chelator—bathocuproine—specific for Cu ions.^23,24 Thus, both ZnO and CuO NPs, like Ag NPs, act as point sources for metal release in the environment that are important in bacterial cell interactions.

To understand factors from the soil environment that may alter the NP-bacterial response, biosensor assays have been performed in the presence of the mixture of components washed from plant roots. This material contains sugars and organic acids used as a carbon source for the microbe so that immediate elevation of light output is seen from the biosensor cells due to enhanced metabolic activity (Fig. 2D). Although exposure to ZnO NPs alone eventually leads to loss in light output, luminescence is maintained when the root wash is present. Similarly, the components in the root wash protect against the toxic effect of the CuO NPs (Fig. 2D). AFM imaging reveals that the materials in root wash coat the NPs (Fig. 1B). However, the compounds in the root wash do not preclude a plant's response to CuO and ZnO NPs, as discussed later in this paper.

Bacterial Metabolism Is Altered At Sublethal Levels

Much of the concern about the environmental risk of NPs has focused on lethal levels for microbes. However, we see major alterations to metabolism at levels that do not produce lethality. The production of three classes of metabolites important to the function of isolate PcO6 in the soil and plant rhizosphere is changed; the metabolites are the plant growth regulator IAA, the fluorescent siderophore pyoverdine (PVD), and the phenazine antibiotics.^16,25,28 IAA levels produced by microbes associating with the root surface are implicated in improving plant growth within a concentration range.^47,48 Siderophores are involved in enhancing Fe levels in Fe-deficient environments, such as alkaline soils, not only in the bacterium, but also in plants.^49

–53 In addition, siderophores help the plant grow in environments with heavy metal contamination.^49,54

–57 Furthermore, the PVD-type siderophores are among effector molecules from beneficial rhizosphere colonists that stimulate plant defenses.⁵⁸ Phenazines decrease the growth of sensitive fungal plant pathogens, are effectors for stimulation of plant defenses, and promote bacterial survival under stress conditions, such as within a biofilm.^59,60 Thus, NPs have the potential to alter metabolic products important in the survival of the bacterium, its interaction with other soil microbes, and in the ability of the bacterium to affect plant stress responses. The findings add to the complexity of predicting in agriculture the outcome of contamination of soils with NPs, since not all NPs act in the same way.

Iaa

IAA production level and timing are affected differentially by Ag (Fig. 3A), ZnO, and CuO NPs.²⁵ Cell growth was not altered. With all NP treatments, increases in IAA levels occur earlier in culture than with control cultures. The significance of interaction between tryptophan in the medium and the ZnO NPs in the observed effects is not known.^61,62 Treatment of cells with Cu and Zn ions shows that the release of ions from CuO NPs, but not ZnO NPs, at the levels observed in bulk solution, could contribute to the response.²⁵

Fig. 3.

Effects of sub-lethal concentrations of NPs on production of metabolites in PcO6. Stimulation of IAA production in PcO6 by Ag NPs occurs without effects on cell growth, measured as optical density (OD_600nm). In contrast, 1 mg/L Ag ions initially show a bacteriostatic effect, yet even when growth is resumed, IAA production is inhibited (A). Dose-dependent inhibition of the production of phenazines in PcO6 by addition of ZnO NPs to the growth medium compared with treatment with Zn ions. Loss in phenazines is seen visually by the reduction in orange pigmentation and by measurement of A_366nm of extracts from the culture medium (B).

Phenazines

ZnO NPs cause dose-dependent reduction in phenazines produced by PcO6 observed visually as a loss in orange pigmentation and confirmed by characteristic absorbance at A_366nm (Fig. 3B).⁶³ Compositional analysis illustrates inhibition for phenazine produced by PcO6 (Table 2). Amendment with Zn ions, replicating soluble Zn from the NPs (up to 30 mg/L soluble Zn), does not fully account for impaired phenazine production (Fig. 3B). However, Zn ion inhibition of the first enzyme involved in phenazine synthesis in another bacterium suggests this as one factor in the change in metabolism.⁶⁴

Table 2.

Effect of ZnO NPs on the Production of Specific Phenazine Structures Produced by PcO6.^a

PHENAZINE	CULTURES LACKING ZnO NPs (PEAK AREA RELATIVE VALUES)	CULTURES WITH ZnO NPs (PEAK AREA RELATIVE VALUES)	% INHIBITION BY ZnO NPs
2-hydroxy-phenazine	8,400,000	0	100
2-hydroxyphenazine 1-carboxylic acid	192,000,000	14,500,000	92
Phenazine-1-carboxylic acid	325,000,000	7,300,000	98
Phenazine-1-carboxamide	304,00	81,000	73

PcO6 was grown in cultures to stationary phase (48 h) with and without 500 mg/L Zn provided by ZnO NPs. Secreted phenazines were extracted into acidified ethyl acetate, taken to dryness and dissolved in ethanol. These samples were analyzed on an Agilent (Santa Clara, CA) LC/TOF-MS system in reverse-phase HPLC and electrospray-ionization mode. The data were reduced using the Masshunter Qualitative Workstation (Agilent).

Pvd

CuO NPs cause strong inhibition of secreted PVD levels from PcO6 cells, whereas higher production occurs with the ZnO NPs.¹⁶ Cu ions used at the level solubilized from the NPs did not duplicate the response or cause repression seen with the NPs of discrete genes in the PcO6 genome responsible for PVD synthesis.^16,28 This transcriptional analysis shows that the response is not a shut-down of all gene expression as anticipated from a general toxic response.

Clearly, at sublethal levels, Ag, CuO, and ZnO NPs affect important metabolic pathways in the bacterium but with different results. This finding has industrial implications. Supplementation of growth medium with NPs may be cost-effective for producing metabolites with commercial value without the need for genetic engineering of the microbe. This would be especially valuable for secreted products in which both cells and NPs could be eliminated by centrifugation of the cultures. However, the metabolic findings also indicate that there may be consequences for soil and water contamination with NPs. The electrostatically driven loading of bacterial cell surfaces with ions released from the particles also will increase the metal burden of a biome. Several manuscripts already address the possibility of Ag NPs altering the microbial-catalyzed nitrogen cycle important in water processing systems through impacts on bacterial populations and inhibition of specific enzymes.^65
–67

Plant Responses to Ag, ZnO, and CuO NPs

In addition to having antimicrobial properties, Ag, ZnO, and CuO NPs are phytotoxic; responses are dependent on the NP and dose, the plant species, and the application site (e.g., root or shoot), and duration of the challenge. Ag NPs are highly toxic toward cucumber, silage grass (Lolium multiflorum), onion, rice, mung bean, sorghum, and zucchini.^68

–74 CuO and ZnO NPs are among the many other metal oxide NPs (e.g., cerium oxide (CeO₂), ytterbium oxide (Yb₂O₃), lanthanum oxide (La₂O₃), gadolinium (Gd₂O₃) that are phytotoxic for cabbage, carrot, cucumber, lettuce, maize, radish, rape, rye, soybean, tomato, wheat, and zucchini.^{26,68,71,75

–85}

Most of the studies reported above demonstrate the effects of NPs only in hydroponic conditions, where metal availability and root structure are quite different from growth in a solid matrix. We use sand as a simple solid growth matrix with only short-term (up to 14 d) growth. Soil factors and NP aging would more likely alter NP activity than in sand. In soil, there would be the potential for effects of ionic strength and an array of compounds including complex organics such as the humic acids. Depending on the NP, coating of NP surfaces with organic materials from the root or other sources would alter aggregation and affect metal solubility.^18,19,86

Amendment of sand with Ag, CuO, and ZnO NPs reduces wheat (Triticum aestivum L.) growth, confirming NP-phytotoxicity (Fig. 4).^{70,71,74

–80} AFM analysis reveals that there are still NPs of original size present in the aqueous fractions of the microcosm as well as agglomerated/coated NPs (Fig. 1A); whether either or both are the active form remains to be determined. Fig. 4C illustrates that NPs cause proliferation in root number and production of lateral roots. Thus, there are clear biochemical effects on plant growth. Metal solubility at levels that peak early followed by reduction occurs (Fig. 4A). Changes in enzymes and levels of oxidized glutathione (GSSG) measured in the NP-exposed plants are in common to the effects of metal ion exposure.^26,87
–89 The different patterns of release may reflect the greater inherent solubility of ZnO compared with CuO and different chemistries of speciation.

Fig. 4.

Effects of NPs on wheat seedlings. Dissolution of CuO and ZnO NPs into Cu or Zn in sand over a 14-d period in relation to effects on wheat growth after 14 d (A). Effects of CuO and ZnO NPs and ions on shoot and root lengths. For each plant response, Cu (3 mg/L) was more phytotoxic than Zn (6 mg/L) over a two-week period (B). Images of wheat plants challenged with CuO and ZnO NPs in sand showing effects on root elongation and proliferation (C).

Although the CuO and ZnO NPs amendments were only contacting the roots, Cu and Zn accumulate in the shoots, meaning that some form of transport from roots to shoots occurs.²⁶ These findings confirm results of metal loading of plant tissues from NPs from other groups; CuO, CeO₂, and ferric oxide accumulate in shoots of plants challenged with the corresponding NPs.^83,90,91 XANES analysis shows that Cu in the shoot is present mainly as CuO, and to a lesser extent complexed as Cu(I)-S, while Zn is found as Zn-phosphate.²⁶ Each of these accumulating products have low solubility, reducing potential toxicity of the ions.

These findings are consistent with low dissolution of CuO NPs, followed by ion uptake and reduction to Cu(I) to complex with sulfhydryl groups present in amino acids or antioxidants such as reduced glutathione (GSH). Cu-S complexes are phytotoxic, being involved in GSH oxidation via interaction with thiol groups.^92,93 Sarret et al., documented transformation of Zn to Zn-phosphate in shoots of Arabidopsis halleri, likely due to initial release of Zn from the NPs.⁹⁴ However, ZnO aggregates from exposure to ZnO NPs are detected in root cells for ryegrass, and onion, and in root xylem vessels of corn.^80,95,96 Root uptake of ZnO NPs is not reported for soybean and velvet mesquite.^87,97 Thus, the form in which Zn accumulates in plants from NP challenge (Zn ions, Zn-phosphate, Zn-nitrate, Zn-COOH/OH, Zn-phytase, Zn-histidine, Zn-citrate, Zn-phyochelatin) varies with plant species and plant physiology.^98.99

Biochemical changes in plants grown with NPs are accompanied by cellular and genetic responses.^69,75,87,95 Lopez-Moreno et al., reported the appearance of additional random amplified polymorphic DNA bands in DNA extracted from soybean roots when treated with 2,000 and 4,000 mg/L of CeO₂ or ZnO NPs.⁹⁷ These novel bands suggest that the NPs cause DNA damage. Similarly, oxidative damage of radish and ryegrass DNA by CuO NPs occurs, evidenced by accumulation of 7,8-dihydro-8-oxoguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 4,6-diamino-5-formamidopyrimidine.⁷⁵ Kumari et al., reported dose-dependent (25–100 mg/L) mitotic, micronucleic, and chromosomal aberration indices in onion roots cells associated with Ag and ZnO NPs.^69,95 These modifications correlate with changes in the number of cells in each mitotic phase.

Bioactivity of NPs on Plants Raised in Soil

Few studies have been reported on the effects of NPs with plants raised in soil, in which one would expect to see effects of organic matter content and ionic strength on NP stability and structure. The definitive factors that influence plant responses to NPs in soil await elucidation.

Exposure of mung bean to Ag NPs (2,000 mg/kg) in soil results in growth effects compared to significant reductions observed in agar at 10–40 mg/L.⁷⁰ This differential effect did not correlate with soluble Ag levels: 1.3 mg/L in agar versus 2.7 mg/L in soil.⁷⁰ However, sorghum, when used in similar 5-d studies in soil with Ag NPs at 200 mg/kg, has growth reduced 20% with more toxicity in the roots than shoots.

Growth studies with ZnO NPs in soil show mixed results with different plants. Reduced wheat growth in soil with ZnO NPs (45 mg/kg soil) correlates with high levels of Zn uptake into stems, seed shells, and grain.⁷⁶ In contrast, growth of cucumber at 2,000 mg/kg soil ZnO NPs is not affected.⁷⁷ Soybean raised in soil accumulated Zn from ZnO NPs into the shoot and seed tissues, with improved overall plant growth.¹⁰⁰ A potentially negative consequence is that the tissues from the ZnO NPs had lowered water content, suggesting problems in drought-associated growth conditions. Lower yields and overall growth occur when the soybean is grown in soil containing CeO₂ NPs. Ce is retained in the roots, where reduced efficacy of nitrogen fixation through root nodulation is observed. How the function of the root nodule bacteroids is affected is not known. However, this finding suggests that CeO₂ contamination of soils would be serious for legumes that rely on nitrogen fixed in the root nodules for growth. Currently, there are no reports of the impact of NPs on other plant microbial endophytes that improve plant nutrition (e.g., arbuscular mycorrhizal fungi for phosphate and mineral nutrition).

Potential of Nanoparticles for Enhancing Crop Performance

As demonstrated above, not all plant responses on exposure to NPs are negative. The soybean field studies found enhanced plant growth with ZnO NPs.¹⁰⁰ Pandey et al., described improved seed germination and root growth of chickpea by ZnO NPs through a mechanism thought to involve increased production of the growth regulator IAA.¹⁰¹ Whether this IAA response is due to metabolic changes in plant-associated bacteria or due to changes in plant metabolism is not resolved. Other metal oxide NPs also have shown potential to enhance plant performance. Magnetite (Fe₃O₄ NPs; 500 mg/L) did not affect pumpkin growth in a 20-d hydroponic challenge, although there is bioaccumulation of Fe from the NPs in the shoot.⁹¹ The Fe₃O₄ NPs (100 mg/L) enhanced root growth of rye grass and pumpkin in an 18-d study.¹⁰² These studies suggest that certain NPs could be beneficial depending on dosage and chemistry.

With titanium dioxide (TiO₂) NPs (2.5%), photosynthesis rate in plants is promoted partially due to an increase in chlorophyll A synthesis and improved activities of RuBisCO activase that enhances CO₂ incorporation.^103
–105 TiO₂ NPs possess considerable photocatalytic properties that may be important in stimulation of photosynthesis, and Ti ions have low cellular toxicity compared with Ag, Zn, or Cu ions.^82,106

In addition to the direct effects on plant metabolism discussed above, NPs may improve plant productivity through effects on interacting microbes. For instance, elevated microbial siderophore production could dissolve hematite (Fe₂O₃) NPs, releasing Fe to be assimilated by the plant.^107,108 Although siderophores are produced primarily to scavenge Fe, they also bind other metals.^{49
–51,55,109
–111} Indeed, microbial siderophores promote plant growth and health by mitigating abiotic stress, while facilitating metal uptake in soils that are heavily contaminated with metals.^{49,51

–57} Such accumulation of metals into plant tissues could be valuable for phytoremediation; selected plants that are tolerant to metal accumulation could be grown in soils contaminated with NPs.

Another means of improving plant growth, plant health, and post-harvest quality through NP applications could arise through growth inhibition of potential plant pathogens, reviewed in more detail by Gogos et al. and Khot et al.^9,11 Field tests show the efficacy of Ag NP sprays in controlling powdery mildew on cucumber and pumpkin.¹¹² The Ag NPs reduce conidial germination and mycelial growth. There also is a surge of interest in the use of ZnO NPs as antimicrobials for plant diseases. He et al. found that ZnO NPs (≥3 mM), over a period of 12 d, significantly reduced growth of post-harvest molds Penicillum expansum and Botrytis cinerea.¹¹³ Microscopy analysis shows serious morphological alterations in the fungal mycelia in the presence of the NPs, with a suppression of conidial germination and development. Furthermore, growth of post-harvest molds Aspergillus flavus and A. niger are reduced in vitro by ZnO NPs (25 mg/L).¹¹⁴ The use of ZnO NPs is attractive because Zn has limited human and environmental toxicity.

Potential for NP Use for Crop Biofortification with Essential Elements

The bioavailability of many essential metals to plants is low in alkaline soils due to pH-induced insolubility.^115,116 The 30% of the earth's land mass that is alkaline is unevenly distributed around the globe. Crops grown on these soils have extremely low levels of essential elements such as Zn, Cu, and Fe. Use of these crops for food consequently leads to problems in human nutrition. According to the US National Institutes of Health, human adults (≥19 years) and teens (9–18 years) require a daily average Zn dose of 9.5 mg, while children (0–8 years) require a daily average dose of 3.5 mg. Nanotechnology may help solve the problem of essential micronutrients deficiency in plants used for food.

Although NPs become aggregated in the presence of plants (Fig. 1), metals are still released. The dissolution of Zn from ZnO NPs and ZnO microparticles is similar in both liquid and solid systems, with pH rather than particle size being the determining factor.^23,26,117 Slow release of soluble Zn from NPs under alkaline conditions would provide a prolonged source of Zn ions at nonphytotoxic levels.^23,26,77,117 Fig. 4A-B demonstrates that dissolution of Zn from ZnO NPs, though higher than that of Cu from CuO NPs, was less phytotoxic, yet a great deal more Zn was taken up by the plant than Cu.²⁶ In other practical studies, Milani et al. formulated ZnO NPs with the fertilizers mono ammonium phosphate (MAP) and urea.¹¹⁷ Owing to pH differences in the formulations, release of Zn is higher with MAP (7%) than urea (<0.5%), indicating that tuning of solubility is feasible. The authors indicate that these release levels are too low to be effective in boosting Zn levels for human nutrition, but did not consider that dissolution of Zn from the amendments will be a continuous, albeit slow, process throughout the cropping session.

Other formulations may also provide higher and more beneficial controlled releases. Nanocomposite formulations consisting of Zn-, Fe-, and Cu-based NPs and biochelators such as microbial siderophores, could be candidates for novel formulations. As noted previously, use of biochelators rather than synthesized chemicals (e.g., ethylenediaminetetraacetic acid, EDTA), to induce metal solubility is not only cost effective, but also environmentally friendly because they are biodegradable and synthesized using “green” chemistry.⁵¹

Green Synthesis of NPs in Plants and Bacteria

In some cases, metal loading of tolerant plants results in synthesis of NPs. Brassica and Medicago spp accumulate Ag and Au as NPs to about 0.4% mass after 24-h treatment of seeds or pre-germinated plantlets with Au or Ag ions.^118

–121 Preferential accumulation in the chloroplast correlates with the potential for reduction of the ions to elemental NPs.¹¹⁹ Although no data shed light on the effects of synthesis of the NPs on plant growth, plant cell wall damage is observed microscopically.¹¹⁹ By lowering the concentrations of the metal treatment while increasing the duration of challenge, it is possible that significantly higher bioaccumulations of the metals could be realized with little or no adverse effects on plant growth. Brassica species are well-known metal hyperaccumulators and so would be good candidates for use in phytoremediation of metallic NP contamination.

Phytoremediation of NP-polluted soils may allow recovery of the NPs from the plant tissues. One example would be the use of magnets in the recovery of iron-based NPs from homogenates of plants in which the NPs have accumulated. Localization of NPs to vacuoles, typically used for storing unwanted toxins, or to chloroplasts suggests that the isolation of these plant organelles would benefit the purification process.^119,122,123

A major industry already exists in green synthesis of Ag NPs using bacterial, fungal, or algal preparations as reductants of Ag ions.^{124

–129} Reduction by the activity of microbial nitrate reductases appears to be involved as one mechanism. For fungi, this enzyme is secreted, so that the process uses cell-free filtrates of pre-grown cultures. The use of cell-free preparations boosts the ease of purification of the NPs. Similarly, novel quantum dots can be raised using the reductive power of the bacterial cell when supplied with ions that the cells' metabolites/enzymes can reduce.^130

–133 However, as determined by FTIR analyses, these NPs are found coated with cellular materials. Whether these contaminating structures alter the effectiveness of the NPs awaits rigorous investigation. The potential differences in shape, size, and coating of the green NPs compared with those generated through other processes may yield products with special technological value. Thus, the studies of NPs and plants can extend to commercialization outside of agriculture.

Conclusions

Our discussion highlights biotic and abiotic environmental factors that influence the bioactivities of Ag, CuO, and ZnO NPs, with a focus on the metabolic responses of plants and their beneficial pseudomonad root colonizers. Fig. 5 summarizes the various fates of NPs in an agricultural setting, illustrating the many dynamic interactions they can undergo. Aggregation, stabilization, surface charge alterations, and dissolution to ions are key changes with the NPs. Association of the NPs with plant or microbial surfaces also adds complexity. Microbes and plants in contact with the NPs are faced with both particle-specific and ion release-related challenges. Ultimately, the plants' response will be strongly dependent on NP type, dose, and speciation and on the plant species involved. As we continue to exploit the special properties of NPs in the production of value-added industrial, medical, and household products, we have the responsibility to continue to evaluate their short- and long-term impacts on varied agro-ecosystems.

Fig. 5.

Illustration of potential fates of NPs in agricultural soil. Interactions between soil microbes (shaded rectangles) and plant roots are shown. M indicates soluble metal, which could be an ion or a chelated species.

Footnotes

Acknowledgments

This work was supported by NIFA grant 2011-03581, the Utah Agricultural Experiment Station (UAES Journal Paper Number 8452), and the Utah Water Research Laboratory. We thank NCL (Salt Lake City) for the gift of Attostat Ag NPs for our studies. We gratefully acknowledge collaborator Dr. Drew Latta of Argonne National Laboratories for his excellence in imaging XANES analyses. We thank our undergraduate researchers, especially Alyssa Calder, Jordan Goodman, and Nicole Martineau, for their assistance in this work.

Author Disclosure Statement

No competing financial interests exist.

References

Roco

. Environmentally responsible development of nanotechnology. Environ Sci Technol, 2005; 39:106–112.

Nohynek

, Lademann

, Ribaud

, Roberts

. Grey goo on the skin? Nanotechnology, cosmetic and sunscreen safety. Crit Rev Toxicol, 2007; 37:251–277.

Brayner

. The toxicological impacts of nanoparticles. Nano Today, 2008; 3:48–54.

Ren

, Hu

, Cheng

EWC

et al. Characterisation of copper oxide nanoparticles for antimicrobial applications. Int J Antimicrob Ag, 2009; 33:587–590.

Xie

, He

, Irwin

et al. Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni. Appl Environ Microb, 2011; 77:2325–2331.

Colvin

. The potential environmental impact of engineered nanoparticles. Nat Biotechnol, 2003; 21:1166–1170.

The Royal Society. Nanoscience and Nanotechnologies: Opportunities and Uncertainties. 2004. www.nanotec.org.uk/finalReport.htm. 2012 November .

Nel

, Xia

, Mädler

, Li

. Toxic potential of materials at the nanoscale level. Science, 2006; 311:622–627.

Gogos

, Knauer

, Bucheli

. Nanomaterials in plant protection and fertilization: Current state, foreseen applications, and research priorities. J Agr Food Chem, 2012; 60:9781–9792.

10.

Khot

, Sankaran

, Maja

et al. Applications of nanomaterials in agricultural production and crop protection: A review. Crop Prot, 2012; 35:64–70.

11.

Pérez-de-Luque

, Rubiales

. Nanotechnology for parasitic plant control. Pest Manag Sci, 2009; 65:540–545.

12.

El Badawy

, Luxton

, Silva

et al. Impact of environmental conditions (pH, ionic strength, and electrolyte type) on the surface charge and aggregation of silver nanoparticles suspensions. Envir Sci Tech, 2010; 44:1260–1266.

13.

Prathna

, Chandrasekaran

, Mukherjee

. Studies on aggregation behaviour of silver nanoparticles in aqueous matrices: Effect of surface functionalization and matrix composition. Colloid Surface A, 2011; 390:216–224.

14.

Jiang

, Oberdorster

, Biswas

. Characterization of size, surface charge, and agglomeration state of nanoparticle dispersions for toxicological studies. J Nanopart Res, 2009; 11:77–89.

15.

Auffan

, Rose

, Wiesner

, Bottero

J-Y

. Chemical stability of metallic nanoparticles: A parameter controlling their potential cellular toxicity in vitro. Environ Pollut, 2009; 157:1127–1133.

16.

Dimkpa

, McLean

, Britt

, Anderson

. CuO and ZnO nanoparticles differently affect the secretion of fluorescent siderophores in the beneficial root colonizer, Pseudomonas chlororaphis O6. Nanotoxicology, 2012; 6:635–642.

17.

Liu

, Chen

, Su

. Effects of material properties on sedimentation and aggregation of titanium dioxide nanoparticles of anatase and rutile in the aqueous phase. J Colloid Interf Sci, 2011; 363:84–91.

18.

Calder

, Dimkpa

, McLean

et al. Soil components mitigate the antimicrobial effects of silver nanoparticles towards a beneficial soil bacterium, Pseudomonas chlororaphis O6. Sci Total Environ, 2012; 429:215–222.

19.

Fabrega

, Fawcett

, Renshaw

, Lead

. Silver nanoparticle impact on bacterial growth: Effect of pH, concentration, and organic matter. Envir Sci Tech, 2009; 43:7285–7290.

20.

Haichar

, Marol

, Berge

et al. Plant host habitat and root exudates shape soil bacterial community structure. ISME J, 2008; 2:1221–1230.

21.

Jin

, He

, Tang

et al.

Mechanisms of microbially enhanced Fe acquisition in red clover (Trifolium pratense L.)

Plant Cell Environ, 2006; 29:888–897.

22.

Yang

, Crowley

. Rhizosphere microbial community structure in relation to root location and plant iron nutritional status. Appl Environ Microb, 2000; 66:345–351.

23.

Dimkpa

, Calder

, McLean

et al. Responses of a soil bacterium, Pseudomonas chlororaphis O6 to commercial metal oxide nanoparticles compared with responses to metal ions. Environ Pollut, 2011; 159:1749–1756.

24.

Dimkpa

, Calder

, Gajjar

et al. Interaction of silver nanoparticles with an environmentally beneficial bacterium, Pseudomonas chlororaphis. J Hazard Mater, 2011; 188:428–435.

25.

Dimkpa

, Zeng

, McLean

et al. Production of indole-3-acetic acid via the indole-3-acetamide pathway in the plant-beneficial bacterium, Pseudomonas chlororaphis O6 is inhibited by ZnO nanoparticles but enhanced by CuO nanoparticles. Appl Environ Microb, 2012; 78:1404–1410.

26.

Dimkpa

, McLean

, Latta

et al. CuO and ZnO nanoparticles: Phytotoxicity, metal speciation and induction of oxidative stress in sand-grown wheat. J Nanopart Res, 2012; 14:1125.

27.

Child

, Miller

, Liang

et al. Polycyclic aromatic hydrocarbon-degrading Mycobacterium isolates: Their association with plant roots. Appl Microbiol Biot, 2007; 75:655–663.

28.

Dimkpa

, McLean

, Britt

et al. Nano-specific inhibition of pyoverdine siderophore production in Pseudomonas chlororaphis O6 by CuO nanoparticles. Chem Res Toxicol, 2012; 25:1066–1074.

29.

Parker

, Norvell

, Chaney

. GEOCHEM-PC—A chemical speciation program for IBM and compatible personal computers. Loeppert

, Schwab

, Goldberg

. Chemical Equilibrium and Reaction Models. Madison, WI: Soil Science Society of America, 2005; 253–270.

30.

Wang

, Wick

, Xing

. Toxicity of nanoparticulate and bulk ZnO, Al₂O₃ and TiO₂ to the nematode Caenorhabditis elegans. Environ Pollut, 2009; 157:1171–1177.

31.

Jackson

, Taylor

. Plant-microbe interactions: Life and death at the interface. Plant Cell, 1996; 8:1651–1668.

32.

Berg

. Plant–microbe interactions promoting plant growth and health: Perspectives for controlled use of microorganisms in agriculture. Appl Microbiol Biot, 2009; 84:11–18.

33.

Spencer

, Ryu

, Yang

K-Y

et al. Induced defence in tobacco by Pseudomonas chlororaphis strain O6 involves at least the ethylene pathway. Mol Plant Microbe In, 2003; 63:27–34.

34.

Cho

, Kang

, Han

et al. 2R, 3R-butanediol, a bacterial volatile produced by Pseudomonas chlororaphis O6, is involved in induction of systemic tolerance to drought in Arabidopsis thaliana. Mol Plant Microbe In, 2008; 8:1067–1077.

35.

Ryu

, Kang

, Han

et al. Tobacco cultivars vary in induction of systemic resistance against cucumber mosaic virus and growth promotion by Pseudomonas chlororaphis O6 and its gacS mutant. Eur J Plant Pathol, 2007; 119:383–390.

36.

Nelson

, Weinel

, Paulsen

et al. Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol, 2002; 4:799–808.

37.

Gajjar

, Pettee

, Britt

et al. Antimicrobial activities of commercial nanoparticles against an environmental soil microbe, Pseudomonas putida KT2440. J Biol Eng, 2009; 3:9.

38.

Lok

, Ho

, Chen

et al. Proteomic analysis of the mode of antibacterial action of silver nanoparticles. J Proteome Res, 2006; 5:916–924.

39.

McQuillan

, Groenaga

, Stokes

, Shaw

. Silver nanoparticle enhanced silver ion stress response in Escherichia coli K12. Nanotoxicology, 2012; 6:857.

40.

Xiu

, Zhang

, Puppala

et al. Negligible particle-specific antibacterial activity of silver nanoparticles. Nano Lett, 2012; 12:4271–4275.

41.

Kahru

, Dubourguier

H-C

, Blinova

et al. Biotests and biosensors for ecotoxicology of metal oxide nanoparticles: A mini review. Sensors, 2008; 8:5153–5170.

42.

Lewinson

, Lee

, Rees

. A P-type ATPase importer that discriminates between essential and toxic transition metals. Proc Natl Acad Sci, 2009; 106:4677–4682.

43.

, Mahendra

, Lyon

et al. Antimicrobial nanomaterials for water disinfection and microbial control: Potential applications and implications. Water Res, 2008; 42:4591–4602.

44.

Padmavathy

, Vijayaraghavan

. Enhanced bioactivity of ZnO nanoparticles—an antimicrobial study. Sci Technol Adv Mat, 2008; 9:035004.

45.

Loper

, Hassan

, Mavrodi

et al. Comparative genomics of plant-associated pseudomonas spp.: Insights into diversity and inheritance of traits involved in multitrophic interactions. PLoS Genet, 2012; 8:e1002784.

46.

Miller

, Pettee

, Zhang

et al. Copper and cadmium: Responses in Pseudomonas putida KT2440. Lett Appl Microbiol, 2009; 49:775–783.

47.

Beyeler

, Keel

, Michaux

, Haas

. Enhanced production of indole-3-acetic acid by a genetically modified strain of Pseudomonas fluorescens CHA0 affects root growth of cucumber, but does not improve protection of the plant against Pythium root rot. FEMS Microbiol Ecol, 1999; 28:225–233.

48.

Patten

, Glick

. Role of Pseudomonas putida indoleacetic acid in development of the host plant root system. Appl Environ Microb, 2002; 68:3795–3801.

49.

Dimkpa

, Merten

, Svatoš

et al. Hydroxamate siderophores produced by Streptomyces acidiscabies E13 bind nickel and promote growth in cowpea (Vigna unguiculata L.) under nickel stress. Can J Microbiol, 2008; 54:163–172.

50.

Dimkpa

, Svatoš

, Dabrowska

et al. Involvement of siderophores in the reduction of metal-induced inhibition of auxin synthesis in Streptomyces spp. Chemosphere, 2008; 74:19–25.

51.

Dimkpa

, Merten

, Svatoš

et al. Siderophores mediate reduced and increased uptake of cadmium by Streptomyces tendae F4 and sunflower (Helianthus annuus), respectively. J Applied Microbiol, 2009; 107:1687–1696.

52.

Kloepper

, Leong

, Teintze

, Schroth

. Enhanced plant growth by siderophores produced by plant growth-promoting rhizobacteria. Nature, 1980; 286:885–886.

53.

Lodewyckx

, Vangronsveld

, Porteous

et al. Endophytic bacteria and their potential applications. Crit Rev Plant Sci, 2002; 21:583–606.

54.

Vansuyt

, Robin

, Briat

J-F

et al. Iron acquisition from Fe-pyoverdine by Arabidopsis thaliana. Mol Plant Microbe Interact, 2007; 20:441–447.

55.

Dimkpa

, Merten

, Svatoš

et al. Metal-induced oxidative stress impacting plant growth in contaminated soil is alleviated by microbial siderophores. Soil Biol Biochem, 2009; 41:154–162.

56.

Glick

. Using soil bacteria to facilitate phytoremediation. Biotechnol Adv, 2010; 28:367–374.

57.

Rajkumar

, Sandhya

, Prasad

MNV

, Freitas

. Perspectives of plant-associated microbes in heavy metal phytoremediation. Biotechnol Adv, 2012; 30,6:1562–1574.

58.

De Vleesschauwer

, Djavaheri

, Bakker

, Höfte

. Pseudomonas fluorescens WCS374r-induced systemic resistance in rice against Magnaporthe oryzae is based on pseudobactin-mediated priming for a salicylic acid-repressible multifaceted defense response. Plant Physiol, 2008; 148:1996–2012.

59.

Kang

, Han

, Zdor

et al. Inhibition of seed germination and induction of systemic disease resistance by Pseudomonas chlororaphis O6 requires phenazine production regulated by the global regulator, GacS. J Microbiol Biotechnol, 2007; 17:586–593.

60.

Wang

, Kern

, Newman

. Endogenous phenazine antibiotics promote anaerobic survival of Pseudomonas aeruginosa via extracellular electron transfer. J Bacteriol, 2010; 192:365–369.

61.

Mandal

, Bhattacharya

, Ganguly

. Nature of interactions of tryptophan with zinc oxide nanoparticles and L-aspartic acid: A spectroscopic approach. Chem Phys Lett, 2009; 472:128–133.

62.

Joshi

, Shewale

, Pandey

et al. Site-specific interaction between ZnO nanoparticles and tryptophan: A first principles quantum mechanical study. Phys Chem Chem Phys, 2011; 13:476–479.

63.

Fang

, Watson

J-L

, Jordan Goodman

et al.

Does doping with aluminum alter the effects of ZnO nanoparticles on the metabolism of soil pseudomonads?

Microbiol Res, 2012, 10.1016/j.micres.2012.09.001

64.

, Mavrodi

, Thomashow

et al. Ligand binding induces an ammonia channel in 2-amino-2-desoxyisochorismate (ADIC) synthase PhzE. J Biol Chem, 2011; 286:18213–18221.

65.

Jeong

, Chae

, Kang

, Shin

. Effects of silver nanoparticles on biological nitrogen removal processes. Water Sci Technol, 2012; 6:1298–1303.

66.

Liang

, Das

, Hu

. Bacterial response to a shock load of nanosilver in an activated sludge treatment system. Water Res, 2010; 44:5432–5438.

67.

Radniecki

, Stankus

, Neigh

et al. Influence of liberated silver from silver nanoparticles on nitrification inhibition of Nitrosomonas europaea. Chemosphere, 2011; 85:43–49.

68.

Barrena

, Casals

, Colon

et al. Evaluation of the ecotoxicity of model nanoparticles. Chemosphere, 2009; 75:850–857.

69.

Kumari

, Mukherjee

, Chandrasekaran

. Genotoxicity of silver nanoparticles in Allium cepa. Sci Total Environ, 2009; 407:5243–5246.

70.

Lee

W-Mi

, Kwak Jin

, An

. Effect of silver nanoparticles in crop plants Phaseolus radiatus and Sorghum bicolor: Media effect on phytotoxicity. Chemosphere, 2012; 86:491–499.

71.

Stampoulis

, Sinha

, White

. Assay-dependent phytotoxicity of nanoparticles to plants. Environ Sci Technol, 2009; 43:9473–9479.

72.

Mazumdar

, Ahmed

. Phytotoxicity effect of silver nanoparticles on Oryza sativa. Inter J ChemTech Res, 2011; 3:1494–1500.

73.

Panda

, Acharya

VMM

, Krishnaveni

et al. In vitro biosynthesis and genotoxicity bioassay of silver nanoparticles using plants. Toxicol in Vitro, 2011; 25:1097–1105.

74.

Yin

, Cheng

, Espinasse

et al. More than the ions: The effects of silver nanoparticles on Lolium multiflorum. Environ Sci Technol, 2011; 45:2360–2367.

75.

Atha

, Wang

, Petersen

et al. Copper oxide nanoparticle mediated DNA damage in terrestrial plant models. Environ Sci Technol, 2012; 46:1819–1827.

76.

, Sun

, Ji

et al. TiO₂ and ZnO nanoparticles negatively affect wheat growth and soil enzyme activities in agricultural soil. J Environ Monitor, 2011; 13:822–828.

77.

Kim

, Kim

, Lee

. Effects of Zn and ZnO nanoparticles and Zn²⁺ on soil enzyme activity and bioaccumulation of Zn in Cucumis sativus. Chem Ecol, 2011; 27:49–55.

78.

Kim

, Lee

. Alteration of phytotoxicity and oxidant stress potential by metal oxide nanoparticles in Cucumis sativus. Water Air Soil Poll, 2012; 223:2799–2806.

79.

Lin

, Xing

. Phytotoxicity of nanoparticles: Inhibition of seed germination and root growth. Environ Pollut, 2007; 150:243–250.

80.

Lin

, Xing

. Root uptake and phytotoxicity of ZnO nanoparticles. Environ Sci Technol, 2008; 42:5580–5585.

81.

, Kuang

, He

et al. Effects of rare earth oxide nanoparticles on root elongation of plants. Chemosphere, 2010; 78:273–279.

82.

Asli

, Neumann

. Colloidal suspensions of clay or titanium dioxide nanoparticles can inhibit leaf growth and transpiration via physical effects on root water transport. Plant Cell Environ, 2009; 32:577–584.

83.

Wang

, Xie

, Zhao

et al.

Xylem- and phloem-based transport of CuO nanoparticles in maize (Zea mays L.)

Environ Sci Technol, 2012; 46:4434–4441.

84.

Yang

, Watts

. Particle surface characteristics may play an important role in the phytotoxicity of alumina nanoparticles. Toxicol Lett, 2005; 58:122–132.

85.

Zhang

, Ma

, Zhang

et al.

Comparative toxicity of nanoparticulate/bulk Yb₂O₃ and YbCl₃ to cucumber (Cucumis sativus)

Environ Sci Technol, 2012; 46:1834–1841.

86.

Lin

, Tian

, Wu

, Xing

. Fate and transport of engineered nanomaterials in the environment. J Environ Qual, 2010; 39:1896–1908.

87.

Hernandez-Viezcas

, Castillo-Michel

, Servin

et al. Spectroscopic verification of zinc absorption and distribution in the desert plant Prosopis juliflora-velutina (velvet mesquite) treated with ZnO nanoparticles. Chem Eng J, 2011; 170:346–352.

88.

Gratao

, Polle

, Lea

, Azevedo

. Making the life of heavy metal-stressed plants a little easier. Funct Plant Biol, 2005; 32:481–494.

89.

Potters

, Pasternak

, Guisez

et al.

Stress-induced morphogenic responses: growing out of trouble?

Trends Plant Sci, 2007; 12:98–105.

90.

Zhang

, He

, Zhang

et al. Uptake and distribution of ceria nanoparticles in cucumber plants. Metallomics, 2011; 3:816–822.

91.

Zhu

, Han

, Xiao

, Jin

. Uptake, translocation, and accumulation of manufactured iron oxide nanoparticles by pumpkin plants. J Environ Monitor, 2008; 10:713–717.

92.

Rigo

, Corazza

, di Paolo

et al. Interaction of copper with cysteine: Stability of cuprous complexes and catalytic role of cupric ions in anaerobic thiol oxidation. J Inorg Biochem, 2004; 98:1495–1501.

93.

Schmidt

, Lauckner

, Lindner

. CZE of sulfur-containing amino acids and peptides and its application to the quantitative study of heavy metal-caused thiol oxidations. Chromatographia, 2012; 75:661–670.

94.

Sarret

, Laprade

, Bert

et al. Forms of zinc accumulated in the hyperaccumulator Arabidopsis halleri. Plant Physiol, 2002; 130:1815–1826.

95.

Kumari

, Khan

, Pakrashi

et al. Cytogenetic and genotoxic effects of zinc oxide nanoparticles on root cells of Allium cepa. J Hazard Mater, 2011; 190:613–621.

96.

Zhao

, Peralta-Videa

, Ren

et al. Transport of Zn in a sandy loam soil treated with ZnO NPs and uptake by corn plants: Electron microprobe and confocal microscopy studies. Chem Eng J, 2012; 184:1–8.

97.

López-Moreno

, de la Rosa

, Hernández-Viezcas

et al. Evidence of the differential biotransformation and genotoxicity of ZnO and CeO₂ nanoparticles on soybean (Glycine max) plants. Environ Sci Technol, 2010; 44:7315–7320.

98.

Kopittke

, Menzies

, de Jonge

et al. In situ distribution and speciation of toxic copper, nickel, and zinc in hydrated roots of cowpea. Plant Physiol, 2011; 156:663–673.

99.

Saraswat

, Rai

JPN

. Complexation and detoxification of Zn and Cd in metal accumulating plants. Rev Environ Sci Biotechnol, 2011; 10:327–339.

100.

Priester

, Ge

, Mielke

et al. Soybean susceptibility to manufactured nanomaterials with evidence for food quality and soil fertility interruption. Proc Natl Acad Sci., 2012; 109:2451–2456.

101.

Pandey

, Sanjay

, Yadav

. Application of ZnO nanoparticles in influencing the growth rate of Cicer arietinum. J Exp Nanosci, 2010; 5:488–497.

102.

Wang

, Kou

, Pei

et al. Physiological effects of magnetite (Fe₃O₄) nanoparticles on perennial ryegrass (Lolium perenne L.) and pumpkin (Cucurbita mixta) plants. Nanotoxicology, 2011; 5:30–42.

103.

Zheng

, Hong

, Lu

, Liu

. Effects of nano-TiO₂ on strength of naturally aged seeds and growth of spinach. Biol Trace Elem Res, 2005; 104:83–91.

104.

Gao

, Hong

, Liu

et al. Mechanism of nano-anatase TiO₂ on promoting photosynthetic carbon reaction of spinach-inducing complex of rubisco-rubisco activase. Biol Trace Elem Res, 2006; 111:239–253.

105.

Gao

, Liu

, Qu

et al.

Was improvement of spinach growth by nano-TiO₂ treatment related to the changes of rubisco activase?

Biometals, 2008; 21:211–217.

106.

, Cook

, Wang

, Hwang

. In vitro evaluation of cytotoxicity of engineered metal oxide nanoparticles. Sci Total Environ, 2009; 407:3070–3072.

107.

Dehner

, Barton

, Maurice

, Dubois

. Size-dependent bioavailability of hematite (alpha-Fe₂O₃) nanoparticles to a common aerobic bacterium. Environ Sci Technol, 2011; 45:977–983.

108.

Barton

, Quicksall

, Maurice

. Siderophore-mediated dissolution of hematite (alpha-Fe₂O₃): Effects of nanoparticle size. Geomicrobiol J, 2012; 29:314–322.

109.

Chen

, Jurkewitch

, Bar-Ness

, Hadar

. 1994. Stability constants of pseudobactin complexes with transition metals. Soil Sci Soc Am J, 1994; 58:390–396.

110.

Brandel

, Humbert

, Elhabiri

et al. Pyochelin, a siderophore of Pseudomonas aeruginosa: Physicochemical characterization of the iron(III), copper(II) and zinc(II) complexes. Dalton T, 2012; 41:2820–2834.

111.

Del Olmo

, Caramelo

, SanJose

. Fluorescent complex of pyoverdin with aluminum. J Inorg Biochem, 2003; 97:384–387.

112.

Lamsal

, Kim

S-W

, Jung

et al. Effects of silver nanoparticles against powdery mildews on cucumber and pumpkin. Mycobiol, 2011; 39:26–32.

113.

, Liu

, Mustapha

, Lin

. Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum. Microbiol Res, 2012; 166–207,215.

114.

Jayaseelan

, Abdul Rahuman

, Kirthi

et al. Novel microbial route to synthesize ZnO nanoparticles using Aeromonas hydrophila and their activity against pathogenic bacteria and fungi. Spectrochim ACTA A, 2012; 90:78–84.

115.

Alloway

. Soil factors associated with zinc deficiency in crops and humans. Environ Geochem Health, 2009; 31:537–548.

116.

White

, Broadley

. Biofortification of crops with seven mineral elements often lacking in human diets—iron, zinc, copper, calcium, magnesium, selenium and iodine. New Phytol, 2009; 182:49–84.

117.

Milani

, McLaughlin

, Stacey

et al. Dissolution kinetics of macronutrient fertilizers coated with manufactured zinc oxide nanoparticles. J Agr Food Chem, 2012; 60:3991–3998.

118.

Gardea-Torresdey

, Gomez

, Peralta-Videa

et al. Alfalfa sprouts: A natural source for the synthesis of silver nanoparticles. Langmuir, 2003; 19:1357–1361.

119.

Beattie

, Haverkamp

. Silver and gold nanoparticles in plants: Sites for the reduction to metal. Metallomics, 2011; 3:628–632.

120.

Harris

, Bali

. On the formation and extent of uptake of silver nanoparticles by live plants. J Nanopart Res, 2008; 10:691–695.

121.

Haverkamp

, Marshall

. The mechanism of metal nanoparticle formation in plants: Limits on accumulation. J Nanopart Res, 2009; 11:1453–1463.

122.

Kubis

, Lilley

, Jarvis

. Isolation and preparation of chloroplasts from Arabidopsis thaliana plants. Method Mol Biol, 2008; 425:171–186.

123.

Robert

, Zouhar

, Carter

, Raikhel

. Isolation of intact vacuoles from Arabidopsis rosette leaf–derived protoplasts. Nature Protoc, 2007; 2:259–262.

124.

Dhillon

, Brar

, Kaur

, Verma

. Green approach for nanoparticle biosynthesis by fungi: Current trends and applications. Crit Rev Biotechnol, 2012; 32:49–73.

125.

Mandal

, Bolander

, Mukhopadhyay

et al. The use of microorganisms for the formation of metal nanoparticles and their application. Appl Microbiol Biot, 2006; 69:485–492.

126.

Prasad

TNVKV

, Naidu

. A critical review on biogenic silver nanoparticles and their antimicrobial activity. Curr Nanosci, 2011; 7:531–544.

127.

Fayaz

, Balaji

, Girilal

et al. Biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: A study against gram-positive and gram-negative bacteria. Nanomedicine, 2010; 6:103–109.

128.

Nanda

, Saravanan

. Biosynthesis of silver nanoparticles from Staphylococcus aureus and its antimicrobial activity against MRSA and MRSE. Nanomedicine, 2009; 5:452–456.

129.

Ingle

, Gade

, Pierrat

et al. Mycosynthesis of silver nanoparticles using the fungus Fusarium acuminatum and its activity against some human pathogenic bacteria. Curr Nanosci, 2008; 4:141–144.

130.

Lee

J-H

, Han

, Choi

, Hur

H-G

. Effects of temperature and dissolved oxygen on Se(IV) removal and Se(0) precipitation by Shewanella sp. HN-41. Chemosphere, 2007; 68:1898–1905.

131.

Bao

, Lu

, Cui

et al. Extracellular microbial synthesis of biocompatible CdTe quantum dots. Acta Biomater, 2010; 6:3534–3541.

132.

Bai

, Zhang

, Guo

et al. Biosynthesis of cadmium sulfide nanoparticles by photosynthetic bacteria, Rhodopseudomonas palustris. Colloid Surface B, 2009; 70:142–146.

133.

Pearce

, Coker

, Charnock

et al. Microbial manufacture of chalcogenide-based nanoparticles via the reduction of selenite using Veillonella atypica: An in situ EXAFS study. Nanotechnol, 2008, 10.1088/0957-4484/19/15/155603.