Ecotoxicology DOI 10.1007/s10646-015-1505-x
Pesticidal activity of metal oxide nanoparticles on plant pathogenic isolates of Pythium Zac Zabrieski1 • Elliot Morrell1 • Joshua Hortin2 • Christian Dimkpa1,4 Joan McLean2 • David Britt3 • Anne Anderson1
•
Accepted: 30 May 2015 Ó Springer Science+Business Media New York 2015
Abstract CuO and ZnO nanoparticles (NPs) have antimicrobial effects that could lead to formulations as pesticides for agriculture or medicine. The responses of two soil-borne plant pathogenic Pythium isolates to the NPs were studied to determine the potential of these metal oxide NPs as pesticides. Growth of the P. ultimum isolate was more sensitive to CuO NPs than the P. aphanidermatum isolate. Growth in liquid medium with CuO NPs eliminated culturability whereas exposure to ZnO NPs resulted in stasis with growth resuming on transfer to medium lacking NPs. The citrate in the medium used for the growth assays was involved in enhanced release of the toxic metals from the NPs. Both CuO and ZnO NPs affected processes involved in Fe uptake. The NPs reduced levels of Fe-chelating siderophore-like metabolites produced by Pythium hyphae. CuO NPs inhibited, but ZnO NPs increased, ferric reductase activity detected at the mycelial surface. These findings illustrate that the toxicity of the metal oxide NPs towards Pythium was influenced by
Electronic supplementary material The online version of this article (doi:10.1007/s10646-015-1505-x) contains supplementary material, which is available to authorized users. & Anne Anderson
[email protected] 1
Biology Department, Utah State University, Logan, UT 84322 5305, USA
2
Utah Water Research Laboratory, Utah State University, Logan, UT 84322 8200, USA
3
Biological Engineering, Utah State University, Logan, UT 84322 4105, USA
4
Virtual Fertilizer Research Center, 1331 H Street, NW, Washington, DC 20005, USA
the medium, especially by the presence of a metal chelator. Environmental factors are likely to alter the pesticide potential of the metal oxide NPs when formulated for agricultural use in soils. Keywords Pythium Nanoparticles Solubility Iron Siderophore
Introduction While the antibacterial activities of nanoparticles (NPs) have been explored, the effects of NPs on fungi and fungallike organisms are lesser studied (Navarro et al. 2008). These interactions however are important in considering whether formulations of NPs could be used as pesticides in agriculture and in antimicrobial preparations for medical use (e g Lipovsky et al. 2011; Kanhed et al. 2014; Mishra et al. 2014; Servin et al. 2015). Recent papers demonstrate the toxicity of ZnO NPs toward plant pathogenic fungi including Penicillium expansum and Botrytis cinerea (He et al. 2011), and an Aspergillus isolate (Jayaseelan et al. 2012). The extent of tolerance for Aspergillus is variable between isolates (Gondal et al. 2012; Jain et al. 2013). ZnO NPs also are toxic to the wheat pathogen, Fusarium graminearum both in medium and in a solid sand matrix (Dimkpa et al. 2013). Foliar applications of CuO NPs limit growth of the oomycete Phytopthora (Giannousi et al. 2013). The experiments in this paper were directed towards determining the potential toxic activity of CuO and ZnO NPs on two pathogenic strains of the oomycete Pythium, P. ultimum (Pu) and P. aphanidermatum (Pa). Soil-borne Pythium isolates are ubiquitous and cause die-back, rot and other symptoms in a wide range of crop plants (Sharma
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et al. 2007; Cheung et al. 2008). The Pu isolate was obtained from diseased wheat through the American Type Culture Collection (# 32939). The Pa isolate was from diseased tomatoes isolated from the Disease Diagnosis Clinic at Utah State University. Attack of wheat by P. aphanidermatum isolates is documented to occur under field conditions (Al-Sheikh 2010). Accordingly, inoculation of wheat or tomato seed in laboratory tests with either of these isolates eliminated seedling stand (Anderson et al. unpublished data). The approach to determine the toxicity of CuO and ZnO NPs involved examination of growth after amendment of a solid medium of potato dextrose agar (PDA), and the liquid medium, potato dextrose broth (PDB). Release of metal from these metal oxide NPs is viewed as one of the mechanisms important in microbial toxicity (Kasemets et al. 2009; Dimkpa et al. 2012a). Consequently, because of their potential toxicity the level of soluble metal was measured in the liquid cultures and experiments were conducted to determine factors influencing NP solubility. Another potential factor affecting mycelial growth could be through NP-triggered alterations in Fe metabolism. Growth of Pythium isolates is highly sensitive to iron levels; previous findings implicate the involvement of siderophores from other microbes in the inhibition of growth of Pythium isolates (Becker and Cook 1988; Matthijs et al. 2007). Fe could be supplied to the mycelia through chelation by siderophores and/or uptake involving a plasma-membrane bound ferric reductase (Howard 1999). Our studies with the soil and rhizosphere-associated bacterium, Pseudomonas chlororaphis O6, demonstrate that CuO NPs inhibit production of a pyoverdine-like Fe siderophore whereas ZnO NPs increased its level (Dimkpa et al. 2012b, c). To the best of our knowledge the production of siderophores by Pythium isolates is not reported in the literature. However, ferric reductase activity is anticipated because the genome of Pu DAOM BRI44, locus PYU1_T008334 is predicted to encode a ferric reductase-like transmembrane protein (data base: pythium.plantbiology.msu.edu). Consequently we conducted experiments to examine the impact of NPs on potential siderophore production and ferric reductase activity for Pythium.
Methods Sources of chemicals CuO and ZnO NPs powders were purchased from SigmaAldrich Chicago USA. These particles have been previously characterized for shape and chemical composition (Dimkpa et al. 2012b, c, d).
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Microbial cultures The Pu isolate, a wheat pathogen, was obtained from the American Type Culture Collection (ATCC #32939). Isolate Pa was isolated from tomatoes grown under greenhouse culture that showed die-back symptoms. The isolate was identified by microscopy and the intervening sequence between ribosomal genes using primers specific for P. aphanidermatum in polymerase chain reactions performed by the Plant Diagnostic Laboratory at Utah State University. Pathogenicity of both strains was confirmed by dieback of wheat and tomato seedlings after the seed was planted into sand bearing mycelial plugs of the isolates as inocula. Cultures were maintained frozen at -80 °C as mycelial plugs previously grown on PDA immersed in 15 % glycerol. PDA, or its broth, PDB, are common media used for fungal growth. Cultures were regrown on PDA and inoculum removed from growing edge of hypha from plates less than 1 week-old. Isolate P. chlororaphis O6 (Spencer et al. 2003), used as a control for production of a secreted siderophore, was grown from freezer stocks at -80 °C in 15 % glycerol plated onto Luria Broth medium to generate inoculum. Assessment of growth inhibition by NPs Stocks of NPs were generated by suspension of the ‘‘as made’’ particles in water, with vortexing for 30 s, followed by immediate addition to either sterile PDA at 46 °C prior to pouring into plates, or to liquid PDB (Midsci, USA). The medium was amended with doses of NPs corresponding to 50,100, 250 and 500 mg [M]/L. The highest concentration was chosen based on previous studies in liquid medium where 500 mg/L ZnO NPs changed the metabolism of the root-associated bacterium, P chlororaphis O6 (Fang et al. 2013) and 500 mg/L CuO NPs eliminated growth (Dimkpa et al. 2011).Thus, the choice of treatment levels would allow comparison of data for toxic affects on different soil microbes. For growth on agar the Pythium inoculum was transferred as a 0.5 cm2 plug to the center of each plate. Growth was recorded 24 and 48 h after incubation under laboratory lights at 26 °C. Growth was determined on the plate medium by measuring, in cm, from the edge of the inoculum plug to the edge of the growing mycelial mat in four directions around the mycelial mat. Mean and standard deviation of these values were reported. Each experiment was performed at least twice, with three replicates of each treatment. Growth in liquid medium was at 26 °C in laboratory lighting with shaking at 150 rpm with 50 mL medium in 250 mL flasks unless otherwise noted. Cultures were
Pesticidal activity of metal oxide nanoparticles on plant pathogenic isolates of Pythium
amended with CuO or ZnO NPs at 100, 250 and 500 mg/L and shaken for 5 days with or without Pythium inocula. The observed adsorption of NPs to the mycelial hyphae was quantified by measurement of the differences in light scattering properties (OD600 nm) of the suspensions between inoculated and noninoculated cultures after 5 days after physical removal of the mycelial mats. The pH of the culture filtrates was measured after filtration through 0.2 lm filters. The mycelial masses were transferred to PDA plates and growth from these inocula determined visually with incubation at 26 °C. Because the Pythium isolates would be responding not only to the NPs but also to metal released from these particles, the soluble metal concentrations were measured at 2 and 5 days after inoculation. Supernatants were obtained by centrifugation of the cultures, from which the mycelial mass had been physically removed, at a force calculated to pellet NPs (Dimkpa et al. 2012b, c). Metals were determined using element identification by ICP-MS (Dimkpa et al. 2012b, c). Solubility of metals from CuO and ZnO NPs in water and PDB medium To understand the role of the components in PDB in metal release from the NPs, the levels of soluble metal was measured after suspension in noninoculated PDB and the values compared to that generated in water suspensions. Suspension of the NPs at 100 and 500 mg [M]/L in water or PDB were shaken at 150 rpm at 26 °C for 2 and 5 days. These times corresponded to the times of assessment of growth in solid and liquid medium respectively. The suspensions were centrifuged following the procedures in Dimkpa et al. (2012b, c) before measuring the pH of the solution and performing analysis of soluble Cu and Zn by element detection with ICP-MS. The composition of PDB was investigated to determine likely metal chelators, such as organic acids, present in the medium that could influence solubility from the NPs. The organic acids were assessed by ion chromatography (IC; Dionex 3000) using KOH-gradient elution as recommended by Dionex, Application Note 123 (Martineau et al. 2014). Because citrate was detected as the major organic acid, at 300 mg/L in the PDB, metal solubility experiments also were conducted using citrate, at 300 mg/L and pH 5.0 as described above. Examination of morphology of mycelia exposed to NPs Microscopy was used to determine whether the NPs at doses that inhibited mycelial growth (500 mg/L) changed the morphology of the Pythium mycelia. Mycelia were grown in
1 mL PDB with and without NPs (500 mg/L) for 3 days in 12-well plates. Biomass was collected from the wells and spread onto glass slides. Glycerol (20 lL, 15 % diluted with sterile water) was added and each sample was covered with a cover slip and the glass edges sealed with nail polish. The mycelia were examined on a confocal laser scanning microscope (LSM 710, Carl Zeiss) with imaging using a 63 9 objective. Autofluorescence was used to examine the samples with excitation at 488 nm and emission at 495–581 nm. The samples shown are one of at least ten fields of view examined from two replicates of each treatment. Evaluation of iron metabolism by Pythium: production of siderophores and ferric reductase activity To our knowledge there are no published reports of secreted siderophores from Pythium isolates. Thus, the potential of the strains to produce siderophores was evaluated using the chrome azurol S (CAS) colorimetric method where the removal of Fe from the CAS complex and its transfer into the siderophore chelate changes the color of the reactant solution from blue to orange (Alexander and Zuberer 1991; Schwyn and Neilands 1987). Briefly, cultures grown for 3 days in low-Fe SIM with and without NP amendments at 500 mg/L (Dimkpa et al. 2012b) were centrifuged (4000 g for 10 min). Because Fe availability in sufficient amounts suppresses siderophore production, control cultures were established with 200 lM Fe added as FeCl3 into the medium. Supernatants from the cultures were filter-sterilized using 0.22 lm membranes, and mixed with the CAS assay solution (1:1, v/v). After 14 h incubation, the intensity of the orange color, indicative of the formation of the siderophore-Fe complex, was read at 630 nm. To confirm siderophore detection by the assay procedure, the wild type strain of P. chlororaphis O6, which secretes a pyoverdinetype siderophore (Dimkpa et al. 2012b, c), was grown in a low Fe SIM medium (Dimkpa et al. 2012b) and the filtrate, generated by centrifugation and sterilized by filtration, was assayed with CAS reagent. As another control, the Pythium filtrates were amended with 200 lM Fe ions just prior to assay with the CAS reagent. The blank for these assays was the CAS reagent solution diluted (1:1, v/v) with noninoculated SIM. Siderophore production was calculated by the formula [(Ar - As)/Ar] 9 100 %, where Ar is the absorbance at 630 nm of the reaction generated with the CAS reagents, and As the absorbance at 630 nm of the Pythium or PcO6 samples with the CAS reagents. Subsequently, data were normalized using the color change with the filtrates from PcO6 to represent 100 %. To determine the ferric reductase activity associated with the mycelia, Pythium was grown for 2 days on PDB.
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Zn/L from the NPs, and was at about 50 % with 500 mg/L ZnO NPs. The addition of CuO NPs at 250 and 500 mg Cu/ L caused the plates to become blue in color, indicative of the release of Cu2? ions from the NPs. The CuO NPs inhibited growth of Pu more than Pa: at the 500 mg/L treatment there was reduction to about 35 % of control for Pa compared to 10 % of the control for Pu (Fig. 1). With both strains, growth was in the form of a dense, aerial, white mycelial mat of equal intensity over the surface of nonamended agar. Growth in the presence of ZnO NPs was as a dense, inner ring of mycelia surrounded by secondary ring of more sparse growth. The growth in the inner ring was uneven. Pythium grown in the presence of CuO NPs was sparse and wispy throughout its radius with less growth as the concentration of NPs increased. However, the inhibition of mycelial growth upon exposure to the NPs was not accompanied by gross changes in hyphal morphology as viewed by confocal microscopy using autofluorescence (Fig. 2).
The mycelia was washed with sterile water, blotted with sterile paper to remove excess water and weighed. The mycelia were transferred to 15 mL plastic tubes and 10 mL suspensions of CuO NPs or ZnO NPs (500 mg [M]/L). Control treatments without metal challenge involved incubation of live mycelia in sterile distilled water or mycelial biomass heat-denatured by an autoclave treatment (121 °C for 10 min). After 2 h of incubation at 24 °C without shaking, the biomass was removed from the treatments, and washed several times in sterile water. The washed mycelia were transferred to the ferric reductase assay solution (3 mL) containing 10 mM MES to buffer at pH 5, 300 lM Ferrozine (Acros Organics, NJ, USA), and 100 lM of freshly prepared Fe-EDTA [FeCl36H2O:Na2EDTA (1:1)]. After 2 h incubation, samples were collected, centrifuged at 10,0009g for 5 min to remove biomass and suspended NPs, and absorbance at 562 nm was measured. Data were normalized per gram of fresh weight of mycelia.
Growth inhibition by NPs in PDB
Results When inoculum plugs were added to the suspended NPs in liquid PDB, the NPs rapidly adhered to the hyphae so that the strands within mycelial mats became coated by the NPs. Mycelia were blackened for the amendements with CuO NPs and had a white coating with ZnO NPs. The suspension of PDB amended with 500 mg/L CuO NPs had an OD600 nm = 1.83 ± 0.20. This OD600 nm was reduced to 0.40 ± 0.10 after 12 h shaking with addition of the inoculum, a 1 cm square of Pythium mycelium. With the ZnO NPs, OD600 nm was reduced from 1.37 ± 0.14 to 0.68 ± 0.18 in the presence of mycelium after 12 h.
Growth inhibition by NPs on potato dextrose agar CuO and ZnO NPs caused dose-dependent growth inhibition of the mycelium of Pa and Pu on PDA (Fig. 1). Both isolates grew rapidly on nonamended PDA such that growth was to the edge of the 4.5 cm diameter-agar plates by 48 h. Consequently the data shown are for 24 h growth. The extent of growth inhibition caused by ZnO NPs was similar between the two strains (Fig. 1). Decreased radial growth compared to the control was observed with 50 mg 3.5 A
A
3 B C
2.5
Growth cm
Fig. 1 The effect of CuO or ZnO NPs on mycelial growth of P. aphanidermatum (Pa) or P. ultimum (Pu) on PDA at 24 h after inoculation. The NPs were added at the doses shown: 50,100, 250 and 500 mg [M]/L. The mean of the extent of growth in centimeter beyond the edge of the inoculum plug is provided. Data are from two independent studies each with three replicates. Bars denoted by different letters indicate significant difference at p = 0.05
B B, C
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CuO
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Pesticidal activity of metal oxide nanoparticles on plant pathogenic isolates of Pythium Fig. 2 Confocal imaging of hyphae of P. ultimum and P. aphanidermatum with and without exposure to CuO and ZnO NPs (500 mg/L) in PDB for 3 days (Color figure online)
Amendment of the flasks with noninoculated agar plugs showed that the mycelia was required for sorption of the NPs. Rapid adsorption of NPs to the filaments of a nylon cloth also was seen thus mimicking the effect of the mycelia; the cloth filaments became white with ZnO NPs, and black in the case of CuO NPs. With the CuO NPs treatments, the PDB developed a blue color, indicative of the presence of Cu ions. The formation of blue color did not require mycelia, indicating that factors from the medium supported ion release. Removal of the inoculum plugs after 5 days followed by transfer to new PDA without NPs showed immediate regrowth of hyphae from the control and the ZnO NPsexposed mass, independent of the dose in the original culture. With the CuO NPs-exposed inoculum plug, growth was resumed when transferred from medium containing 100 and 150 mg/L CuO NPs but with the 500 mg/L exposure, there was no growth at 1 week. The medium around the plugs from the 500 mg/L exposure became blue on the transfer agar, indicating release of Cu ions from NPs trapped on the mycelial mass. Soluble metal levels under culture conditions Studies of the solubility of Cu or Zn from the metal oxide NPs, measured after 2 or 5 days of culture with Pa, showed that mg/L levels were present in the medium compared
with lg/L levels in water (Fig. 3a, b). Solubility of Zn with the dose of 100 mg/L ZnO NPs showed that total breakdown of the complex was achieved in the PDB by 48 h (Fig. 3a). The presence of the mycelia had no affect on soluble Zn levels (Fig. 3a). However, Cu release to mg/L levels was dependent on time, dose and the presence of mycelia (Fig. 3b). To understand how PDB enhanced NP solubility, assessment of the composition of PDB was performed and results are shown in Table S1. Examination of organic acids revealed that citrate was present. When citrate at the pH and concentration found in PDB (300 mg/L) to suspend the NPs, both Cu and Zn were solubilized from the NPs at levels (Tables 1, 2) similar to those in the PDB cultures (Fig. 2). Of interest was that the high levels of solubility occurred even though the pH of the suspensions was more alkaline (Table 2). Fe metabolism in Pythium The production of secreted siderophore by Pythium was assayed using the CAS assay (Fig. 4). The chromogenic change in the CAS reaction mixture from blue to orange was typical for the presence of a siderophore, such as with the pyoverdine-like siderophore produced by PcO6 that chelated available Fe(III) (Fig. 4a). Partial decolorization was observed with the culture medium harvested after Pa
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a
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0 water 300
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Fig. 3 a and b Release of soluble metal from NPs in water and PDB with and without inoculation by Pa. Data are the means of three separate studies each with three replicates in cultures amended with 0, 100 or 500 mg/L NPs. Bars with different letters denote differences at p = 0.05. a Release of Zn from ZnO NPs. Data with ZnO NPs were independent of NP concentration and time and isolate. b Release of Cu from CuO NPs. Time, concentration and medium had differential affects Table 1 pH of solutions from PDB shaken for 2 and 5 days with defined concentrations of CuO and ZnO NPs with and without inoculation of P. aphanidermatum (Pa) With Pa 2 days
Without Pa 5 days
2 days
5 days
CuO NPs (mg/L) 0
5.04 ± 0.14
5.31 ± 0.04
5.04 ± 0.14
5.60 ± 0.02
100
5.02 ± 0.03
5.32 ± 0.05
5.01 ± 0.02
5.11 ± 0.01
500
5.32 ± 0.15
5.49 ± 0.10
5.25 ± 0.01
5.65 ± 0.01
ZnO NPs (mg/L) 0
5.04 ± 0.14
5.61 ± 0.04
5.04 ± 0.14
5.60 ± 0.02
100
6.27 ± 0.18
6.05 ± 0.08
6.28 ± 0.05
5.85 ± 0.05
500
6.06 ± 0.03
6.74 ± 0.05
6.94 ± 0.06
7.06 ± 0.01
and Pu growth (Fig. 4a). The data shown in Fig. 4b are based on the A630 nm values generated with the culture filtrates after Pa and Pu growth with normalization against siderophore production by PcO6 by setting this value to
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100 %. The % change was less when the filtrates from Pythium growth were assayed (treatments Pyt –Fe) and this value was reduced by growth of the Pythium strains with 200 lM Fe [treatments Pyt(?Fe)] (Fig. 4b). Similarly, color change was low when Fe (200 lM) was added to the culture filtrate prior to the CAS assay (treatment -Fe ? Fe). Growth of Pythium on the SIM medium in the presence of 500 mg/L CuO NPs and to a greater extent the ZnO NPs also lowered color change from that of the Pythium isolates without amendments with NPs (treatments Pyt ? NPs) (Fig. 4b). To investigate a second mechanism for Fe uptake into Pythium mycelia, ferric reductase activity associated with intact mycelia was measured. Both Pythium strains demonstrated ferric reductase activity when mycelia were incubated for 2 h in water (Fig. 5). This activity was destroyed by heat treatment of the mycelial mass [Fig. 5a, b (control K)], indicating the biotic nature of the ferric ion reduction. Exposure of mycelia which had been grown without challenge to CuO NPs for 2 h prior to assay reduced the ferric reductase activity from the control levels, significantly for Pu. In contrast, there was a trend for exposure to ZnO NPs to enhance reductase activity, significant at p = 0.05 for Pa.
Discussion Pathogenic isolates of the oomycete, Pythium, were sensitive to dose-dependent growth inhibition by CuO and ZnO NPs with greater sensitivity to the CuO NPs. Exposure to ZnO NPs was reversible because the mycelia regained growth after transfer to new medium. Recent work on the responses of Pseudomonas fluorescens to Zn (Alhasawi et al. 2014) showed that multiple targets are involved in Zn toxicity in bacterial cells. The responses included lowered sulfhydryl levels, and inhibition of complexes in the electron transfer chain and specific enzymes of the tricarboxylic acid cycle. Similarly, Cu toxicity is reported to be multifaceted (Festa and Thiele 2012). Because the response to CuO NPs strongly limited regrowth of the mycelia, the formulation of CuO NPs as a pesticide might be preferred to that of the ZnO NPs. Growth inhibition occurred without obvious morphological difference in the Pythium hyphae exposed to the NPs in contrast to studies with the fungus, B. cinerea where hyphal swelling was reported with ZnO NP treatments(He et al. 2011). Similarly, abnormal hyphal tip growth after challenge of another isolate of Pa was observed in challenges with a biocontrol pseudomonad (Deora et al. 2008). The findings of growth inhibition were generated using an acidic growth medium, PDB. The role of transformation involving aggregation/agglomeration of the NPs in this
Pesticidal activity of metal oxide nanoparticles on plant pathogenic isolates of Pythium Table 2 Metal solubilization and pH changes in suspensions of CuO and ZnO NPs in citrate at 300 mg/L with an initial pH of pH 5
Citrate 2 days metal (mg/L)
2 days pH
5 days metal (mg/L)
5 days pH
0.03 ± 0.02
4.97 ± 0.04
0.05 ± 0.03
4.99 ± 0.06
83 ± 30
5.1 ± 0.1
117 ± 18
5.5 ± 0.2
6.50 ± 0.02
145 ± 6
6.29 ± 0.10 5.00 ± 0.05
CuO NPs (mg/L) 0 100 500
148 ± 5
ZnO NPs (mg/L) 0
0.07 ± 0.01
4.97 ± 0.04
0.09 ± 0.05
100
111 ± 7
7.9 ± 0.5
108 ± 0.5
8.1 ± 0.2
500
170 ± 5
8.42 ± 0.13
139 ± 23
8.58 ± 0.09
Fig. 4 Observations with the CAS assay to detect siderophore secretion by Pythium isolates. a Comparison of the colorations in the CAS assay generated with filtrates from cultures of Pythium and PcO6. 1. Reference (CAS reagents with noninoculated SIM); 2. CAS reagents with filtrate from P. ultimum; 3. CAS reagents with filtrate from P. aphanidermatum; 4. CAS reagents with filtrate from PcO6. b Normalized absorbance measurements (A630 nm) from the CAS assay with filtrates from P. ultimum P. aphanidermatum and PcO6.
Data shown are for growth on a Fe-deficient medium [Pyt(-)Fe], for the medium supplemented with Fe [Pyt (-Fe ? Fe)] for the medium with 500 mg/L CuO NPs (Pyt ? CuO NPs) and for medium supplemented with 500 mg/L ZnO NPs (Pyt ? ZnO NPs). Treatment of the filtrate with Fe prior to assay is shown as Pyt(?Fe). The values are averages of three assays with standard deviations. The different letters indicate significant differences between the test samples (p = 0.05; n = 3) (Color figure online)
medium and with fungal growth is under investigation because changes in surface functions will influence bioactivity (Navarro et al. 2008; Schultz et al. 2015). However, our current findings point to the importance of environmental factors that influenced solubilization of metals from the NPs. The PDB medium promoted dissolution of the NPs to levels much higher than in water, even with increasing pH. Examination of the composition of PDB indicated that the presence of citrate was involved in
the increased dissolution of the NPs. The formation of citrate metal complexes increased solubility of the metal from the NPs. Additionally such metal citrate complexes could have influenced bioavailability and resulting toxicity; metal citrate complexes are bioactive in bacteria (JoshiTope and Francis 1995; McLean et al. 2013). In soils, a plants’ rhizosphere may be acidified relative to the bulk soil and organic acids could be present in the root exudates (Rudrappa et al. 2008; Marschner 2012; Martineau et al.
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Absorbance 562 nm /g fresh weight mycelium
0.35 0.3
a
Control (K) Control CuO NPs ZnO NPs
a
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0.15
bc
0.1 0.05
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0 P. ulmum
P. aphanidermatum
Fig. 5 Effects of NPs on ferric reductase activity of Pythium mycelium. Ferric-chelate reductase activity from the mycelia of P. ultimum and P. aphanidermatum, and the influence of CuO and ZnO NPs. Mycelia was exposed for 2 h prior to assay using NPs at 500 mg [M]/L. Heat-killed mycelia were used in control (K). Absorbance at 562 nm was recorded after 2 h incubation and physical removal of the mycelial mass. Data were adjusted by the fresh weight of the mycelial mass. Values are averages of six replications per treatment and the standard deviations are shown. The different letters on bars denote significant differences at p = 0.05
2014). Thus, these findings suggested that pesticide activity of metal oxide NPs with Pythium could be more pronounced in an acid than alkaline soil. Indeed, mitigation of the phytotoxicity of ZnO NPs in alkaline but not acid soils was observed (Watson et al. 2015). The phytotoxicity was correlated with the levels of soluble metal in the soil pore waters (Watson et al. 2015). In examining the response to the NPs, our experiments provided novel information about Fe metabolism in Pythium, suggesting that these oomycetes utilized siderophores and a mycelial ferric oxide reductase for uptake of Fe. Use of the CAS assay system evidenced that the Pythium isolates secreted metabolites with siderophore activity during growth on PDB. As with other microbes, the presence of Fe in the growth medium repressed siderophore production from the Pythium isolates (Howard 1999). Hydroxamate siderophores are among the types of siderophores commonly secreted by fungi (Howard 1999; Winkelmann 2007). In studies where PDA was amended with desferrioxamine B, a commercially-available hydroxamate-type siderophore, there was no effect on growth of the Pythium isolates (data not shown). We interpreted these observations to mean that Pythium was able to use Fechelated with hydroxamate siderophores. Production of siderophore was inhibited more by the ZnO NPs than the CuO NPs, in contrast with the effects of the NPs on growth
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inhibition. Published studies (e.g. Di Fiore et al. 2012; Zhu et al. 2011) indicated that hydroxamates formed complexes with both Zn and Cu, suggesting that when such metal ions were available they would compete with Fe for binding and disrupt Fe homeostasis in Pythium mycelium. We found no evidence that Pythium produced zincophores to specifically bind Zn ions, as has been speculated for fungi (Wilson et al. 2012), because Zn solubility was not increased by the presence of Pa mycelia in PDB. Effects on Fe metabolism in wheat, which also uses a secreted siderophore-mechanism to uptake Fe into roots, were recently noted when the seedlings were grown with CuO NPs (Dimkpa et al. 2015). The NPs modified a second process involved with Fe uptake in Pythium mycelia, the ferric reductase activity. There were trends for inhibited activity with CuO NPs and enhanced activity with ZnO NPs. Studies on Fe uptake involving ferric reductase systems in other organisms (Howard 1999) suggested that Cu2? ions are also substrates for reduction by ferric reductases. Additionally divalent Zn ions could compete with the transportation of Fe2? through the divalent metal transporter coupled to the reductase (Howard 1999). A proposed consequence of this effect would be stimulation in expression of the co-regulated reductase/transporter system in response to lower Fe levels, as observed in other microbes (Dancis et al. 1992). Such effects of the NPs on Fe metabolism could have an additional effect on Pythium in the rhizosphere. Greenshields et al. (2007) discussed how changes to siderophore levels or ferric reductase activities impaired the virulence of several fungal pathogens. Whether the NPs also modulate Pythium virulence, in addition to causing mycelial growth repression, awaits experimentation. In summary, our findings portend to the formulation of the metal oxide NPs as pesticides active against Pythium. A novel observation was the physical entrapment of NPs by Pythium hyphae that would result in intense challenge from ions as the NPs solubilize on the mycelial surface. However, the pesticide activity would be dependent on factors that promoted release of the toxic metal; toxicity in these studies was influenced by acidic pH and the presence of a metal chelator, citrate, in the PDB medium. Thus in soil, pH and the presence of chelators or materials to absorb Zn or Cu ions would modify the efficacy of the NPs as a pesticide active against soil-borne Pythium inocula. Acknowledgments The support of the Agricultural Experiment Station to AJA and the Water Research Laboratory to JM and of a grant from the USDA (Grant Number 10867118) is gratefully acknowledged. This is Utah Agricultural Research Paper Number: 8740. Conflict of interest The authors declare that they have no conflict of interest for this work.
Pesticidal activity of metal oxide nanoparticles on plant pathogenic isolates of Pythium
References Alexander DB, Zuberer DA (1991) Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. Biol Fertil Soils 12:39–45 Alhasawi A, Auger C, Appanna VP, Chahma M, Appanna VD (2014) Zinc toxicity and ATP production in Pseudomonas fluorescens. J Appl Microbiol 117:65–73 Al-Sheikh H (2010) Two pathogenic species of Pythium: P. aphanidermatum and P. diclinum from a wheat field. Saudi J Biol Sci 17:347–352 Becker OJ, Cook RJ (1988) Role of siderophores in suppression of Pythium species and production of increased growth response of wheat by fluorescent pseudomonads. Phytopathology 78:778–782 Cheung F, Win J, Lang JM, Hamilton J, Vuong H, Leach JE, Kamoun S, Levesque CA, Tisserat N, Buell CR (2008) Analysis of the Pythium ultimum transcriptone using Sanger and pyrosequencing approaches. BMC Genomics 9:542. doi:10.1186/1471-2164-9542 Dancis A, Roman DG, Anderson GJ, Hinnebusch G, Klausner RD (1992) Ferric reductase of Saccharomyces cerevisiae: molecular characterization, role in iron uptake and transcriptional control by iron. Proc Natl Acad Sci USA 89:3869–3873 Deora A, Hashidoko Y, Tahara S (2008) Antagonistic effects of Pseudomonas jessenii against Pythium aphanidermatum: morphological, ultrastructural and cytochemical aspects. J Basic Microbiol 48:71–81 Di Fiore A, Maresca A, Supuran CT, De Simone G (2012) Hydroxamate represents a versatile zinc binding group for the development of new carbonic anhydrase inhibitors. Chem Commun (Camb) 48:8838–8840. doi:10.1039/c2cc34275h Dimkpa CO, Calder A, Britt DW, McLean JE, Anderson AJ (2011) Responses of a soil bacterium, Pseudomonas chlororaphis O6 to commercial metal oxide nanoparticles compared with responses to metal ions. Environ Pollut 159:1749–1756 Dimkpa CO, McLean JE, Britt DW, Anderson AJ (2012a) Bioactivity and biomodification of Ag, ZnO and CuO nanoparticles with relevance to plant performance in agriculture. Ind Biotechnol 8:344–357 Dimkpa CO, McLean JE, Britt DW, Anderson AJ (2012b) CuO and ZnO nanoparticles differently affect the secretion of fluorescent siderophores in the beneficial root colonizer, Pseudomonas chlororaphis O6. Nanotoxicology 6:635–642 Dimkpa CO, McLean JE, Britt DW, Johnson WP, Arey B, Lea AS, Anderson AJ (2012c) Nanospecific inhibition of pyoverdine siderophore production in Pseudomonas chlororaphis O6 by CuO nanoparticles. Chem Res Toxicol 25:1066–1074 Dimkpa CO, McLean JE, Latta DE, Manango´n E, Britt DW, Johnson WP, Boyanov MI, Anderson AJ (2012d) CuO and ZnO nanoparticles: phytotoxicity, metal speciation and induction of oxidative stress in sand-grown wheat. J Nanopart Res 14:1125. doi:10.1007/s11051-012-1125-1129 Dimkpa CO, McLean JE, Britt DW, Anderson AJ (2013) Antifungal activity of ZnO nanoparticles and their interactive effect with a biocontrol bacterium on growth antagonism of the plant pathogen Fusarium graminearum. Biometals 26:913–924 Dimkpa CO, McLean JE, Britt DW, Anderson AJ (2015) Nano-CuO and interaction with nano-ZnO or soil bacterium provide evidence for the interference of nanoparticles in metal nutrition of plants. Ecotoxicology 24:119–129 Fang T, Watson JL, Goodman J, Dimkpa CO, Martineau N, Das S, McLean JE, Britt DW, Anderson AJ (2013) Does doping with aluminum alter the effects of ZnO nanoparticles on the metabolism of soil pseudomonads? Microbiol Res 168:91–98
Festa RA, Thiele DJ (2012) Copper at the front line of the host pathogen battle. PLoS Pathog 8(9):e1002887. doi:10.1371/ journal.ppat.1002887 Giannousi K, Avramidis I, Dendrinou-Samara C (2013) Synthesis, characterization and evaluation of copper based nanoparticles as agrochemicals against Phytophthora infestans. RSC Adv 3:21743–21752 Gondal MA, Alzahrani AJ, Randhawa MA, Siddiqui MN (2012) Morphology and antifungal effect of nano-ZnO and nano-Pddoped nano-ZnO against Aspergillus and Candida. J Environ Sci Health A 47:1413–1418 Greenshields DL, Liu G, Wei Y (2007) Roles of iron in plant defence and fungal virulence. Plant Signal Behav 2(4):300–302 He L, Liu Y, Mustapha A, Lin M (2011) Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum. Microbiol Res 166:207–215 Howard DH (1999) Acquisition, transport, and storage of iron. Clin Microbiol Rev 12(3):394–404 Jain N, Bhargava A, Tarafdar JC, Singh SK, Panwar J (2013) A biomimetic approach towards synthesis of zinc oxide nanoparticles. Appl Microbiol Biotechnol 97:859–869 Jayaseelan C, Rahuman AA, Kirthi AV, Marimuthu S, Santhoshkumar T, Bagavan A, Gaurav K, Karthik L, Rao KV (2012) Novel microbial route to synthesize ZnO nanoparticles using Aeromonas hydrophila and their activity against pathogenic bacteria and fungi. Spectrochim Acta A 90:78–84 Joshi-Tope G, Francis AJ (1995) Mechanisms of biodegradation of metal-citrate complexes by Pseudomonas fluorescens. J Bacteriol 177:1989–1993 Kanhed P, Birla S, Gaikwad S, Gade A, Seabra AB, Rubila O, Duran N, Rai N (2014) In vitro antifungal efficacy of copper nanoparticles against selected crop pathogenic fungi. Mater Lett 115:13–17 Kasemets K, Ivask A, Dubourguier HC, Kahru A (2009) Toxicity of nanoparticles of ZnO, CuO and TiO2 to yeast Saccharomyces cerevisiae. Toxicol In Vitro 23:1116–1122 Lipovsky A, Nitzan Y, Gedanken A, Lubart R (2011) Antifungal activity of ZnO nanoparticles—the role of ROS mediated cell injury. Nanotechnology 22:105101 Marschner P (2012) Marschner’s mineral nutrition of higher plants, 3rd edn. Elsevier, Oxford Martineau N, McLean JE, Dimkpa CO, Britt DW, Anderson AJ (2014) Components from wheat roots modify the bioactivity of ZnO and CuO nanoparticles in a soil bacterium. Environ Pollut 187:65–72 Matthijs S, Tehrani KA, Laus G, Jackson RW, Cooper RM, Cornelis P (2007) Thioquinolobactin, a Pseudomonas siderophore with antifungal and anti-Pythium activity. Environ Microbiol 9: 425–434 McLean JE, Pabst MW, Miller CD, Dimkpa CO, Anderson AJ (2013) Effect of complexing ligands on the surface adsorption, internalization and bioresponse of copper and cadmium in a soil bacterium, Pseudomonas putida. Chemosphere 91:374–382 Mishra S, Singh BR, Singh A, Kesqanit C, Naqvi AH, Singh HB (2014) Biofabricated silver nanoparticles act as a strong fungicide against Bipolaris sorokiniana causing sport blotch disease in wheat. PLoS One 9(5):e97881 Navarro E, Baun A, Behra R, Hartmann NB, Filser J, Miao AJ, Quigg A, Santschi PH, Sigg L (2008) Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology 17:372–386 Rudrappa T, Czymmek KJ, Pare´ PW, Bais HP (2008) Root-secreted malic acid recruits beneficial soil bacteria. Plant Physiol 148:1547–1556
123
Z. Zabrieski et al. Schultz C, Powell K, Crossley A, Jurkschat K, Kille P, Morgan AJ, Read D, Tyne W, Lahive E, Svendsen C, Spurgeon DJ (2015) Analytical approaches to support current understanding of exposure, uptake and distributions of engineered nanoparticles by aquatic and terrestrial organisms. Ecotoxicology 24:239–261 Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56 Servin A, Elmer W, Mukherjee A, De la Torre-Roche R, Hamdi H, White JC, Bindraban P, Dimkpa C (2015) A review of the use of engineered nanomaterials to suppress plant disease and enhance crop yield. J Nanopart Res 17:92 Sharma A, Wray V, Johri BN (2007) Rhizosphere Pseudomonas sp. strains reduce occurrence of pre- and post-emergence dampingoff in chile and tomato in Central Himalayan region. Arch Microbiol 187:321–335 Spencer M, Ryu C-M, Yang K-Y, Kim YC, Kloepper JW, Anderson AJ (2003) Induced defence in tobacco by Pseudomonas chlororaphis strain O6 involves at least the ethylene pathway. Physiol Mol Plant Pathol 63:27–34
123
Watson JL, Fang T, Dimkpa CO, Britt DW, McLean JE, Jacobson A, Anderson AJ (2015) The phytotoxicity of ZnO nanoparticles on wheat varies with soil properties. Biometals 28:101–112. doi:10. 1007/s10534-014-9806-8 Wilson D, Citiulo F, Hube B (2012) Zinc exploitation by pathogenic fungi. PLoS Pathog 8(12):e10003034 Winkelmann G (2007) Ecology of siderophores with special reference to the fungi. Biometals 20:379–392 Zhu F, Wu J, Chen G, Lu W, Pan J (2011) Biosynthesis, characterization and biological evaluation of Fe(III) and Cu(II) complexes of neoaspergillic acid, a hydroxamate siderophore produced by co-cultures of two marine-derived mangrove epiphytic fungi. Nat Prod Commun 6:1137–1140
