Citrate-Coated Silver Nanoparticles Growth-Independently Inhibit

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Citrate-coated silver nanoparticles growth- independently inhibit aflatoxin synthesis in Aspergillus parasiticus Chandrani Mitra, Phani M Gummadidala, Kamelia Afshinnia, Ruth Corrin Merrifield, Mohammed A Baalousha, Jamie R Lead, and Anindya Chanda Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 15 Jun 2017 Downloaded from http://pubs.acs.org on June 16, 2017

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Environmental Science & Technology

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Citrate-coated silver nanoparticles growth-

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independently inhibit aflatoxin synthesis in

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Aspergillus parasiticus

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Chandrani Mitra1, Phani M. Gummadidala1, Kamelia Afshinnia2 , Ruth C. Merrifield2,

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Mohammed Baalousha2, Jamie R. Lead2 and Anindya Chanda1*

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1

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South Carolina, Columbia, USA

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2

Department of Environmental Health Sciences, Arnold School of Public Health, University of

Center for Environmental Nanoscience and Risk, Arnold School of Public Health, University of

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South Carolina, Columbia, USA

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Corresponding author, [email protected]

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Phone: 803-777-8263

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Fax: 803-777-3391

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ACS Paragon Plus Environment

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Abstract

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Manufactured silver nanoparticles (Ag NPs) have long been used as antimicrobials. However,

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little is known about how these NPs affect fungal cell functions. While multiple previous studies

20

reveal that Ag NPs inhibit secondary metabolite syntheses in several mycotoxin producing

21

filamentous fungi, these effects are associated with growth repression and hence need sub-lethal

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to lethal NP doses, which besides stopping fungal growth, can potentially accumulate in the

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environment. Here we demonstrate that citrate coated Ag NPs of size 20 nm, when applied at a

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selected nonlethal dose, can result in a > 2 fold inhibition of biosynthesis of the carcinogenic

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mycotoxin and secondary metabolite, aflatoxin B1 in the filamentous fungus and an important

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plant pathogen, Aspergillus parasiticus, without inhibiting fungal growth. We also show that the

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observed inhibition was not due to Ag ions, but was specifically associated with the mycelial

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uptake of Ag NPs. The NP exposure resulted in a significant decrease in transcript levels of five

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aflatoxin genes and at least two key global regulators of secondary metabolism, laeA and veA,

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with a concomitant reduction of total reactive oxygen species (ROS). Finally, the depletion of Ag

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NPs in the growth medium allowed the fungus to regain completely, its ability of aflatoxin

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biosynthesis. Our results therefore demonstrate the feasibility of Ag NPs to inhibit fungal

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secondary metabolism at nonlethal concentrations, hence providing a novel starting point for

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discovery of custom designed engineered nanoparticles that can efficiently prevent mycotoxins

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with minimal risk to health and environment.

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Keywords: Aflatoxin biosynthesis, Secondary metabolism, Citrate coated nanoparticles, reactive

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oxygen species, oxidative stress

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Conflict of interest statement: The authors declare no conflict of interest


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Introduction

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Silver nanoparticles (Ag NPs) are one of the most studied manufactured nanoparticles 1-5

41

due to their antimicrobial properties

42

resistant microbes

43

cytotoxicity 9, 10, along with their ease of synthesis and use. However, as popularity of Ag NPs

44

continues to increase in medicine, pharmaceutical, cosmetics, electronics and diverse other

45

consumer product industries

46

essential to judge comprehensively, their beneficial effects on, for instance, food preservation as

47

well as their potential harmful impact on environmental health.

48

6-8

and their efficacy in controlling diverse and multi-drug

. They are generally preferred to silver ions due to their lower mammalian

11

, additional studies on their effects on microbial metabolism are

To this end, a significant number of studies have shown that Ag NPs can alter the 12

13

14, 15

49

metabolite profiles in bacteria

50

effects

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antifungal effects

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well. However, while these studies have all focused on growth-dependent repression of

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secondary metabolism using high NP concentrations, the growth-independent effects of Ag NPs,

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typically at low NP concentrations, on fungal secondary metabolism remain unclear. We

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emphasize here that environmentally relevant concentrations of Ag NPs are much lower than

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those typically used in toxicological and ecotoxicolgoical studies, are largely variable depending

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on the environmental compartment of concern, the local conditions and weather events such as

58

tidal flooding

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4 µg kg−1 to 14 mg kg−1 in soils

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concentrations below 50 µg L-1 have not been reported to the best of our knowledge. While NP

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exposure pathways for fungi are still unclear at this point, it is possible that multiple exposure

16

and algae

either through growth inhibitory

or hormetic

. Multiple studies conducted on fungal strains suggest that Ag NPs also demonstrate

25

9, 17-20

and as a result, can repress their secondary metabolite synthesis

21-24

as

, and can range from 10 pg L-1 to 1 µg L-1 in water columns and from 26, 27

. However, effects of Ag NPs on fungal cells at


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pathways exist in fungi for high and low concentrations leading to differential biological

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outcomes. Hence, studying biological behaviors of fungi at sub-lethal doses are critical. In

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particular it is essential to know whether fungi, and specifically, the mycotoxin producers,

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which are very resistant to harsh conditions in the environment 28, alter their metabolic profiles in

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response to Ag NPs. This information is critical in the realistic evaluation of the impact of NP

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accumulation on ecological physiology and human health. Here we address this knowledge gap

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using the plant pathogen, Aspergillus parasiticus, our laboratory’s model for studying fungal

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secondary metabolism

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mycotoxin that contaminates billions of dollars’ worth of crops worldwide including corn,

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peanuts, treenuts and cotton

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investigating molecular mechanisms underlying fungal secondary metabolite syntheses that are

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connected with significant economic and health implications.

29-31

. This filamentous fungus synthesizes aflatoxin B1, a carcinogenic

32, 33

. Hence, it is a well characterized and established model for

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The primary goal of this study was to investigate whether and how aflatoxin production

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in A. parasiticus is impacted by 20 nm sized citrate-coated silver nanoparticles (cit-Ag NPs).

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The cit- Ag NP s has been routinely used as model manufactured nanoparticles in several

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previous studies34-38 that have examined their uptake and toxicity in microbes, aquatic animals

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and other eukaryotic cells. The specific aims of this study were: (a) to investigate the effect of a

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working cit-Ag NP concentration (50 nM) on mycelial growth and aflatoxin biosynthesis at the

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levels of transcription activation of the aflatoxin gene cluster and (b) to investigate the possible

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mechanism for inhibition of aflatoxin biosynthesis.

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MATERIALS AND METHODS:

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Nanoparticle synthesis


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The cit-Ag NPs were synthesized by a standard chemical reduction method described previously

86

39, 40

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sodium citrate (0.31 mM) and 10 mL of 0.25mM of sodium borohydride was prepared and stored

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at 4oC in absence of light for 30 min. The silver nitrate and sodium citrate solutions were mixed

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together in a conical flask and stirred vigorously. Subsequently, 6 mL of the sodium borohydride

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was added to this reaction mix as the reducing agent. After 10 min of stirring, the reaction mix

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was heated slowly to boiling. The solution was then heated for an extra 90 min, left overnight

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and cooled (4 °C, in the dark). The Ag NPs thus formed (as indicated by the change of solution

93

color from colorless to yellow) were then cleaned, to remove the excess reagents, by

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ultrafiltration (Amicon, 1 kDa regenerated cellulose membrane, Millipore) in a diafiltration

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mode to prevent NP aggregation and drying. Ag NPs were re-dispersed in 0.31 mM sodium

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citrate solution to avoid further growth and the washing process was repeated at least three times

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and was performed each time before use. The concentration of Ag NPs was finally measured by

98

inductively coupled plasma-optical emission spectroscopy.

. Briefly, 100 mL aqueous solution of silver nitrate (0.25 mM), 100 mL aqueous solution of

99 100

Nanoparticle characterization

101

Presence of Ag NP suspension was determined in the NP suspension using surface plasmon UV-

102

Vis spectrophotometry (wavelength 200nm to 800nm). The z-average hydrodynamic diameter

103

and polydispersity index (PDI) were determined using dynamic light scattering (DLS) using a

104

Malvern Zetasizer (Nanoseries, Malvern Instruments) as described previously 41. The z-average

105

diameter and PDI were measured five times to calculate the standard error of mean. Particle size

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distribution, and shape were analyzed using transmission electron microscopy (TEM) as

107

described previously

41 42

. Briefly, NPs were drop deposited onto Formvar/carbon coated 50


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mesh Cu grids (S162-3, AGAR Scientific) and allowed to adsorb to the carbon coating for

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10 min (without drying). The grids were then gently immersed in ultrapure water to remove un-

110

adsorbed particles, and air-dried under protective cover. Electron micrographs (10 micrographs

111

from various locations of the grids) were acquired using TEM (Hitachi H-8000) at 200 keV. The

112

cit-Ag NP aggregation in the test media was qualitatively monitored by UV-vis

113

concentration of the synthesized Ag NPs was measured by by inductively coupled plasma-atomic

114

emission spectroscopy (ICP-OES).

43

. Finally the

115 116

Determination of total and dissolved silver concentrations

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Mycelia after NP exposure (50 mg) and fungal growth media (1 mL) samples were digested at

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room temperature (with 10 mL of 70 % nitric acid (AR grade; Fisher scientific) for 24 h. This

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reaction mixture was diluted with 15 mL double distilled water and heated at 80oC for 15min

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prior to cooling to room temperature and filtration (Whatman filter paper 2). The samples were

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finally diluted in 100 mL volumetric flask and then analyzed using an inductively coupled

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plasma-optical emission spectrometer (ICP-OES; Varian 710-ES). The detection limit was 5 µg

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L-1. Dissolved silver in the growth media was determined by centrifugation (4000xg, 45min) and

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ultrafiltration (Amicon; 1Kda cutoff) of the media and subsequent ICP-OES analyses of the

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filtrate. Nanoparticle concentration was determined by difference.

126 127

Fungal strain, medium and growth conditions:

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The wild-type aflatoxin producer, Aspergillus parasiticus, SU-1 (NRRL 5862) was used for this

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study. For NP exposure experiments, 106 spores of A. parasiticus were inoculated in 100 mL

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yeast extract sucrose (YES) liquid growth medium containing cit- Ag NP (50 ng mL-1 ), and


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grown under our standard growth conditions (in an incubator shaker at 29oC in absence of light,

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with rpm 150). The choice of 50 ng mL-1 as the ideal working concentration for this study was

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based on an initial dose-response experiment (see results), which was used to determine the cit-

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Ag NP concentration that only inhibited aflatoxin biosynthesis without affecting fungal growth.

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Untreated control groups were grown under same growth conditions but in absence of cit- Ag

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NPs.

137 138

Growth and aflatoxin measurements:

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Quantitative comparisons of growth was conducted using dry weight measurements of harvested

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mycelia as described previously

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quantified using Enzyme-Linked Immunosorbant Assay (ELISA) as described previously

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Since previous studies

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study have shown that 40 h cultures demonstrate aflatoxin biosynthesis and export into the

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growth medium at peak levels, a comparison of 40h aflatoxin levels in the presence and absence

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of cit- Ag NP s was conducted. Aflatoxin levels were measured at 48h time-point as well, to

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examine whether aflatoxin biosynthesis could revert to control levels after an almost complete

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removal (by uptake) of most or all cit- Ag NP s in the growth medium by the mycelium.

33

44

. Aflatoxin B1 accumulation in the growth medium was 45

.

that have employed the fungal growth conditions used in the current

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NP-aflatoxin interaction assay:

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Interaction of cit-Ag NPs with aflatoxin was determined by incubating total aflatoxin extracted

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from a 40h A. parasiticus culture with the NPs at 50 ng mL-1 concentrations for 4 hours. This

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aflatoxin-cit-Ag NP mixture was ultra-filtered using amicon ultra-centrifugal filter unit (3Kda).


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The concentration aflatoxin in the filtrate was compared with an unfiltered mix and an aflatoxin-

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only control using ELISA. Significance of difference in aflatoxin measurements between the

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filtrate and the unfiltered mix was used to evaluate NP-aflatoxin interaction and the significance

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of difference in aflatoxin measurements between the unfiltered mix and the aflatoxin-only

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control was used to evaluate interference of the NPs in the aflatoxin measurements.

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Quantification of ROS:

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Total ROS generated by mycelia was quantified using a 2’,7’-dichlorofluorescein diacetate

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(DCFH-DA) protocol described previously

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mycelia in the presence of absence of cit- Ag NPs, equal weights of mycelia from each sample

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were placed into 1 mL freshly made 1 µM DCFH-DA in PBS. The yield of the fluorescent

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product dichlorofluorescin (DCF) upon oxidation of DCFH-DA due to generation of ROS was

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measured after a 5hour reaction at 37 °C using an excitation/emission wavelength of 490/525

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nm.

46

. For comparison of total ROS generated by

167 168

Gene expression studies:

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a) RNA extraction, purification and cDNA synthesis. Total RNA was extracted from cells

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harvested in triplicates using a TRIzol (TRIzol Reagent; Invitrogen, Carlsbad, CA) based method

171

previously described

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Qiagen® RNEasy Cleanup Kit (Qiagen, Valencia, CA), and then stored at -80°C. The cDNA

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was synthesized from RNA using a reverse transcription kit (New England BioLabs Inc.,

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Ipswich, MA) according to manufacturer’s instructions.

33

. Within 24 hours of extraction, RNA cleanup was conducted using a


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b) Real-time PCR assays: The expressions of aflatoxin and superoxide dismutase genes were

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measured using quantitative real-time PCR assays using SsoAdvanced universal SYBR Green

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supermix (BioRad Laboratories, Hercules, CA) and gene specific forward and reverse primers

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(Table 1). The gene specific primers were designed using Primer3 online software

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(http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The aflatoxin gene primers were identical to

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those described previously

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Laboratories, Hercules, CA). Similar to the previous gene expression studies in A. parasiticus 47,

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β-tubulin gene was used as the housekeeping gene. Expression value of each gene was obtained

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from the threshold cycle values that were normalized against 18SrRNA expression in each

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sample. All RT-PCRs were performed in triplicates for each gene per sample. Data analysis of

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gene expression studies were performed using CFX Manager software (Bio-Rad Laboratories).

47

. Reactions were performed in a CFX96 thermal cycler (Bio-Rad

186 187

Statistical Analysis:

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Statistical analyses for this study were conducted using the GrpahPad Prism software (Graphpad,

189

CA, USA). Statistical significance of two-tailed p-values were determined using an unpaired t-

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test, with n=3 and p < 0.05.

191 192

Results and Discussion:

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Properties of pristine Ag NPs.

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The absorption spectrum of cit-Ag NPs ( figure 1a) showed a single peak centered on λmax 395

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nm (implicative of successful synthesis of the 20 nm Cit- Ag NP s 1, 48) and showed no signs of


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Ag NP aggregates in the synthesized suspension. TEM micrographs confirmed that cit- Ag NP s

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were present predominantly as single particles (representative TEM image shown in figure 1b).

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The average core size of the NPs as revealed by TEM was ca. 13.8 ± 2.3 nm (mean ± standard

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deviation, n= 500). The average hydrodynamic diameter and size PDI as revealed by DLS were

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ca. 20.1 ± 1.0 nm (figure 1c) and 0.11 ± 0.03 respectively, implicating a narrow size distribution.

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The total concentration of the Ag NP stock solution was 9.6 mg L-1.

202 203

Choice of cit-Ag NP dose for studying the Ag NP effect on aflatoxin biosynthesis.

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Dose-response experiment demonstrated that cit- Ag NP did not inhibit A. parasiticus growth

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within 10-50 ng mL-1 concentration range (figure 2a). However, higher concentrations up to 500

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ng mL-1 (highest concentration measured), resulted in significant growth inhibition (~2 fold

207

reduction of dry-weight). Also concentrations < 50 ng mL-1 did not result in significant reduction

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in aflatoxin levels in the growth medium. At 50 ng mL-1, cit- Ag NP resulted in significant

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reduction (~2 fold) of aflatoxin accumulation in the growth medium (figure 2b). Aflatoxin

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measurement and growth analyses in experiment was conducted with 40h cultures based on

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previous studies, which show that under our growth conditions aflatoxin is synthesized at peak

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levels at 40h. To confirm that reduction in of aflatoxin accumulation in the growth medium was

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not due to growth effects at time points earlier than 40 h time-point, the growth rates of the

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fungus (cit- Ag NP treated versus untreated) were compared during the 40h period. No

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significant difference in growth rate, expressed as dry weight accumulation per unit time (mg h-

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1

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suggesting that at the applied concentration, cit- Ag NP associated inhibition of aflatoxin

), was observed between the cit- Ag NP treated mycelia and untreated mycelia (figure 2c),


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accumulation in the growth medium was not a fungal growth inhibition effect. Also, NP-

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aflatoxin interaction assays indicated no significant differences in aflatoxin measurements

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between aflatoxin-NP mixtures, ultrafiltered aflatoxin-NP mixtures and aflatoxin-only controls

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indicating that the reduced aflatoxin measurements were not an artifact from aflatoxin-NP

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interactions (SI Fig 1). Hence the cit- Ag NP concentration of 50 ng mL-1 was chosen in this

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study as the ideal dose to examine the possibility of specific inhibitory effects of the NPs on

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aflatoxin synthesis.

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Interaction of cit- Ag NP with the growth medium.

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The possibility of whether the observed cit- Ag NP - mediated reduction in aflatoxin

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accumulation in the growth medium was caused by dissolution of the NPs into the growth

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medium was also explored. Residual silver in the growth medium obtained upon

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ultracentrifugation and ultrafiltration of the cit- Ag NP -containing medium was below the ICP-

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OES detection limit (5 ng mL-1, i.e. 2-fold reduction in transcripts of both laeA and

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veA, thereby demonstrating the ability of cit- Ag NP to inhibit secondary metabolism in A.

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parasiticus. Our results therefore suggest that inhibition of aflatoxin biosynthesis by cit- Ag NP

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was mediated at least in part, by the transcription inhibition of laeA and veA. These genes encode

315

the protein LaeA and VeA, both of which are a part of the regulatory complex of secondary

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metabolism regulatory Velvet complex43, and which in addition to aflatoxin biosynthesis,

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regulate other secondary metabolite pathways of filamentous fungi as well 43. Hence our results

318

demonstrate the feasibility of cit- Ag NP s to inhibit secondary metabolism in filamentous fungi

319

without inhibiting fungal growth.

320 321

Effect of cit- Ag NP on total mycelial ROS

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Quantitative comparison of total ROS generated by cit- Ag NP treated mycelia and the untreated

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control cells demonstrated that cit- Ag NP exposure resulted in a significant reduction (by

324

~30%) of total ROS at 24h (figure 4b). In addition, we also explored whether the cit- Ag NP

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mediated drop in total ROS was associated with a concomitant reduction of transcriptional

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activation of superoxide dismutase genes (SOD genes), which are enzymes that remove

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superoxides (one class of ROS) through dismutation reactions

328

significant reduction was observed in the transcriptional activation of the cytosolic Cu/Zn-SOD

329

gene. No effect was observed on the transcriptional activations of the other SOD genes. Multiple

330

studies

61-69

60

. As shown in SI figure 4,

suggest that activation of secondary metabolism is a response to an increase in


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intracellular oxidative stress. Hence it is possible that cit- Ag NP

mediated reduction in

332

intracellular oxidative stress (as implicated by the drop in total ROS) and cytosolic Cu/Zn-SOD

333

gene could result in the reduction in transcriptional activation of aflatoxin genes (observed in

334

figure 4a). We reason that since only the cytosolic Cu/Zn-SOD gene demonstrated

335

transcriptional downregulation, the cit- Ag NP possibly resulted in the reduction of ROS build-

336

up in the cytosol. Interestingly these findings suggest an intracellular behavior (antioxidant

337

effects) of Ag NP, which are contradictory to the effects of Ag NPs when applied at higher doses

338

to induce cell toxicity 9, 17-20, and may partly explain hormetic responses found in other biological

339

systems 16. It is possible that prior to uptake, the NPs- growth medium interaction resulted in a

340

protein coating that resulted in the subsequent antioxidant effects. The molecular mechanisms

341

that enable the NPs to reduce cellular ROS will be investigated in greater detail in our future

342

studies.

343 344

Aflatoxin biosynthesis upon cit- Ag NP removal from the growth medium.

345

Finally, to confirm that the fungus is able to recover its ability of aflatoxin biosynthesis upon

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complete removal of cit- Ag NPs in the growth medium, a quantification of aflatoxin

347

accumulation at 48h was conducted. As shown in figure 5a, both growth (figure 5a-i) and

348

aflatoxin accumulation (figure 5a-ii) demonstrated no significant difference between the treated

349

and control samples, thereby demonstrating that upon depletion of cit- Ag NP s in the growth

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medium (by ~24-30h), mycelia were able to regain their full ability of aflatoxin biosynthesis.

351

Analysis of the total ROS at both 30h and 40h also confirmed the build of ROS in mycelia upon


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depletion of cit- Ag NP in the growth medium (figure 5b). Transcript levels of aflatoxin genes

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(SI figure 5) also increased to those observed in the control.

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Environmental Implications

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Filamentous fungi are found ubiquitously throughout all ecosystems on the planet and

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interact with a wide-range of different organisms that include other microbes, plants, animals and

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humans; a excellent recent review from Braga et al

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interactions in establishment and maintenance of an environment. Fungal secondary metabolites

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play significant regulatory roles in shaping the community profiles, chemical interactions and

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ecological physiology of their ecosystems28. Hence, to fully understand the effects of effluent

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Ag NPs on the environment and human health, it is essential to understand their impacts on

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fungal secondary metabolism, especially when the NPs are present at concentrations that do not

363

inhibit growth of the fungal cells. Our results clearly demonstrate the potential of NPs to

364

interfere with the production of secondary metabolites that are enormously significant to

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environmental and public health.

70

sheds light on the importance of such

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Our results also provide the first demonstration of the feasibility of Ag NPs to interfere in

367

the biosynthesis of natural products in filamentous fungi without affecting their growth effect.

368

We showed that mycelial uptake of cit- Ag NP, resulted in reduction in intracellular ROS that

369

associated with a down-regulation of the transcriptional activation of secondary metabolism and

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aflatoxin biosynthesis. This establishes a novel research direction that can generate custom

371

designed engineered nanoparticles, which can efficiently control contamination of crops with

372

aflatoxins and other mycotoxins with low NP doses that pose minimal human hazard and risk to

373

the environment. Our future studies will explore the molecular mechanisms through which


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nanoparticles modulate fungal cellular stress and syntheses of mycotoxins that are enormously

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relevant to the safety of environmental and public health.

376 377 378

Figure 1. Characterization of synthesized cit- Ag NP . a) UV-Vis spectral profile of the cit- Ag

379

NP stock solution. b) Transmission electron microscopy image of a representative cit- Ag NP

380

sample (scale bar, 50 nm) c) Dynamic Light Scattering of a representative cit- Ag NP sample.

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Figure 2. Effect of cit- Ag NP on A. parasiticus growth and aflatoxin biosynthesis. a) Analysis

382

of different doses of cit- Ag NP exposures on A. parasiticus growth. Effect of growth upon

383

exposure to each dose was compared with the untreated set of samples. Statistical significance of

384

two-tailed p-values were determined using an unpaired t-test, with n=3 and p < 0.05 as

385

significance level. b) Effect on aflatoxin biosynthesis. A dose of 50 ng mL-1 cit- Ag NP was

386

spiked into the growth medium during the start of growth. Aflatoxin B1 accumulation in the

387

growth medium upon cit- Ag NP exposure was compared with untreated samples at 40h.

388

Statistical significance of two-tailed p-values were determined using an unpaired t-test, with n=3

389

and p < 0.05 as significance level. ‘*’ denote significance. c) Effect on growth rates. A dose of

390

50 ng mL-1 cit- Ag NP was spiked into the growth medium during the start of growth. Dry

391

weights were monitored during a time span of 40h to derive the growth rates (ng h-1) by

392

quantifying the slope of the growth curves during the exponential growth phase. The growth

393

rates were compared with the untreated samples. Statistical significance of two-tailed p-values

394

were determined using an unpaired t-test, with n=3 and p < 0.05 as significance level. ‘*’ denote

395

significance.


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Figure 3. NP behavior and uptake during fungal growth. a. Time-course UV-Vis spectral

398

profiles of the growth medium. Growth medium spiked with 50ng mL-1 cit- Ag NP (N+M)

399

spectral profiles and the growth medium spiked with cit- Ag NP containing the growing mycelia

400

(N+M+F) were compared with the 50ng mL-1 cit- Ag NP stock solution (N) to conduct a

401

qualitative analysis of the cit- Ag NP absorbance peaks under each condition. b. Reduction of

402

total [Ag] in the growth medium. A dose of 50 ng mL-1 cit- Ag NP was spiked into the growth

403

medium during the start of growth. A time-course inductively coupled plasma optical emission

404

spectroscopy (ICP-OES) was performed until minimum detection limit (3 ng mL-1 denoted by

405

the dotted line) was reached at 30h. These readings were compared with total silver levels at the

406

corresponding time-points in the ‘growth medium only’ control flasks. Statistical significance of

407

two-tailed p-values were determined using an unpaired t-test, with n=3 and p < 0.05 as

408

significance level. ‘*’ denote significance. c. Increase in total silver in mycelia. Inductively

409

coupled plasma optical emission spectroscopy (ICP-OES) was performed with spores (0h) and

410

mycelia (at 24h and 30h). No detectable silver was found in untreated mycelia (not shown)

411 412

Figure 4. Effect of cit- Ag NP on aflatoxin biosynthesis regulatory gene transcripts and total

413

ROS generation. a. mRNA levels at 24h for aflatoxin pathway genes (aflR, nor-1, vbs, ver-1 and

414

omtA) and the global secondary metabolism regulators (laeA and veA) upon cit- Ag NP uptake

415

were compared with the untreated controls. Statistical significance of two-tailed p-values were

416

determined using an unpaired t-test, with n=3 and p < 0.05 as significance level. ‘+’, mycelia

417

treated with cit- Ag NP , ‘-’ , untreated samples, ‘*’ denote significance. b. Quantitative

418

comparison of total ROS at 24h. Total ROS was quantified using a DCFH-DA based protocol as


19


419

described in A. parasiticus 46. Statistical significance of two-tailed p-values were determined

420

using an unpaired t-test, with n=3 and p < 0.05 as significance level. ‘+’, mycelia treated with

421

cit- Ag NP , ‘-’ , untreated samples, ‘*’ denote significance.

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Figure 5. Aflatoxin synthesis and total ROS generation post cit- Ag NP uptake. a. i) Aflatoxin

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B1 accumulation in the growth medium at 48h in the treated flasks was compared with untreated

425

samples and ii) comparison of the dry weights of the treated and untreated mycelia. Statistical

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significance of two-tailed p-values were determined using an unpaired t-test, with n=3 and p

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