Production of silver nanoparticles using yeasts and evaluation of their antifungal activity against phytopathogenic fungi

Accepted Manuscript Title: Production of silver nanoparticles using yeasts and evaluation of their antifungal activity against phytopathogenic fungi Author: Jorge G. Fern´andez Mart´ın A. Fern´andez-Baldo Elias Berni Gerardo Cam´ı Nelson Dur´an Julio Raba Mar´ıa I. Sanz PII: DOI: Reference:

S1359-5113(16)30144-1 http://dx.doi.org/doi:10.1016/j.procbio.2016.05.021 PRBI 10695

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

27-10-2015 24-4-2016 20-5-2016

Please cite this article as: Fern´andez Jorge G, Fern´andez-Baldo Mart´ın A, Berni Elias, Cam´ı Gerardo, Dur´an Nelson, Raba Julio, Sanz Mar´ıa I.Production of silver nanoparticles using yeasts and evaluation of their antifungal activity against phytopathogenic fungi.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.05.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Production of silver nanoparticles using yeasts and evaluation of their antifungal

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activity against phytopathogenic fungi

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Jorge G. Fernández, 1,3 Martín A. Fernández-Baldo, 2 Elias Berni, 3 Gerardo Camí, 2,4 Nelson Durán, 1,3 Julio Raba, 1,3 María I. Sanz*

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INQUISAL, CONICET. Universidad Nacional de San Luis, Chacabuco 917.

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D5700BWS. San Luis, Argentina. 2

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Institute of Chemistry, Biological Chemistry Laboratory, Universidade Estadual de Campinas, CEP 13083-970, Caixa Postal 6154, Campinas, SP, Brazil.

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Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, San

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Luis, Argentina. 4

Center of Natural and Human Sciences, Department of Biochemistry and Biophysics, Universidade Federal do ABC, CEP 09210-170, Santo André, SP, Brazil.

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*Author to whom correspondence should be addressed: (e-mail) [email protected]

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(phone) +54-2652-425385; (Fax) +54-2652-430224.

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INQUISAL, Departamento de Química. Universidad Nacional de San Luis, CONICET.

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Chacabuco 917. D5700BWS. San Luis, Argentina.

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Facultad de Química Bioquímica y Farmacia, Universidad Nacional de San Luis, San

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Luis, Argentina. 1

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Highlights Production of Ag NPs was carried out using the yeasts C. laurentii and R. glutinis. Ag NPs from both yeasts showed differences in their physicochemical properties. Ag NPs from both yeasts showed antifungal activity against pathogens of postharvest. Ag NPs from R. glutinis showed the major antifungal activity. Ag NPs from R. glutinis were similarly effective than Iprodione.

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Abstract

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The present study investigated the biological synthesis, characterization and

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antifungal activity of silver nanoparticles (Ag NPs). The production of Ag NPs was

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performed using the culture supernatants of two yeasts: Cryptococcus laurentii and

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Rhodotorula glutinis. These yeasts were chosen for their nitrate reductase activity. The

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role of nitrate reductase in the production of nanoparticles was assessed using inhibitors.

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The characterization of Ag NPs was made by UV-visible spectrophotometry, photon

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correlation spectroscopy, transmission electron microscopy and X-ray diffraction.

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Moreover, the FTIR spectrum identified the possible stabilizing agents present in

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supernatants. The Ag NPs obtained from both yeasts were disperse and stable and

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exhibited differences in sizes, zeta potential, concentration and stabilizing compounds.

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The antifungal activity of the Ag NPs was evaluated against the relevant

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phytopathogenic fungi, the common producers of postharvest diseases in pome fruits.

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Susceptibility tests on agar demonstrated that the antifungal activity of the nanoparticles

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from R. glutinis was higher than that from the ones from C. laurentii, and both were

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significantly more effective for inhibiting the fungi than the nanoparticles made from

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chemical synthesis. At 3 ppm, the nanoparticles from R. glutinis had similar efficacy to

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iprodione, a fungicide commonly utilized for combating postharvest diseases.

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Keywords Cryptococcus laurentii; Rhodotorula glutinis; Silver nanoparticles;

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Antifungal activity; Postharvest phytopathogen fungi 4

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1. Introduction

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The synthesis of nanomaterials with a specific composition and size and distinct

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properties has expanded the scope of their applications in several industries, including

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agriculture, textile, food, biomedical, optical, and electronic fields. In recent years,

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silver nanoparticles (Ag NPs) have gained significance due to their potential

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applicability in many fields [1-5].

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The development of environmentally friendly, benign and green technologies for

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the production of nanoparticles with a range of chemical and physical properties is one

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of the challenges in the newly emerging field of nanobiotechnology [6-8]. The

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microbiological synthesis of Ag NPs presents several important advantages over

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chemical synthesis, such as higher production and lower costs, and overall, it is an eco-

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friendly method [9]. In fact, a number of different species of bacteria and fungi are able

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to reduce silver ions, producing Ag NPs with antimicrobial properties [4, 10-14].

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To date, there has been a continued search for novel microorganisms that

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synthesize Ag NPs. In this regard, owing to the promising application of Ag NPs for

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plant disease management, researchers have begun to use biological agents for Ag NP

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synthesis. The involvement of agriculturally important microbes for Ag NP synthesis

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could lead to more environmentally friendly and biocompatible nanoparticles for the

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efficient application in agroecosystems [15]. In this context, most recently, a well-

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known plant growth-promoting Serratia sp. has been used to synthesize Ag NPs with

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antifungal activity against Bipolaris sorokiniana, the spot blotch pathogen of wheat

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[15].

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Cryptococcus laurentii and Rhodotorula glutinis are two yeasts that have been

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reported as biological control agents [16, 17]. These yeasts were chosen in this case to 5

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produce Ag NPs. The aim of this work was to produce Ag NPs using these epiphytic

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yeasts isolated from apple peel, to compare their characteristics and to investigate their

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potential application to the control of fungi that cause postharvest diseases in pome

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fruits.

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2. Materials and Methods 2.1. Microorganisms

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Cryptococcus laurentii (BNM 0525) and Rhodotorula glutinis (BNM 0524)

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previously isolated from apple peel and identified in our laboratory [16] were selected

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for the synthesis of Ag NPs. Both strains are deposited in the National Bank of

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Microorganisms (WDCM938) of the “Facultad de Agronomía, Universidad de Buenos

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Aires (FAUBA)”, Argentina. The yeasts were grown on potato dextrose agar (PDA) at

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25°C for 48 to 72 h and were maintained on PDA slants. These yeasts were chosen for

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the nitrate reductase activities that they displayed. The antifungal activity was assessed

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against the phytopathogenic fungi: Botrytis cinerea (BNM 0528), Penicillium expansum

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(CEREMIC 151-2002), Aspergillus niger (NRRL 1419), Alternaria sp. (NRRL 6410),

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and Rhizopus sp. (NRRL 695). The isolates were maintained on PDA at 4°C for further

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studies.

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2.2. Culture

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C. laurentii and R. glutinis were cultured in Muller-Hinton broth (MHB), the

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composition of which was (g L-1): beef infusion, 2; acid casein peptone, 17.5; corn

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starch, 1.5; pH 7.4. The medium was inoculated with a suspension of 2.0×106 yeast

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cells mL-1. Next, the culture flasks were incubated at 28ºC and 100 rpm in an orbital

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shaker. After 24 h of incubation, the cultures were centrifuged at 11,000  g for 10 min 6

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in a Sorvall SS-3 (DuPont Instruments), and the supernatants were reserved for the

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synthesis of Ag NPs. Before their use, the supernatants were analyzed immediately,

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evaluating the cells by microscopic analysis, and a portion was subsequently seeded on

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potato dextrose agar (PDA) to search for viable cells.

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2.3. Nitrate reductase assay

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The enzyme-nitrate reductase was assayed in the supernatant from culture yeast

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according to the procedure followed by Harley [18] and Saifuddin et al. [19]. A 5 mL

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aliquot of culture supernatant of the yeast was mixed with 5 mL of assay medium (30

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mM KNO3 and 5% propanol in 0.1 M phosphate buffer of pH 7.5) and incubated in the

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dark for 1 h. After incubation, the nitrites formed in the assay mixture were estimated

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by adding 2.5 mL of sulphanilamide and N-(1-naphthyl) ethylene diamine

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dihydrochloride solutions to it. The color developed was measured in an UV–Vis

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spectrophotometer at 540 nm. The enzyme activity was finally expressed in terms of

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nmol of nitrite h-1 mL-1.

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2.4. Synthesis of silver nanoparticles

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For the synthesis of the Ag NPs, one milliliter of an aqueous silver nitrate

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solution (1 mM) was added to Erlenmeyer flasks containing 100 mL of yeast culture

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supernatant free of cells. The resulting mixture was shaken at 100 rpm in an orbital

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shaker for 48 h at 28 ± 4ºC in dark. Appropriate controls (uninoculated MH medium

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plus silver nitrate and yeast culture supernatant without silver nitrate) were run

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simultaneously. The experiment was carried out in triplicate. 7

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2.5. Nitrate reductase inhibition

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The inhibition study was performed to confirm the mechanism of the Ag NP

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biosynthesis. The inhibitors used were phenyl methane sulfonyl fluoride (PMSF),

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ethylene diamine tetra acetic acid (EDTA) and sodium azide. Experiments were carried

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out similarly to those described in 2.4.

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milliliter of AgNO3 (1 mM) were added to Erlenmeyer flasks with 100 ml of the

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supernatant. Three separate trials were performed for each inhibitor. The concentration

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of Ag NPs produced was measured for each case. The nitrate reductase activity was

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measured prior the addition of the silver nitrate solution.

One milliliter of inhibitor (10 mM) and one

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2.6. Physicochemical characterization of silver nanoparticles

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To characterize the Ag NPs, standard protocols were used. Spectroscopic

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analyses of surface plasmon resonance (SPR) and fluorescence were measured in an

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UV-visible spectrophotometer model UV-1650 PC, Shimadzu, and Perkin-Elmer (LS-

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55) luminescence spectrophotometer, respectively. The average diameter of the

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nanoparticles was determined by Zetasizer Nanoseries ZEN3600, Malvern Instruments,

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through the technique of photon correlation spectroscopy (PCS). Zeta potential was

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measured in the same equipment. The structure and morphology were studied by means

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of transmission electron microscopy (TEM; Carl Zeiss CEM902) and X-ray diffraction

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(Shimadzu XRD6000).

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The determination of Ag NPs concentration in culture supernatant from yeasts was

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carried out by UV-visible spectrophotometry at 420 nm. A calibration curve was made

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with the Ag NPs (Sigma-Aldrich St. Louis, MO, USA) between 5 and 20 mg.L-1. For

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the spectrophotometric assay validation, the method of adding an internal standard 8

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(spiking) was used. A known concentration of Ag NPs (Sigma-Aldrich St. Louis, MO,

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USA) was added to suspensions of Ag NPs (Sigma-Aldrich St. Louis, MO, USA) of 5,

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10 and 20 mg.L-1 and samples (biosynthesized Ag NPs from culture supernatant of C.

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laurentii and R. glutinis). The concentration results were plotted against spiking levels

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and interpolated using weighted linear regression (Table 1). The determination of the

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residual ionic silver concentration was carried out by potentiometry in a potentiostat

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PGSTAT 302N, AUTOLAB.

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2.7. Fourier transform infrared (FTIR) analysis

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The Fourier transformed infrared (FTIR) spectrum of Ag NPs was obtained

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from Nicolet Protégé 460 spectrometer provided with a CsI beam splitter in the 4000-25

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cm-1 range, 64 scans/sample with a spectral resolution of 4 cm-1 using the KBr pellet

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technique. The sample was prepared by adding supernatant with Ag NPs onto a KBr

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pellet in a drop-wise manner and drying at 100°C in an oven for 15 min.

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2.8. Assessment of antifungal activity

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For the assessment of antifungal activity, 200 µL of fungal spores suspension

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(2.0×106 spores mL-1) were aseptically spread onto plates of PDA. Next, wells of 3 mm

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were made aseptically and were filled with 60 µL of extracts containing biosynthesized

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Ag NPs or chemically synthesized Ag NPs (Sigma-Aldrich St. Louis, MO, USA). The

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concentrations of the Ag NPs were adjusted to 3 mg L-1. Yeast culture supernatants and

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sterile distilled water were used as controls. The plates were incubated at 28 ± 4ºC for 7

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days. After incubation, the zones of inhibition (mm) were measured. The assays were

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performed in triplicate.

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The chemical, iprodione “Rovral 50 WP” (Rhone-Poulenc Agrochimie) was

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dissolved in an acetone–water mixture. A concentrated stock solution was prepared and

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progressively diluted in sterile water to provide an appropriate concentration for the

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assay. The concentration used was 500 mg L−1, the normal concentration recommended

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for standard postharvest treatments [17]. Controls with dilutions of acetone–water

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mixture were made.

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The minimal concentration of the Ag NPs, that causes 100% of spores

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germination inhibition (MIC), was determined using a microdilution method in Potato

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Dextrose Broth (PDB).

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spores. mL-1), different volumes of extracts containing nanoparticles (50, 100, 150 and

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200 l) and PBD until final volume of 300 l. The mixture was incubated for 24 h at

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28 ºC. The final concentrations of the Ag NPs were

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silver nanoparticles from R. glutinis and C. laurentti respectively. Approximately 50 µl

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of the samples were placed on microscope slides and 100 spores per slide were

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evaluated. Controls were performed without nanoparticles. Experiments were repeated

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three times.

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2.9. Assessment of toxicity

In a vial were put 100 l of the spores suspension (2 X 106

0-2 mg.L-1 and 0-4 mg.L-1 for

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Standardized bioassays using seeds of the lettuce (Lactuca sativa) were

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performed to assess the toxicity of biosynthesized and Commercial AgNPs and also of

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the yeast culture supernatants without AgNPs. To these assays, 10 seeds were placed in

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a Petri dish with filter paper in the bottom as the support; afterwards, 2.5 ml of each

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treatment were applied. Distilled water was used as a negative control and a Cu+2

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solution as the positive control. After 5 days of incubation at 25 ± 1°C, the number of 10

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germinated seeds was counted, and expressed as the percentage of germination (G%).

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Also root elongation (RE) was analyzed and the results were expressed in mm.

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Experiments were done in triplicate and were repeated three times. The results were

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analyzed using analysis of variance. Statistical analyses were done with OriginPro 7.0

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(MicroCal Software, Inc. 2002).

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3. Results and Discussion

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The primary objective of this work was to study the production of Ag NPs using

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epiphytic yeasts isolated from fruits, compare their characteristics and investigate their

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potential application for the control of phytopathogenic fungi that cause rotting in pome

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fruits.

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The selection of the yeasts was based in their nitrate reductase activity because

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this enzyme seem be involved in the Ag NP synthesis [18-20]. Then, the nitrate

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reductase activity was assayed in the culture supernatant of various epiphytic yeasts

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previously isolated in our laboratory from different fruits (data not shown). The higher

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values of nitrate reductase activities were found in the supernatant from C. laurentii and

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R. glutinis, which were 266.56±13.11 nmol h-1 mL-1 and 216.85±11 nmol h-1 mL-1,

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respectively.

Therefore, these yeasts were selected for the synthesis of the

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nanoparticles.

Both yeasts are considered GRAS microorganisms and are classified

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among “risk group 1” by the World Health Organization (WHO).

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When the supernatants that were free of cells, derived from the yeast cultures,

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were incubated with silver nitrate, the production of silver nanoparticles was evidenced

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by the appearance of a color change from pale yellow to dark brown in the reaction

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flasks. The reaction mixture remained a dark brown color for various months. On the

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other hand, the control reaction mixtures did not show any change in color. 11

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The Ag NP formation was verified by surface plasmon resonance (Figure 1). In

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both cases, a wide spectrum, characteristic of a high distribution of size nanoparticles,

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was observed. These samples were analyzed for size distribution and zeta potential by

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photon correlation spectroscopy. The Ag NPs produced by C. laurentii showed two

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populations of different sizes (Figure 2a), one centered at 400 nm (13%) and another

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around of 35 nm (74%).

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confirmed by the transmission electron microscopy images (Figure 2b). The

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quantification of the size distribution of the Ag NPs from R. glutinis was as follow:

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65% at 15 nm, 17% at 160 nm and 18% at 220 nm (Figure 3a). The TEM images are

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shown in Figure 3b.

The presence of these two populations of particles was

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A spectroscopic analysis by fluorescence emission was also performed. Figure 4

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shows the fluorescence emission spectra of the Ag NPs from R. glutinis and C.

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laurentii. An emission band centered at 345-355 nm (λexc 295 nm) was observed. The

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nature of the emission indicated presence of proteins in the supernatant containing the

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nanoparticles. Similar results were obtained by Durán et al. (2005) [20].

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Further studies using X-ray diffraction were carried out to confirm the

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crystalline nature of the particles produced by the yeasts.

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supernatants (Figure 5) shows the four intense peaks ((111), (200), (220) and (311)) at

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2θ° values, confirming that the silver particles that were formed have a nanocrystalline

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nature.

The XRD pattern of both

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The concentrations of Ag NPs determined by UV-visible spectrophotometry in

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culture supernatants from C. laurentii and R. glutinis were 6 ± 0.2 and 3 ± 0.3 mg/L-1,

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respectively. The method was validated by adding an internal standard to discard the

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matrix effect in measuring the nanoparticle concentration in the supernatants. As can be 12

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seen in Table 1, the recovery percentage was satisfactory; therefore the UV-visible

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spectrophotometry was considered a valid method for the estimation of the nanoparticle

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concentration. The Ag NP concentration obtained from R. glutinis was half of the Ag

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NP concentration of the other yeast. These results were coincident with the first

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estimation of concentration performed by UV-visible spectrophotometry (Figure 1).

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The concentrations of residual ionic silver measured in the supernatants were in the

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range of 0,001 mg.L-1.

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FTIR spectroscopy was carried out to investigate the chemical nature of the

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compounds present in supernatants, which could be involved in the stabilization of

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nanoparticles. The FTIR spectrum of synthesized Ag NPs from C. laurentii (Figure 6a)

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showed a pattern of peaks similar to R. glutinis (Figure 6 b), although with a number of

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differences.

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biosynthesized Ag NPs, the presence of proteins and polymeric carbohydrates can be

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inferred, the latter to a greater extent because the peaks of the absorption of C=O and

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CN (characteristics of proteins) appear with less intensity (band amide 2 and band

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amide 3) with respect to the other vibrational modes. Another interesting point is the

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important intensity of the peaks C-OH and C-O-C, together to the peaks aliphatic C-C

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and C-H, which justifies the presence of polymeric carbohydrates. The bands observed

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in the range between 902 and 462 cm-1 in the spectrum of C. laurentii and between 909

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and 523 cm-1 in that of R. glutinis correspond to vibrational modes of the structural net

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of biological polymers present in the supernatants. The most interesting peaks appear at

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902 and 837 cm-1 for Ag NPs from C. laurentii and 909 y 840 cm-1 for Ag NPs from R.

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glutinis, indicating glycosidic linkages and type, respectively, in coincidence with

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the presence of polymeric carbohydrates, perhaps of arabinogalactan type for C.

According to the pattern of peaks, for the two supernatants containing

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laurentii and arabinan and galactoglucomannan types for R. glutinis. The peak at 3382

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cm-1, corresponding to the –OH and NH groups of Ag NPs from C. laurentii, is very

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wide and difficult to resolve. However, the deconvolution of this peak shows two peaks

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of low intensity at 3252 and 3237 cm-1, which would indicate the presence of NH

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groups of amide (band amide 1), corresponding to peptides, while the peak 3394 cm-1,

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corresponding to Ag NPs from R. glutinis, does not show other peaks after performing

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the deconvolution. Furthermore, the decrease in the intensity of the carbonyl bands (C

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= O) and CN bands show that the proportion of proteins was lower in the supernatant

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from R. glutinis [21, 22, 23]. The FTIR spectrum of chemical Ag NPs (Figure 6c) only

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showed a pattern corresponding to peaks of acetic acid-acetate buffer.

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As previously described, both systems exhibited particles of different sizes with

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a high percentage of small particles. It is believed that biogenic systems at certain

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conditions produced small Ag NPs, and then after a certain period an agglomeration is

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initiated due to coalescence and Ostwald ripening processes. However, the

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agglomeration phenomena can be reduced by the use of templates that can offer

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electrostatic and steric barriers [24]. In the case of biogenic synthesis, the compounds

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present in supernatants are probably able to provide these two barriers. The electrostatic

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barrier is easily measured through the zeta potential, which determines the surface

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charge of biosynthesized Ag NPs. In this case, the results were ξ=-24.9 ± 0.5 mV and

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ξ=-13.5 ± 0.8 mV for Ag NPs from C. laurentii and R. glutinis, respectively. Although

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it is considered that the optimum zeta potential to avoid the formation of agglomerates

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is close to ± 30 mV, in this case the steric barriers provided by polymeric carbohydrates

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and proteins must be taken into account because structures compatible with

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agglomerates were not observed. However, the steric barrier is not easy to measure, and 14

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in this instance, it would be more efficient for the nanoparticles from R. glutinis because

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the predominant population was smaller nanoparticles, and their size was approximately

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half that of the nanoparticles obtained with C. laurentii.

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To confirm whether nitrate reductase was involved in the synthesis of Ag NPs,

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the effect of the inhibitors on the enzyme activity and the ability to synthesize

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nanoparticles was studied. The results are shown in Figures 7 and 8. Several

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conclusions were obtained from these studies. First, it was clear that the concentration

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of nanoparticles was dependent on the activity of the enzyme. Second, the effect of

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inhibitors is not the same for both cases. Third, according to the relationship between

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enzyme activity and the concentration of the nanoparticles, the supernatant of C.

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laurentii seemed more effective for the synthesis. Other authors have reported the

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correlation between enzyme activity and the synthesis of nanoparticles and also the

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influence the physiological state of the culture in the process, particularly the redox state

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[25]. The mechanism for the synthesis of Ag NPs, proposed by Duran et al [20], is

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shown in Scheme 1. There, the involvement of cofactors that are dependent on the redox

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state of the culture at the harvest time can be observed.

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For investigating the applicability of biosynthesized Ag NPs to the control of

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postharvest diseases, their antifungal activity was evaluated against the most important

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pathogenic fungi that rot apples, pears and grapes.

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expansum, A. niger, Alternaria sp. and Rhizopus sp. cause diseases that limit the storage

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period and marketing life of fruits and vegetables. For this reason, these fungi were

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selected for these assays. Tests (Table 2) showed that the antifungal activity of Ag NPs

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from R. glutinis was higher than the ones from C. laurentii, and both were significantly

333

more effective in inhibiting the fungi than the nanoparticles of chemical synthesis. 15

Fungi such as B. cinerea, P.

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Moreover, at 3 ppm, the nanoparticles from R. glutinis were similarly effective to

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iprodione against all fungi with the exception of Rhizopus. Iprodione is a fungicide

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commonly utilized for combating postharvest diseases, which was assayed at 500 ppm,

337

which is the concentration recommended for standard postharvest treatments. Controls,

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with yeast crude extracts alone and an acetone-water mixture did not show antifungal

339

activity. This fact allowed us to discard the presence of antifungal compounds produced

340

by the yeasts and also the effect of the solvent employed for the dissolution of

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iprodione. The MIC for silver nanoparticles from R. glutinis against all fungi tested was

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2 mg. L-1 while the MIC for nanoparticles from C. laurentii was 4 mg. L-1. The

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antifungal activity of Ag NPs produced by both yeasts was effective for one year.

344

From the results of the antifungal activity tests, it can be inferred that the size of

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the nanoparticles clearly influenced their antifungal activity. According to different

346

authors [26, 27], silver nanoparticles have the ability to anchor the microbial cell wall

347

and then penetrate it, thereby causing structural changes that affect the permeability of

348

the cell membrane and cause cell death. The size and surface area of the nanoparticles

349

are closely related because a decreasing size increases the relative surface area of Ag

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NPs, leaving a greater number of atoms exposed on the surface, which will be available

351

to redox reactions, photochemical reactions and physicochemical interactions with cells.

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The antifungal activity of the nanoparticles from C. laurentii and R. glutinis could be

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explained by the previously described mechanisms, because in both cases, small

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particles are predominant. Moreover, the polymeric carbohydrates and proteins that

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accompany to nanoparticles would favor the interactions with the cell membrane.

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The preliminary assays demonstrated the antifungal activity of fruit packaging

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paper imbibed with Ag NPs from R. glutinis and C. laurentii and also a better 16

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absorption onto the paper of the biosynthesized AgNPs (Figure 9). With respect the

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toxicity, bioassays using seeds of the lettuce (Lactuca sativa) [28] showed that AgNPs

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from chemical synthesis resulted more toxics than Ag NPs from R. glutinis or C.

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laurentii (Table 3). Despite these auspicious results, further studies are necessary for

362

investigate the feasibility of incorporating Ag NPs into films and other packaging

363

materials of fruits.

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4. Conclusions

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In this study, the two assessed yeasts were adequate for the biosynthesis of Ag

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NPs. As the epiphytic yeasts, such as C. laurentii and R. glutinis, are harmless, and they

368

are considered GRAS microorganisms, the production of nanoparticles using these

369

yeasts possesses significant advantages. Further studies are needed to optimize the

370

production of nanoparticles, which should be based on increasing the concentration of

371

nitrate reductase, optimizing the redox state in the supernatant and study and improving

372

the production of exocellular polymeric carbohydrates involved in the stabilization of

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the nanoparticles. The antifungal activity of these nanoparticles and the potential

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application as an effective fungicide against some phytopathogenic fungi that affect

375

pome fruits was demonstrated. Additional studies are needed to investigate the

376

feasibility of incorporating Ag NPs into films and other packaging materials of these

377

fruits.

378 379

Contributors

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Jorge G. Fernández performed the experiments and prepared the manuscript.

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Martín Fernández-Baldo performed the experiments, contributed to the silver

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nanoparticles’ characterization and prepared the manuscript. Elias Berni contributed to 17

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the silver nanoparticles’ characterization and analyzed the characterization data.

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Gerardo Camí contributed to the silver nanoparticles’ characterization and tested in

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vitro antifungal activity. Nelson Duran helped perform the experiments and contributed

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significantly to manuscript preparation. Julio Raba analyzed results and contributed to

387

manuscript preparation. María I. Sanz conceived and designed the experiments,

388

analyzed results and contributed to manuscript preparation.

389 390

Acknowledgments

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Support from CNPq, FAPESP, INOMAT (MCT/CNPq) (Brazil), NanoBioss

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(MCTI/CNPq), the Brazilian Network on Nanotoxicology (MCTI/CNPq) and

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Universidad Nacional de San Luis, the Agencia Nacional de Promoción Científica y

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Tecnológica, and Consejo Nacional de Investigaciones Científicas y Técnicas

395

(CONICET) (Argentina) are acknowledged.

396 397

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[27] Ortega F, Fernández-Baldo M, Fernández G, Serrano M, Sanz MI, Diaz-

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Mochón J, Lorente J, Raba J. Study of antitumor activity in breast cell lines using

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silver nanoparticles produced by yeast. International Journal of Nanomedicine 2015;

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0:2021–2031.

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CRMN, 1H-RMN, IR,

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[28] Charles J, Sancey B, Morin-Crini N, Badot P, Degiorgi, Trunfio G, Crini G.

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Evaluation of the phytotoxicity of polycontaminated industrial effluents using the

478

lettuce plant (Lactuca sativa) as a bioindicator. Ecotoxicol Environ Safety, 2011;74:

479

2057-2064

480

481

482

483

484

485

486

487

488

489

490

491

492

493 22

494

495

496

Figures

497

Scheme 1: Possible mechanism of silver reduction by nitrate reductase. From Duran et

498

al (2005). [20]

499

Figure 1: UV–Vis spectra recorded after the addition of 1 ml of silver nitrate solution

500

(1 mM) to 100 ml of culture supernatants of C. laurentii or R. glutinis. The mixture

501

was shaken at 100 rpm in an orbital shaker for 48 h at 28±4ºC in the dark.

502

Figure 2: Size distribution (a) and TEM images (b) of Ag NPs produced with the

503

supernatant from C. laurentii. The average diameter size of the nanoparticles was

504

determined by Zetasizer Nanoseries ZEN3600, Malvern Instruments, through the

505

technique of photon correlation spectroscopy (PCS) and the structure and morphology

506

were studied by means of transmission electron microscopy (TEM; Carl Zeiss

507

CEM902).

508

Figure 3: Size distribution (a) and TEM images (b) of Ag NPs produced by using

509

supernatant from R. glutinis. The average diameter size of the nanoparticles was

510

determined by Zetasizer Nanoseries ZEN3600 – Malvern Instrument, through the

511

technique of photon correlation spectroscopy (PCS) and the structure and morphology

512

were studied by means of transmission electron microscopy (TEM; Carl Zeiss CEM

513

902).

514

Figure 4: Fluorescence emission spectra recorded from the Ag NPs-yeasts reaction

515

mixture. The excitation wavelength was 295 nm. Spectra were obtained in a Perkin-

516

Elmer (LS-55) luminescence spectrophotometer. 23

517

Figure 5: X-Ray diffraction pattern of Ag NPs from C. laurentii or R. glutinis. Peaks,

518

((111), (200), (220) and (311)) at 2θ° values, confirmed that the silver particles formed

519

have a nanocrystalline nature. (Shimadzu XRD6000).

520

Figure 6: The FTIR spectra of silver nanoparticles from: (a) C. laurentii, (b) R. glutinis

521

and (c) chemical synthesis. Spectra were obtained from Nicolet Protégé 460

522

spectrometer provided with a CsI beam splitter in the 4000–25 cm-1 range, 64

523

scans/sample, with spectral resolution of 4 cm-1, using the KBr pellet technique

524

Figure 7:

525

synthesis in supernatant from C. laurentii. Phenyl methane sulfonyl fluoride (PMSF),

526

ethylene diamine tetra acetic acid (EDTA) and sodium azide were assayed at a

527

concentration of 10 mM.

528

Figure 8: Effect of inhibitors on nitrate reductase activity and nanoparticles synthesis

529

in supernatant from R. glutinis. Phenyl methane sulfonyl fluoride (PMSF), ethylene

530

diamine tetra acetic acid (EDTA) and sodium azide were assayed at a concentration of

531

10 mM.

532

Figure 9: Antifungal activity of fruit packaging paper imbibed with Ag NPs. Assays

533

were performed using discs of packaging paper imbibed with extracts containing 3 ppm

534

of biosynthesized Ag NPs (R.glutinis, C.laurentii), 3 ppm of chemically synthesized

535

Ag NPs (Ch) or 500 ppm of Iprodione. Discs were put onto plates of PDA with 200 µL

536

a B.cinerea BNM 0527 spores suspension (2.0×106 spores mL-1). Yeast culture

537

supernatants without AgNPs (SR, SC) were used as controls.

538

incubated at 28 ± 4ºC for 7 days. Assays were performed by duplicate.

The effect of inhibitors on nitrate reductase activity and nanoparticles

539 540 541 24

The plates were

542

Table 1

543

Determination of Ag NPs concentration in culture supernatant

544

Nanoparticles

Concentration measureda (mg L-1)

From C. laurentii From R. glutinis 5 mgL-1(Sigma) 10 mgL-1(Sigma) 20 mgL-1(Sigma)

6.0 ± 0.2 3.0 ± 0.3 5.6 ± 0.3 11.0± 0.1 18.0± 0.1

a

Ag NPs spiking level/mg L-1 20 Recovery (%)

27.0 ± 0.2 22.7 ± 0.1 25.7 ± 0.1 29.0 ± 0.2 39.0 ± 0.3

103.8 94.0 100.3 93.5 102.0

X ± SD, mean ± standard deviation (n=3)

Determination of Ag NP concentration in culture supernatant was carried out by UVvisible spectrophotometry at 420 nm. Calibration curve was made with Ag NPs (Sigma-Aldrich St. Louis, MO, USA) in concentrations between 5 and 20 mg.L-1. For validating spectrophotometric assay, a known concentration of Ag NPs (20 mg.L1) (Sigma-Aldrich St. Louis, MO, USA) was added to the samples and to suspensions of Ag NPs (Sigma-Aldrich St. Louis, MO, USA) of 5, 10 and 20 mg.L-1.

545 546 547 548 549 550 551 552 553 554 555 556 557 558 25

577

Table 2

578

Antifungal activity of Biosynthesized AgNPs against Postharvest phytopathogenic fungi

Diameter of inhibition zone (mm)a B. cinereaa

P. expansuma

A. nigera

Alternaria sp.a

Rhizopus sp.a

15.1 ± 1.6a

11.1 ± 1.4b

14.8±2.2a

11.3 ±1.6ab

9.7 ± 2.1b

11.4 ± 1.4b

8.3 ± 1.2b

10.5±1.2b

9.6 ± 2.3b

7.9 ± 1.8b

2.2 ±0.5c

1.9 ± 0.7c

3.0 ± 0.3c

1.7 ± 0.5c

1.3 ± 0.5c

Iprodione

17.1± 1.1a

14 ± 2.1a

15.9±1.4a

10.2 ± 1.5b

13.5 ± 1.1a

distilled water

n.m.b

n.m

n.m

n.m

n.m

Acetone water mixture supernatant from yeasts

n.m.b

n.m

n.m

n.m

n.m

n.m b

n.m

n.m

n.m

n.m

AgNPs from R. glutinis AgNPs from C. laurentii AgNPs (Sigma)

579 580 581 582 583 584 585 586 587 588 589 590 591 592 593

a

X ± SD, mean ± standard deviation.(n=3)

594

b

n.m: non measurable

27

595 596 597 598 599 600

Assays were performed by the method of diffusion on agar. 200 µL of fungal spores suspension (2.0×10 6 spores mL-1) were spread onto plates of PDA. Samples and controls (60 µL ) were put in wells of 3 mm. Concentration of nanoparticles was adjusted at 3 ppm and iprodione was assayed at concentration of 500 ppm. Controls consisted in distilled water, supernatant from yeasts and dilutions of acetone–water mixture. The plates were incubated at 28±4ºC for 7 days. The assays were performed by triplicate. Values with different letters in the same column are significantly different.

601 602 603

28

604

Table 3

605 606

Evaluation of toxicity of biosynthesized and Commercial AgNPs using lettuce

607

seeds

608

Treatment

Germination (%)

Root elongation ( mm) a

AgNPs from C. laurentii

80

27.7 + 2.1a

Supernatant (C.laurentii)

90

29.2 + 1.3a

AgNPs from R.glutinis

90

28.5 ± 1.3a

Supernatant (R. glutinis)

90

30.4 ± 0.9 a

Ag NPs (Sigma)

60

22.5 ± 3.2b

Distilled water

90

31.2 ± 4.2a

Cu2+ Solution

40

12.5 ± 2.2c

609 610

a

X ± SD, mean ± standard deviation.(n=3)

611 612 613 614 615 616

To these assays, 10 seeds were placed in a Petri dish with filter paper in the bottom as the support; afterwards, 2.5 ml of each treatment were applied. Distilled water was used as a negative control and a Cu+2 solution as the positive control. After 5 days of incubation at 25 ± 1°C the percentage of germination (G%) and root elongation (RE) were determined . Experiments were done in triplicate. Values with different letters in the same column are significantly different.

617 618 619 620 621 622 623 624 625 626 29