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Antimicrobial volatile organiccompounds affect morphogenesis-related enzymes in Guignardiacitricarpa, causal agent of citrus blackspotMauricio Batista Fialho a , Luiz Fernando Romanholo Ferreira b ,Regina Teresa Rosim Monteiro b & Sérgio Florentino Pascholati aa Department of Plant Pathology and Nematology, ‘Luiz deQueiroz College of Agriculture’, University of São Paulo, CP 09,CEP 13418-900, Piracicaba, SP, Brazilb Center for Nuclear Energy in Agriculture, University of SãoPaulo, CP 96, CEP 13400-970, Piracicaba, SP, Brazil

Available online: 01 Jun 2011

To cite this article: Mauricio Batista Fialho, Luiz Fernando Romanholo Ferreira, Regina TeresaRosim Monteiro & Sérgio Florentino Pascholati (2011): Antimicrobial volatile organic compoundsaffect morphogenesis-related enzymes in Guignardia citricarpa, causal agent of citrus black spot,Biocontrol Science and Technology, 21:7, 797-807

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RESEARCH ARTICLE

Antimicrobial volatile organic compounds affect morphogenesis-relatedenzymes in Guignardia citricarpa, causal agent of citrus black spot

Mauricio Batista Fialhoa, Luiz Fernando Romanholo Ferreirab, Regina Teresa

Rosim Monteirob and Sergio Florentino Pascholatia*

aDepartment of Plant Pathology and Nematology, ‘Luiz de Queiroz College of Agriculture’,University of Sao Paulo, CP 09, CEP 13418-900, Piracicaba, SP, Brazil; bCenter for NuclearEnergy in Agriculture, University of Sao Paulo, CP 96, CEP 13400-970, Piracicaba, SP, Brazil

(Received 13 December 2010; returned 22 February 2011; accepted 10 April 2011)

Although non-volatile substances toxic to plant pathogenic microorganisms havebeen extensively studied over the years, few studies have focused on microbial volatileorganic compounds (VOCs). The VOCs produced by the yeast Saccharomycescerevisiae strain CR-1, used in fermentative processes for fuel ethanol production,are able to inhibit the vegetative development of the fungus Guignardia citricarpa,causal agent of the disease citrus black spot. How microbial VOCs affect thedevelopment of fungi is not known. Thus, the objective of the present work was tostudy the effect of the artificial mixture of VOCs identified from S. cerevisiae onintracellular enzymes involved in the mycelial morphogenesis in G. citricarpa. Thephytopathogenic fungus was exposed to artificial mixture of VOCs constituted byalcohols (ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol and phenylethyl alcohol)and esters (ethyl acetate and ethyl octanoate) in the proportions naturally found inthe atmosphere produced by the yeast. The VOCs inhibited considerably the mycelialdevelopment and interfered negatively with the production of the morphogenesis-related enzymes. After 72 h of exposure to the VOCs the laccase and tyrosinaseactivities decreased 46 and 32%, respectively, however, the effect on the chitinase andb-1,3-glucanase activities was lower, 17 and 13% of inhibition, respectively. There-fore, the exposure of the fungus to the antimicrobial volatiles can influence bothfungal mycelial growth rate and activity of enzymes implicated in morphogenesis.This knowledge is important to understand the microbial interactions mediated byVOCs in nature and to develop new strategies to control plant pathogens as G.citricarpa in postharvest.

Keywords: antimicrobial activity; biocontrol; Citrus; morphogenesis; plantdisease

Introduction

Citrus black spot, a fungal disease caused by Guignardia citricarpa Kiely

(anamorphic stage: Phyllosticta citricarpa McAlpine) [Ascomycetes: Dothideales],

is one of the most important diseases of citrus worldwide. It has high economic

importance and affects the most important commercial citrus varieties in many

producing areas of Africa, Asia, Australia, and South America (OEPP/EPPO 2009).Several fruit symptoms are associated to the disease and although not showing

apparent symptoms, the infected fruits can develop them at postharvest during

*Corresponding author. Email: sfpascho@esalq.usp.br

Biocontrol Science and Technology,

Vol. 21, No. 7, July 2011, 797�807

ISSN 0958-3157 print/ISSN 1360-0478 online

# 2011 Taylor & Francis

DOI: 10.1080/09583157.2011.580837

http://www.informaworld.com

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transport or storage. The lesions are restricted to the fruit rind, but the fruits become

aesthetically damaged, making them undesirable to the fresh fruit market. In

addition, it is considered an A1 quarentenary disease and infected fruits cannot be

exported especially to the European Community due to phytosanitary restrictions

(OEPP/EPPO 2009).

Even though their effectiveness is limited, the use of fungicides is the main control

method used at pre- and post-harvest. However, the acquisition of resistance by the

pathogen and the consumer perception about the potential impact of traditional

control practices on health and on environment led to an increased demand for

residue-free chemical products. Therefore, farmers and researchers started to consider

the use of alternative methods to control diseases (Punja and Utkhede 2003).

During a plant�pathogen interaction, microbial antagonists may interrupt some

stage of the disease or the pathogen’s life cycle. This may occur by several

mechanisms such as parasitism, competition for nutrients and colonization niches,

production of hydrolytic enzymes and antibiotic compounds (Sharma, Singh, and

Singh 2009), including volatiles (Strobel 2006).

Volatile organic compounds (VOCs) produced by one microorganism could

enhance its status by affecting the physiology of other competitor organisms causing

them disadvantage (Mackie and Wheatley 1999; Wheatley 2002). Typically, such

compounds have low molecular weight, high vapor pressure, are active at very low

concentrations and belong to several chemical groups (Wheatley 2002). The

antagonism caused by these compounds has received limited attention in comparison

to medium-diffusible compounds (Chaurasia et al. 2005), but recently new findings

have focused attention on these volatile metabolism products. Most of the studies

about production of antimicrobial VOCs are related to Muscodor spp., Trichoderma

spp., and Bacillus spp. to control phytopathogenic and wood decay fungi (Humphris,

Bruce, Buultjens, and Wheatley 2002; Grimme, Zidack, Sikora, Strobel, and

Jacobsen 2007; Leelasuphakul, Hemmaneea, and Chuenchitt 2008).

M. albus, an endophytic fungus isolated from cinnamon tree, is a well known

volatile antimicrobial producer. The fungus emits a complex mixture of about 30

VOCs and it has been tested to control several pathogens in infested soils, fruits and

seeds in storage (Strobel 2006). The use of artificial mixtures showed that the

presence of naphthalene, propanoic acid, and 3-methyl-1-butanol was necessary to

keep the inhibitory activity against the pathogens Pythium ultimum, Rhizoctonia

solani, and Sclerotinia sclerotiorum (Ezra, Hess, and Strobel 2004).

The saprophytic fungi Trichoderma spp. have many antagonistic mechanisms which

have contributed to their success as biological control agents. Wheatley, Hackett,

Bruce, and Kundzewicz (1997) demonstrated the production of 2-propanone,

2-methyl-1-butanol, decanal, heptanal, and octanal by T. pseudokoningii and T.

viride as responsible for the antimicrobial activity against wood decay fungi (Wheatley

et al. 1997).

The action mechanisms of antimicrobial volatiles are not fully understood until

now and the discussions have been merely speculatory. It is likely that volatiles act by

changing protein expression (Humphris et al. 2002) and affecting the activity of

specific enzymes (Mackie and Wheatley 1999). The knowledge about how this

mechanisms works is essential to improve the biocontrol effectiveness as well as to

develop innovative control strategies.

798 M.B. Fialho et al.

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Fungal polyphenol oxidases like tyrosinases and laccases are enzymes linked to

mycelial growth. Tyrosinases are directly involved in the melanin biosynthesis.

Melanin is a pigment implicated in the resistance to stress factors such as free

radicals, UV radiation and contributes to the cell wall resistance against hydrolytic

enzymes (Henson, Butler, and Day 1999). The laccases are involved in the

morphogenesis, protection against stress, resistance to fungicides, lignin degradation,

and plant�pathogen interaction (Baldrian 2006).

In fungi, the shape and cell integrity are dependent of the cell wall, a complex

structure that typically has as main components the polysaccharides chitin and 1,3-b-

and 1,6-b- glucan. During the normal growth, chitinases degrades the chitin present

in the hypha tip, with concomitant insertion of chitin oligomers by chitin synthases.

In similar way, b-1,3-glucanases and b-glucan synthases act together removing and

inserting glucan oligomers in the cell wall. Therefore, chitinases and b-1,3-glucanases

have important role in the break and polymer reconstruction leading to cell wall

remodeling during cell division and morphogenesis processes, such as growth and

hyphal branching, differentiation and germination of spores as well as autolytic

processes (Adams 2004).

Potential applications for biological fumigation by microbial antagonists or their

artificial mixtures of VOCs in closed chambers are currently being investigated and

include the control of a wide range of storage pathogens in fresh fruits as well as

other commodities, such as seeds, grains, and nuts. This process does not require

direct contact with the product and minimizes product handling. Another promising

option includes its use to replace methyl bromide fumigation as a means to control

soil-borne plant diseases (Strobel 2006).

The yeast Saccharomyces cerevisiae strain CR-1, isolated from fermentative

processes for fuel ethanol production, is able to inhibit the mycelial growth of G.

citricarpa. The antagonism was attributed to production of a mixture of VOCs

composed mainly of ethanol, constituting 85% of the headspace, the aliphatic alcohols

3-methyl-1-butanol and 2-methyl-1-butanol, the aromatic alcohol phenylethyl alcohol

and the esters ethyl acetate and ethyl octanoate (Fialho et al. 2010).

The biological fumigation of fruits using S. cerevisiae or artificial mixtures of

VOCs is an attractive alternative method to control the citrus black spot at

postharvest during storage and shipment since the traditional control methods has

been ineffective due to resistance to the limited spectrum of fungicides permitted

for the postharvest management (Adaskaveg, Forster, and Sommer 2002). This

process would be safer to human health and environment as the yeast is classified

as Biosafety Level 1 by U.S. Office of Health and Safety (CDC/OHS 2009), since it

is not a human pathogen, does not produces mycotoxins, antibiotics, or other

molecules whose presence is unacceptable in foods. In addition, all VOCs produced

by the yeast are generally recognized as safe (GRAS) by the American Food and

Drug Administration (FDA 2011). Another advantage is the better acceptance by

consumers, who are familiar with S. cerevisiae widely used in the production of

foods and drinks.

Due to the lack of knowledge about the action mechanisms of antimicrobial

VOCs, this study aimed to investigate the activity of morphogenesis-related enzymes

in G. citricarpa exposed to the artificial mixture of VOCs identified from S. cerevisiae.

Biocontrol Science and Technology 799

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Materials and methods

Phytopathogenic fungus

Guignardia citricarpa, isolated from orange fruit lesions, was maintained in potato

dextrose agar (PDA) at 268C, under fluorescent light and a 12 h L:12 h D

photoperiod. The fungus is deposited as isolate IP-92 in the Mycological Culture

Collection of the Laboratory of Plant Pathology in the Department of Phytosanity at

FCAV/UNESP, in Jaboticabal-SP, Brazil.

Antimicrobial activity of the artificial mixture of VOCs

From the information obtained by Gas Chromatography coupled to Mass

Spectrometric Detection (GC�MS) analysis of the gaseous atmosphere produced

by S. cerevisiae strain CR-1 (Fialho et al. 2010) it was produced an artificial

mixture of VOCs, using authentic standard chemicals (99% ACS reagent grade,

Sigma/Aldrich Chemical Co., St. Louis, USA). The mixture contained the six

compounds positively identified and the proportion of each compound was

calculated from the relative peak areas in relation to all other components of the

mixture (Table 1).

Two section-divided polystyrene plates (BD Falcon, USA) were used to the

bioassays as illustrated in the Figure 1. In one side it was added 10 mL of PDA and

over the medium a semi-permeable membrane (5�5 cm) was placed. On top of the

membrane, a mycelium plug (5 mm) of the pathogen was added. The headspace of

the polystyrene plate was 50 mL and this was used to calculate the concentration of

VOCs per mL of air space. After 5 days of growth, on the opposite side of the plate,

24 mL (0.48 mL mL�1 of air space) of the artificial mixture was added on a piece of

sterile cotton wool. The plates were immediately wrapped with parafilm and

maintained at 268C under a 12 h L: 12 h D photoperiod. The control consisted of

plates containing the pathogen in the absence of the artificial mixture.

After 24, 48, and 72 h of exposure to VOCs the membranes containing the

mycelium were removed from the medium and the biomass harvested, weighed and

stored at �208C. The mycelial growth was also evaluated daily based upon the

average between two opposing measurements of the colonies. All experiments were

carried out in triplicate.

Table 1. VOCs produced by S. cerevisiae strain CR-1 on PDA.

Compound1 % Relative (v/v)

1 Ethanol 85.3

2 Unidentified 1.5

3 Ethyl acetate 1.8

4 3-Methyl-1-butanol 6.9

5 2-Methyl-1-butanol 2.4

6 Phenylethyl alcohol 0.7

7 Ethyl octanoate 1.4

1Identification by Gas Chromatography coupled to Mass Spectrometric Detection (GC�MS) (Fialho et al.2010).

800 M.B. Fialho et al.

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Enzyme extraction

The frozen mycelia were grounded in liquid nitrogen in a cooled mortar and added

100 mM potassium phosphate buffer (pH 7.5) containing 1 mM EDTA and 3 mM

dithiotreitol (5 mL g�1 mycelium). The homogenates were centrifuged at 15,000�g

for 20 min at 48C, and the supernatants were collected and kept at �208C prior the

enzyme analysis. The protein concentration was quantified by the Bradford method

(Bradford 1976), using bovine serum albumin as standard, in order to determine the

specific enzyme activities.

Enzyme assays

The laccase assay employed 0.3 mL of 50 mM citrate-phosphate buffer (pH 5.0), 0.1

mL of syringaldazine as substrate (1 mg mL�1) in ethanol and 0.6 mL of enzyme

extract. The oxidation of syringaldazine was measured by monitoring the absorbance

increase at 525 nm after 10 min of reaction at 308C (Szklars, Antibus, Sinsabaugh,and Linkins 1989).

The tyrosinase activity was assayed using 0.650 mL of 5 mM L-DOPA (3,4-

dihydroxyphenylalanine) as substrate in 100 mM sodium phosphate buffer (pH 6.5)

and 0.1 mL of enzyme extract. The dopaminechrome formation was measured by

monitoring the absorbance increase at 475 nm for 5 min.

The chitinase activity was assayed using of 0.2 mL CM-chitin-RBV as substrate

(4 mg mL�1) and 0.6 mL 100 mM sodium phosphate buffer (pH 6.8). The reaction

was started by addition of 0.2 mL of enzyme extract. After 2 h of incubation at 408C,the reaction was stopped by adding 0.2 mL 1 M HCl followed by centrifugation at

10,000�g for 5 min. The supernatant absorbance was measured at 550 nm.

For b-1,3-glucanase activity the reducing sugars (glucose) released from the

substrate laminarin were quantified by the dinitrosalicylic acid (DNS) method

Figure 1. The schematic illustration shows the geometry of the bioassay system. G. citricarpa

was grown for 5 days in PDA medium, containing a semi-permeable membrane, in one side of

a two section-divided polystyrene plate. On the opposite side of the plate was added 24 mL of

the artificial mixture of VOCs on a cotton wool.

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(Miller 1959), using glucose as standard. In a solution containing 0.15 mL laminarin

(4 mg mL�1) in 100 mM sodium acetate buffer (pH 5.0) 0.1 mL of enzyme extract

was added. After 2 h of incubation at 408C, the reaction was stopped by addition of

0.125 mL of DNS reagent. The solution was boiled for 5 min, cooled and the volumeadjusted to 1.5 mL with distilled water. The absorbance was measured at 540 nm.

Results and discussion

The mycelial growth of G. citricarpa stopped when the artificial mixture was added to

the plates and after 72 h of exposure to VOCs the inhibition was 28.5% compared to

the control (Figure 2), mimicking the inhibitory effects of the S. cerevisiae atmosphere

on the phytopatogen (Fialho et al. 2010). The artificial mixture as the natural VOCs

produced by the yeast had no lethal effect, as the fungal cultures recovered when

removed from the influence of the artificial mixture (data not shown).

Laccases and tyrosinases have an important role in fungal morphogenesis andhave been correlated with mycelium growth and conidia formation. In the present

work, when the fungus was exposed to the VOCs the laccase activity was significantly

reduced when compared to the control (Figure 3a). The effect on tyrosinase activity

was similar however without changes in the first 24 h of exposure to the VOCs

(Figure 3b). After 72 h of exposure to the VOCs the laccase and tyrosinase activities

decreased 46 and 32%, respectively. The role of laccase is well documented mainly in

wood-decaying basidiomycetes of which primary function is to be excreted and to

oxidize the lignin. Intracellular laccases have a role in the transformation of lowmolecular weight phenolic compounds, and are involved in the formation of melanin

and other protective compounds of the cell wall (Baldrian 2006).

Duffy, Schouten, and Raaijmakers (2003) showed that laccases in fungi are

implicated in specific steps of the melanin biosynthesis. These enzymes mediate the

polymerization of the immediate precursor 1,8-dihydroxynaphthalene (DHN) in

DHN-melanin. The DHN has antibiotic properties, therefore, could be speculated

that the negative regulation of laccase activity could result in the accumulation of

DHN, causing as consequence the fungal development reduction. In addition, thelaccases in fungal phytopathogens may be important as virulence factor and

0

1

2

3

4

24h 48h 72h

Myc

elia

l gro

wth

(cm

)

Exposure time

Control Volatiles

** ****

Figure 2. Effect of the artificial mixture of VOCs (0.48 mL mL�1 air space) on mycelial

growth of G. citricarpa after 24, 28, and 72 h of exposure. Values are means of six replicates

(9SD). **Indicates values that differ significantly from the control at P 5 0.01, Tukey’s test.

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protection mechanism against plant defense compounds such as stilbenes, isofla-

vones, coumarins and sesquiterpenes (Mayer and Staples 2002).

Most studies report the effect of non-volatile compounds on laccase and

tyrosinase activity. The compound N-hydroxyglycine produced by Penicillium

citrinum do not inhibit tyrosinase, however, it is a potent inhibitor of laccases in

fungi and plants (Zhang, Kjonaas, and Flurkey 1999). On the other hand, kojic acid,

produced by Aspergillus and Penicillium species, inhibit the tyrosinase activity on

several species of basidiomycetes, Aspergillus and Neurospora crassa (Kim and

Uyama 2005).

The only study that evaluated the effect of VOCs on enzyme production reported

that volatiles produced by soil bacteria inhibited totally the laccase activity in

Phanaerochaete magnoliae and decreased significantly the activity in Trichoderma

viride. The tyrosinase activity in T. viride was not affected by any of the bacterial

isolates, but the activity in P. magnoliae was increased, inhibited or unaffected

depending on the bacteria to which it was exposed. Growth rates of some fungi were

inhibited by up to 60% in some cases (Mackie and Wheatley 1999).

In the present work, the degree of inhibition caused by the VOCs was lower on

the enzymes chitinase and b-1,3-glucanase if compared to inhibition caused on the

enzymes laccase and tyrosinase. The chitinase activity was significantly reduced, 15

and 17% after 24 and 72 h of exposure, respectively (Figure 4a). The b-1,3-glucanase

activity increased 33% after 24 h of exposure to VOCs, however the activity

decreased 13% after 72 h (Figure 4b).

0

10

20

30

40

50(a)

(b)

Lacc

ase

act

ivity

(mU

mg–1

pro

tein

)**

**

**

0

20

40

60

80

100

120

24h 48h 72h

Tyr

osin

ase

act

ivity

(U m

g–1 p

rote

in)

Exposure time

Control Volatiles

**

**

Figure 3. Effect of the artificial mixture of VOCs (0.48 mL mL�1 air space) on laccase (a) and

tyrosinase (b) activity of G. citricarpa after 24, 48, and 72 h of exposure. Values are means of

three replicates (9SD). **Indicates values that differ significantly from the control at P 5

0.01, Tukey’s test.

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The cell wall structure is highly dynamic and subject to constant changes, such as

hyphal branching, septum formation and spore germination. The constituents of the

wall polymers, especially chitin and b-1,3-glucan, form a complex cross network so

that plasticity maintenance during morphogenesis is dependent on the activity of

enzymes such as chitinases and b-1,3-glucanases. The function and regulation of

genes related to chitinolitic and glucanolitic activity are well known in S. cerevisiae,

A. fumigatus, Coccidioides posadasii, and C. immitis. Many of these enzymes were

associated with the cell wall remodeling during the morphogenesis (Adams 2004).

Chitinases are found with chitin synthases in the mycelium in active phase of

growth. When the chiA gene, coding for a chitinase in A. fumigatus, was

interrupted, the frequency of sporulation and mycelial growth rate were reduced

(Takaya et al. 1998). The trisaccharide allosamidin, a potent inhibitor of several

fungal chitinases, had fungistatic action on P. chrysogenum, inhibiting the hyphal

tip development (Sami et al. 2001).In S. cerevisiae, the gene gas1 coding for b-1,3-glucanases is expressed during the

vegetative growth. The interruption of the gene reduces the cross-connection between

the b-1,3-glucan polymers and other cell wall constituents and causes several

morphological defects (Popolo and Vai 1999). Disruption of the gene encoding an

enzyme with b-1,3-glucanase activity in C. immitis led to reduction of the mycelial

growth and development rate during the parasitic phase. Furthermore, there is

drastic virulence reduction in its host (Cole and Hung 2001).

0.00

0.05

0.10

0.15

0.20

0.25

0.30(a)

(b)

Chi

tinas

e a

ctiv

ity(A

bs 5

50 h

–1 m

g–1 p

rote

in)

**

*

0.00

0.05

0.10

0.15

0.20

0.25

0.30

24h 48h 72h

Glu

cans

e a

ctiv

ity(µ

g gl

ucos

e h–1

mg–1

pro

tein

)

Exposure time

Control Volatiles

**

**

Figure 4. Effect of artificial mixture of VOCs (0.48 mL mL�1 air space) on chitinase (a) and

b-1,3-glucanase (b) activity by G. citricarpa after 24, 48, and 72 h of exposure. The values are

means of three replicates (9SD). **, * Indicates values that differ significantly from the

control at P 5 0.01 and P 5 0.05, respectively, Tukey’s test.

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In the present work, the increase of the b-1,3-glucanase activity in the first 24 h of

exposure may be related to autolysis processes. During autolysis, the activity of lytic

enzymes rises substantially, particularly b-1,3-glucanases and chitinases, able to

hydrolyze the cell wall polysaccharides. The autolysis may occur due to intrinsic

factors such as culture age and programmed cell death. In addition, extrinsic factors

such as limiting conditions of oxygen and nutrients and physical stress can also

trigger the process (White, McIntyre, Berry, and McNeil 2002).

The exposure of G. citricarpa to alcohols, the main components of the mixture of

VOCs, may reduce glucose assimilation by the cells (Jacobsen 1995). Thus, an initial

response of the fungus to reduced availability of carbon source could be the autolysis

which is also considered a strategy for survival, with parts of the culture surviving by

recycling the lysis products released by hydrolases. Therefore, b-1,3-glucanases can

break the b-1,3-glucan in the cell wall and release glucose monomers, which can be

used as carbon source for some time (White et al. 2002).

As it is known, there is a complex relationship between fungal development and

enzyme production (Mackie and Wheatley 1999). It was verified in the present

work that the exposure of G. citricarpa to the VOCs can influence the mycelial

growth rate and the activity of morphogenesis-related enzymes. More studies are

necessary to know the action mechanisms of inhibitory VOCs to allow an

optimized handling of this characteristic in the alternative control of phytopatho-

gens and to understand the role of the volatile metabolites on the interactions

among microorganisms in the nature.

Acknowledgements

This research was supported by CAPES (Coordination for the Improvement of HigherEducation Personnel), a Brazilian foundation within the Ministry of Education and by CNPq(National Council for Scientific and Technological Development), a Brazilian foundationassociated to the Ministry of Science and Technology.

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