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Biocontrol Science and Technology

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Antimicrobial volatile organic compounds affect morphogenesisrelated enzymes in Guignardia citricarpa, causal agent of citrus black spot
Mauricio Batista Fialho , Luiz Fernando Romanholo Ferreira , Regina Teresa Rosim Monteiro & Srgio Florentino Pascholati
a b a a b

Department of Plant Pathology and Nematology, Luiz de Queiroz College of Agriculture, University of So Paulo, CP 09, CEP 13418-900, Piracicaba, SP, Brazil
b

Center for Nuclear Energy in Agriculture, University of So Paulo, 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 Teresa Rosim Monteiro & Srgio Florentino Pascholati (2011): Antimicrobial volatile organic compounds affect morphogenesis-related enzymes in Guignardia citricarpa, causal agent of citrus black spot, Biocontrol Science and Technology, 21:7, 797-807 To link to this article: http://dx.doi.org/10.1080/09583157.2011.580837

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Biocontrol Science and Technology, Vol. 21, No. 7, July 2011, 797807

RESEARCH ARTICLE Antimicrobial volatile organic compounds affect morphogenesis-related enzymes in Guignardia citricarpa, causal agent of citrus black spot
Mauricio Batista Fialhoa, Luiz Fernando Romanholo Ferreirab, Regina Teresa Rosim Monteirob and Sergio Florentino Pascholatia*
a Department 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 Nuclear Energy in Agriculture, University of Sao Paulo, CP 96, CEP 13400-970, Piracicaba, SP, Brazil

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(Received 13 December 2010; returned 22 February 2011; accepted 10 April 2011) Although non-volatile substances toxic to plant pathogenic microorganisms have been extensively studied over the years, few studies have focused on microbial volatile organic compounds (VOCs). The VOCs produced by the yeast Saccharomyces cerevisiae 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 the development of fungi is not known. Thus, the objective of the present work was to study the effect of the artificial mixture of VOCs identified from S. cerevisiae on intracellular enzymes involved in the mycelial morphogenesis in G. citricarpa. The phytopathogenic fungus was exposed to artificial mixture of VOCs constituted by alcohols (ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol and phenylethyl alcohol) and esters (ethyl acetate and ethyl octanoate) in the proportions naturally found in the atmosphere produced by the yeast. The VOCs inhibited considerably the mycelial development and interfered negatively with the production of the morphogenesisrelated enzymes. After 72 h of exposure to the VOCs the laccase and tyrosinase activities decreased 46 and 32%, respectively, however, the effect on the chitinase and b-1,3-glucanase activities was lower, 17 and 13% of inhibition, respectively. Therefore, the exposure of the fungus to the antimicrobial volatiles can influence both fungal mycelial growth rate and activity of enzymes implicated in morphogenesis. This knowledge is important to understand the microbial interactions mediated by VOCs in nature and to develop new strategies to control plant pathogens as G. citricarpa in postharvest. Keywords: antimicrobial activity; biocontrol; Citrus; morphogenesis; plant disease

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
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 plantpathogen interaction, microbial antagonists may interrupt some stage of the disease or the pathogens 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.

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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 plantpathogen 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-band 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.

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800 Materials and methods Phytopathogenic fungus

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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

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From the information obtained by Gas Chromatography coupled to Mass Spectrometric Detection (GCMS) 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 1 2 3 4 5 6 7
1

% Relative (v/v) 85.3 1.5 1.8 6.9 2.4 0.7 1.4

Ethanol Unidentified Ethyl acetate 3-Methyl-1-butanol 2-Methyl-1-butanol Phenylethyl alcohol Ethyl octanoate

Identification by Gas Chromatography coupled to Mass Spectrometric Detection (GCMS) (Fialho et al. 2010).

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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 articial mixture of VOCs on a cotton wool.

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 (3,4dihydroxyphenylalanine) 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

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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 volume adjusted 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 and have 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 low molecular 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, the laccases in fungal phytopathogens may be important as virulence factor and
4

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Mycelial growth (cm)

3 ** 2 1 0 24h 48h Exposure time

Control Volatiles

**

**

72h

Figure 2. Effect of the articial 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 signicantly from the control at P 5 0.01, Tukeys test.

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Laccase activity (mU mg1 protein)
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(b)
Tyrosinase activity (U mg1 protein)

120 100 80 60 40 20 0 24h 48h Exposure time

Control Volatiles

**

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**

72h

Figure 3. Effect of the articial 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 signicantly from the control at P 5 0.01, Tukeys test.

protection mechanism against plant defense compounds such as stilbenes, isoflavones, 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).

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Chitinase activity (Abs 550 h1 mg1 protein) 0.30 0.25 0.20 0.15 0.10 0.05 0.00

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** *

(b)
Glucanse activity (g glucose h1 mg1 protein) 0.30 0.25 0.20 0.15 0.10 0.05 0.00 24h 48h Exposure time
Control Volatiles

** **

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Figure 4. Effect of articial 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 signicantly from the control at P 5 0.01 and P 5 0.05, respectively, Tukeys test.

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).

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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 phytopathogens 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 Higher Education Personnel), a Brazilian foundation within the Ministry of Education and by CNPq (National Council for Scientific and Technological Development), a Brazilian foundation associated to the Ministry of Science and Technology.

References
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