Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil

International Journal of Food Microbiology 145 (2011) 9–21

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o

Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil Liliana de Oliveira Rocha a,⁎, Gabriela Martins Reis a, Valéria Nascimento da Silva a, Raquel Braghini a, Marta Maria Geraldes Teixeira b, Benedito Corrêa a a b

Department of Microbiology, Biomedical Institute II, University of São Paulo, Prof. Lineu Prestes, 1374, Laboratory 249, São Paulo, Brazil Department of Parasitology, Biomedical Institute II, University of São Paulo, Prof. Lineu Prestes, 1374, Laboratory 107, São Paulo, Brazil

a r t i c l e

i n f o

Article history: Received 19 May 2010 Received in revised form 5 October 2010 Accepted 3 November 2010 Keywords: Fusarium verticillioides Fumonisins Sequencing AFLP Real time RT-PCR

a b s t r a c t Gibberella moniliformis is most commonly associated with maize worldwide and produces high levels of fumonisins, some of the most agriculturally important mycotoxins. Studies demonstrate that molecular methods can be helpful for a rapid identification of Fusarium species and their levels of toxin production. The purpose of this research was to apply molecular methods (AFLP, TEF-1α partial gene sequencing and PCR based on MAT alleles) for the identification of Fusarium species isolated from Brazilian corn and to verify if real time RT-PCR technique based on FUM1 and FUM19 genes is appropriated to estimate fumonisins B1 and B2 production levels. Among the isolated strains, 96 were identified as Fusarium verticillioides, and four as other Fusarium species. Concordant phylogenies were obtained by AFLP and TEF-1α sequencing, permitting the classification of the different species into distinct clades. Concerning MAT alleles, 70% of the F. verticillioides isolates carried the MAT-1 and 30% MAT-2. A significant correlation was observed between the expression of the genes and toxin production r = 0.95 and r = 0.79 (correlation of FUM1 with FB1 and FB2, respectively, P b 0.0001); r = 0.93 and r = 0.78 (correlation of FUM19 with FB1 and FB2, respectively, P b 0.0001). Molecular methods used in this study were found to be useful for the rapid identification of Fusarium species. The high and significant correlation between FUM1 and FUM19 expression and fumonisins production suggests that real time RT-PCR is suitable for studies considering the influence of abiotic and biotic factors on expression of these genes. This is the first report concerning the expression of fumonisin biosynthetic genes in Fusarium strains isolated from Brazilian agricultural commodity. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Fumonisins are a group of mycotoxins discovered in 1988 in cultures of Fusarium verticillioides (syn. F. moniliforme, teleomorph Gibberella fujikuroi mating population A, or Gibberella moniliformis) isolated from corn in South Africa, which are associated with several mycotoxicosis (Bezuidenhout et al., 1988; Gelderblom et al., 1988). Twenty-eight analogs have been described so far, including fumonisins B1 (FB1), B2 (FB2) and B3 (FB3) that are naturally produced. FB1 is the most important of the group, accounting for 70% of all fumonisins found in culture and in naturally contaminated corn (Rheeder et al., 2002). Fumonisins are synthesized from a structure that is characteristic of sphinganine, an intermediate in the biosynthesis of sphingolipids. This similarity of fumonisins with the sphinganine molecule has led some investigators to believe that the mechanism of action of these

⁎ Corresponding author. Departamento de Microbiologia, Instituto de Ciências Biomédicas, n. 1374, Lab. 249, Cidade Universitária, São Paulo, Brazil. Tel.: +55 11 30917295; fax: +55 11 30917354. E-mail address: [email protected] (L. de Oliveira Rocha). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.11.001

toxins is related to the inhibition of sphingolipid mechanism, with serious consequences for the cell (Sweeley, 1991; Wang et al., 1991). Fumonisins cause mycotoxicosis in humans and animals, and FB1 is classified as a group 2B carcinogen by the IARC (1993). In Brazil, studies regarding the occurrence of FB1 and FB2 in freshly harvested corn detected these mycotoxins in approximately 92% and 81% of samples, respectively, with average levels ranging from 0.02 to 79 μg/g for FB1 and from 0.02 to 29 μg/g for FB2, these findings demonstrate the need for fumonisin control in corn grains from Brazil (Moreno et al., 2009; Rocha et al., 2009; Van der Westhuizen et al., 2003; Rodríguez-Amaya and Sabino, 2002). Fumonisins are produced by the G. fujikuroi species complex (Nelson et al., 1991; Leslie et al., 1996; Desjardins et al., 1995). This complex colonizes a series of agriculturally important plants and thus poses a serious risk to food safety. The taxonomy of this complex is extremely controversial and some species cannot be differentiated morphologically (Leslie and Summerell, 2006). An alternative taxonomic system recognizes nine different biological species (mating populations) within the Gibberella fujikuroi complex and it is based on the sexual crossing between two different mating types. Two isolates are cross-fertile if they carry different mating type idiomorphs (MAT-1 and MAT-2) (Kerényi et al.,

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L. de Oliveira Rocha et al. / International Journal of Food Microbiology 145 (2011) 9–21

1999; Leslie, 1999; Steenkamp et al., 2000; Leslie and Summerell, 2006). Some difficulties exist in the application of this concept to Fusarium because of the high frequency of asexual reproduction, the unequal relative frequency of the MAT-1 and MAT-2 alleles, and the limited number of female fertile strains. In heterothallic Fusarium species, the female structure (protoperithecium) is used only for sexual reproduction. This structure remains immature until it is fertilized, and a perithecium is produced. Male structures are not specialized (macroconidia, microconidia, ascospores or hyphal fragments). Successful sexual cross and subsequent induction of perithecial differentiation and development depends on the female and male nucleus carrying the opposite mating types alleles. Hence, the identification of these alleles and of the female fertile strains demonstrates the possibility of crossing in the field (Leslie and Summerell, 2006; Silva et al., 2006; Leslie and Klein, 1996). Traditional methods for the differentiation of Fusarium species are time consuming and require a high level of knowledge. In this respect, DNA-based methods may contribute to the recognition of these isolates and to the identification of morphologically indistinguishable species. Molecular techniques have been used successfully for identification of some fungal species and for detection of mycotoxin biosynthetic genes (Jurado et al., 2010). Brazil is currently the third largest producer of corn in the world (Conab, 2009). In view of this fact, associated with the high frequency of F. verticillioides and their high levels of fumonisins production in maize, this research was intended to analyze molecular methods applied for Fusarium species identification in order to characterize isolated strains from corn of different origins in Brazil, and to verify if real time RT-PCR based on FUM1 and FUM19 genes is appropriate to estimate the level of fumonisins production. The aims of this study were (i) to identify F. verticillioides strains isolated from freshly harvested corn grains in four regions of Brazil by amplified fragment length polymorphism (AFLP) and partial sequencing of the TEF-1α gene; (ii) to characterize the MAT-1 and MAT-2 alleles by PCR; (iii) to analyze the fumonisin-producing ability of F. verticillioides and F. proliferatum in relation to geographic origin, and (iv) to quantify the expression of FUM1 and FUM19 genes by real time RT-PCR and correlate the level of transcripts with fumonisins B1 and B2 production by F. verticillioides and F. proliferatum.

2. Materials and methods 2.1. Isolation of Fusarium species from corn grains A total of 100 Fusarium strains were isolated from 200 samples of corn grains (Agromen 2012 hybrid, AGN 2012) freshly harvested in four different regions of Brazil: Várzea Grande (Mato Grosso — MT), Nova Odessa (São Paulo — SP), Santa Maria (Rio Grande do Sul — RS), and Oliveira dos Campinhos (Recôncavo Baiano, Bahia — BA). The isolates were obtained according to the method of Berjak (1984) (Tables 1–2).Approximately 30 g was removed from each corn subsample (1 kg) for disinfection in 2% sodium hypochlorite solution for 3 min. This procedure is used to eliminate external contaminants. After disinfection, the grains were washed three times with sterile distilled water. Thirty-three grains were selected randomly and seeded directly on a Petri dishes containing DRBC Agar (DichloranRose Bengal Chloramphenicol Agar) (Pitt et al., 1979). Three plates containing 11 grains were used for each sample. The plates were incubated at 25 °C for 5 days. The Fusarium colonies were then transferred to potato agar and Spezieller Nährstoffarmer Agar (SNA) and incubated at 25 °C for 5–7 days under white and black light (12 h/day). The colonies were identified according to the nomenclature proposed by Leslie and Summerell (2006). Colonies belonging to the genus Fusarium were lyophilized and also maintained in SNA medium.

2.2. Genomic DNA extraction from the strains For the production of fungal biomass, 5 × 106 spores of each strain (counted in a Neubauer chamber) were inoculated into an Erlenmeyer flask (250 mL) containing 50 mL of Czapek broth (Oxoid) and incubated for 3 days with shaking. After this period, the content of the flasks was filtered aseptically through a filter paper (Whatman grade 4), washed twice with sterile water, transferred to 1.5 mL microtubes, and stored at −20 °C. Next, genomic DNA was extracted using the Easy-DNA kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. 2.3. Identification of the isolates by AFLP The AFLPTM Microbial Fingerprinting kit (Applied Biosystems, Foster City, CA, USA) was used. The instructions provided by the manufacturer were adapted as proposed by Leslie and Summerell (2006) and Moretti et al. (2004). Genomic DNA was digested at 37 °C for 3 h in a final volume of 10 μL containing 1 U EcoRI, 1 U MseI, 1× MseI buffer, and 250 ng/μL DNA. For ligation of the primer adapters to the digested DNA: 1× MseI buffer, 1× T4 DNA ligase buffer, 0.2 μM EcoRI adapters, 2 μM MseI adapters, 6.8 U/μL T4 DNA ligase, and 0.1 μg/μL BSA in a final volume of 10 μL were added to 10 μL of the restriction reaction and the mixture was incubated at 16 °C for 4 h. After incubation, DNA was diluted 10× with TE buffer (20 mM Tris–HCl, 0.1 mM EDTA, pH 8.0), according to the instructions of AFLP Microbial Fingerprinting kit (Applied Biosystems). The preamplification and selective amplification reactions were carried out according to the instructions of the AFLP Microbial Fingerprinting kit. FAM dye-labeled EcoRI + AC and MseI + CC or CT primers were used for selective amplification. The reaction products (1.5 μL) were mixed with 25 μL deionized formamide and 1 μL GeneScan-500 (ROX) size standard. The samples were heated to 95 °C for 5 min and then immediately placed on ice. The fragments were separated in an ABI Prism 310 Genetic Analyzer using POP-4 polymer (Applied Biosystems, Foster City, CA, USA). An injection time of 12 s, voltage of 7.5 kV and run time of 30 min were used. The data were transferred to the STRand program, version 2.2.30 (Veterinary Genetics Laboratory, University of California, Davis) for visualization of the fragments and analyzed according to the instructions of the program (Toonen and Hughes, 2001). The fragments generated were counted manually for each of the two primer combinations (EcoRI AC+ MseI CC and EcoRI AC+ MseI CT). The genetic similarity between the isolates and the F. verticillioides MATA-1 (MRC 8559) reference strain (South African Medical Research Council) was calculated according to Nei and Li (1979). Mathematically, Sij = 2a/(2a + b + c), where Sij is the similarity between two individuals i and j; a corresponds to the number of fragments shared by i and j; b is the number of fragments present in i and absent in j, and c is the number of fragments absent in i and present in j. 2.4. Identification of the strains by TEF-1α partial gene sequencing The morphological identification was confirmed by TEF-1α partial gene sequencing using the ef-1/ef-2 (≈650 bp) forward and reverse primers as described by Geiser et al. (2004). Sequencing was performed using the BigDye Terminator v3.1. kit (Applied Biosystems, Foster City, CA, USA) according to manufacturer's instructions in a 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences were edited with the BioEdit v7.0.9.0 software (www. mbio.ncsu.edu/BioEdit/BioEdit.html) and then aligned to GenBank sequences (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and to sequences in the Fusarium ID v1.0 database (http://isolate.fusariumdb.org/blast. php) (Geiser et al., 2004). Multiple alignments were performed with the ClustalX2 v2.0.11 program and then manually adjusted to obtain positional homology. The best model of nucleotide substitutions was

Table 1 Fusarium strains isolated in the regions of São Paulo and Rio Grande do Sul and the respective molecular identification of species, fumonisin production, identification of MAT alleles and relative expression of FUM-1 and FUM-19. The values of expression levels represent the times that FUM1 and FUM19 is expressed in each sample compared to 45BA strain. Origin: Rio Grande do Sul

Isolate

Species

Concentration (μg/g) FB1 FB2

MAT alleles

FUM-1

FUM-19

Isolate

Species

Concentration (μg/g) FB1 FB2

01 03 06 08 10 11 14 16 21 22 24 25 27 28 30 32 34 36 37 38 41 42 46 47 48

F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides Fusarium sp F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides

1.0 12 0.8 0.6 1.8 98 0.8 0.2 0.4 0.5 4.6 29 5.4 1.4 16 16 ND 23 0.9 115 0.6 1.1 156 1.5 0.8

MAT-1 MAT-2 MAT-1 MAT-1 MAT-2 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-2 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1

0.9 3.0 0.9 1.3 2.3 4.9 0.9 0.7 0.9 0.7 1.5 3.7 2.0 2.3 3.2 3.7 – 3.3 1.3 6.1 1.9 1.8 6.3 1.2 0.9

1.9 3.3 0.9 2.8 2.6 5.0 0.9 1.3 1.7 1.7 2.0 4.9 0.6 0.7 1.1 0.9 – 4.5 0.6 6.5 0.6 2.8 7.2 2.0 0.9

01 02 03 04 05 08 11 12 17 19 20 21 26 27 28 29 31 33 35 36 38 40 46 47 49

F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides

0.5 1.5 4.1 0.7 246 0.7 3.8 23 10 218 0.8 64 185 1.3 0.2 243 2.1 3.7 294 0.4 8.7 10 2.1 36 0.3

0.02 3.8 ND 0.03 0.3 97 0.2 0.1 0.2 ND 2.4 12 0.1 0.1 6.2 0.9 ND 7.8 0.04 47 0.2 0.1 52 0.2 0.02

0.2 0.5 1.2 0.1 155 0.1 ND 10 3.2 94 0.1 22 101 0.3 0.02 2.5 0.3 1.2 76 0.1 1.7 4.8 0.1 18 0.1

MAT alleles

FUM-1

FUM-19

MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-2 MAT-2 MAT-1 MAT-2 MAT-2 MAT-1 MAT-1 MAT-1 MAT-2 MAT-1 MAT-1 MAT-2 MAT-1 MAT-2 MAT-2 MAT-2 MAT-1 MAT-2 MAT-1 MAT-1

0.8 2.0 3.5 2.0 0.9 3.0 8.6 0.9 0.9 1.6 2.0 2.1 8.7 4.8 3.3 0.6 6.1 1.7 0.7 8.0 2.1 3.0 9.0 3.0 0.8

1.6 2.1 3.0 2.8 0.9 4.0 9.2 0.9 1.6 2.5 2.7 3.5 7.9 5.7 2.6 0.7 7.0 2.1 0.8 8.9 3.2 3.8 9.7 3.8 0.9

L. de Oliveira Rocha et al. / International Journal of Food Microbiology 145 (2011) 9–21

Origin: São Paulo

ND: not detected; detection limit: 0.015 μg/g for FB1 and FB2.

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12 Table 2 Fusarium strains isolated in the regions of Mato Grosso and Bahia and the respective molecular identification of species, fumonsisin production, identification of MAT alleles and relative expression of FUM-1 and FUM-19 .The values of expression levels represent the times that FUM1 and FUM19 is expressed in each sample compared to 45BA strain (set at 1.0). Origin: Bahia

Isolate

Species

Concentration (μg/g) FB1 FB2

03 05 06 08 09 11 12 13 14 15 23 24 30 31 32 33 35 36 40 41 43 44 45 48 49

F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides

0.8 1.1 4.1 1.8 1.1 0.6 265 1.1 76 1.3 19 283 15 1.2 0.8 2.2 1.7 1.3 2.0 63 120 13 1.6 13 2.7

ND 0.04 1.0 ND 0.1 0.2 0.7 0.02 20 0.3 17 19 3.1 0.04 ND ND ND 0.03 ND 15 27 3.1 0.04 3.4 0.02

MAT alleles

FUM-1

FUM-19

Isolate

Species

Concentration (μg/g) FB1 FB2

MAT alleles

MAT-1 MAT-2 MAT-1 MAT-2 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-2 MAT-2 MAT-1 MAT-2 MAT-2 MAT-2 MAT-2 MAT-1 MAT-2 MAT-2 MAT-2 MAT-2

1.2 1.5 1.6 1.5 1.8 0.9 9.9 1.1 4.1 0.8 2.6 10.6 3.1 0.8 0.9 1.4 1.1 1.6 1.4 5.7 7.5 4.3 1.3 4.3 1.4

1.6 2.5 3.2 2.3 2.2 0.8 8.5 0.9 5.6 0.9 3.5 9.1 4.0 1.3 0.8 1.7 1.7 1.9 1.7 6.0 8.6 5.2 1.3 3.5 1.5

02 03 04 12 13 14 15 16 18 19 21 22 26 27 29 35 36 37 39 40 42 43 45a 47 50

F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. proliferatum F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. sacchari F. proliferatum F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides

0.4 70 0.3 2.3 123 18 6.2 300 3.6 38 3.7 4.0 71 227 11 ND 139 5.7 762 282 212 5.7 0.4 50 6.1

MAT-1 MAT-1 MAT-1 MAT-2 MAT-1 MAT-1 MAT-1 MAT-1 MAT-2 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-1 MAT-2 MAT-1 MAT-2 MAT-1

ND: not detected; detection limit: 0.015 μg/g for FB1 and FB2. a Calibrator strain used for relative quantification of FUM1 and FUM19 genes.

0.1 18 ND 0.4 35 4.5 0.2 68 0.1 1.1 0.3 0.4 24 56 4.0 ND 76 3.1 165 110 58 3.1 ND 16 2.0

FUM-1

0.9 4.8 1.2 0.9 5.6 2.8 1.5 9.3 0.9 3.3 2.5 2.8 5.0 8.3 2.3 – 6.3 2.2 21.6 8.6 7.0 2.3 1 4.7 2.8

FUM-19

0.9 4.2 1.6 2.0 5.0 3.0 2.5 8.6 1.7 4.3 2.9 3.6 4.6 9.8 2.4 – 7.6 2.3 22.2 7.9 7.4 3.4 1 5.9 3.4

L. de Oliveira Rocha et al. / International Journal of Food Microbiology 145 (2011) 9–21

Origin: Mato Grosso

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selected using the Modeltest v3.7 program. The partial sequences of the TEF-1α gene of the F. verticillioides, F. proliferatum and F. sacchari isolates were deposited in GenBank of the National Center for Biotechnology Information (NCBI) under the accession numbers: from GU989096 to GU989198. 2.5. Phylogenetic analysis Phylogenetic analysis was performed separately based on the AFLP markers and the partial sequence of the TEF-1α gene using PAUP 4.0b10 program. The strains used in the phylogenetic analysis were isolated from corn grains of different geographic origins (Table 1). F. verticillioides MAT-A1 (MRC 8559) and MAT-A2 (MRC 8560) reference strains obtained from the South African Medical Research Council and a Fusarium oxysporum strain kept at the collection of the Department of Mycology, Institute of Biomedical Sciences, USP, were included. The polymorphic fragments obtained by AFLP were used to construct a binary matrix according to the presence or absence of fragments in the different isolates, with 0 = absence of band and 1 = presence of band. The similarity matrix was analyzed by the distance method using a neighbor-joining algorithm available in the PAUP 4.0b10 phylogenetic analysis package (Swofford, 1998). The support of individual nodes was assessed by bootstrap analysis for 1000 replicates. The maximum likelihood method under an HKY85 + G model was used for analysis of the markers generated by partial sequencing of the TEF-1α gene. The support of individual nodes was assessed by bootstrap analysis for 1000 replicates. 2.6. MAT-1 and MAT-2 genes amplification by PCR The mating types (MAT-1 and MAT-2) of the isolates were identified by PCR using the primers Gfmat1a/Gfmat1b and Gfmat1c/ Gfmat1d as described by Steenkamp et al. (2000). The annealing temperature for hybridization of the primers used for amplification of MAT-2 was adjusted to 55 °C. The MAT-1 allele corresponds to a fragment of approximately 200 bp and the MAT-2 allele to a fragment of 800 bp. 2.7. Production of fumonisins B1 and B2 by the strains A 0.1 mL aliquot of a spore suspension of the respective strains was seeded onto Petri dishes containing potato dextrose agar (Oxoid) and incubated at 25 °C for 7 days. After growth, a disk measuring about 1.5 cm in diameter was cut from the agar, inoculated into an Erlenmeyer flask containing 50 g sterile rice, and incubated at 25 °C for 15 days, followed by an additional 15 days at 15 °C (Ross et al., 1990). Fumonisins were extracted as described by Sydenham et al. (1990) and Shephard et al. (1990), with minor modifications, with 100 mL of methanol and water (3:1, v/v), followed by shaking for 45 min. The samples were filtered through Whatman grade 4 (12 cm) filter paper and the pH was corrected to 5.8–6.5 with 1 N NaOH, if necessary. Fumonisins were purified by transferring 10 mL of the filtrate to a minicolumn containing 500 mg ion-exchange silica (BondElut SAX — Varian, Palo Alto, CA, USA), previously conditioned with 5 mL methanol and 5 mL methanol:water (3:1, v/v), at a flow rate of 1 mL/min. Next, fumonisins were eluted with 15 mL methanol: acetic acid (99:1, v/v) maintaining the same flow rate. The product was evaporated and the residue was separated by HPLC. The residue was resuspended in 1 mL acetonitrile:water (50:50, v/v). A 50 μL aliquot of the extract of the sample was diluted in 50 μL OPA reagent (40 mg ortho-phthalaldehyde dissolved in 1 mL methanol, then diluted in 5 mL 0.1 M sodium tetraborate solution and supplemented with 50 μL mercaptoethanol). The solution was shaken for 30 s. Two minutes after the addition of OPA, the solution was

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injected into a Shimadzu LC-10AD liquid chromatograph equipped with a 20 μL fixed loop injector (Rheodyne). After separation on a C-18 reverse phase column (Phenomenex, 5 ODS-20, 150 × 4.6 mm), fumonisins were detected with a fluorescence detector (model RF-10AXL) of excitation and emission wavelengths of 335 and 440 nm, respectively. A system of acetonitrile:water:acetic acid (480:520:5, v/v/v) was used as the mobile phase. The flow rate was 1.0 mL/min, the column temperature was 30 °C, and room temperature was maintained at 22–23 °C. The retention time of FB1 and FB2 under these conditions is 9 and 20 min, respectively. A calibration curve was used for fumonisin quantification, with a detection limit of the 0.015 μg/g for FB1 and FB2. 2.8. Real time RT-PCR for the detection and quantification of the FUM1 and FUM19 genes in F. verticillioides and F. proliferatum 2.8.1. Isolation of mRNA and reverse transcription Total RNA was extracted from F. verticillioides and F. proliferatum cultures grown in liquid Czapek medium for 7 days at 25 °C using the RNeasy kit (Qiagen, Valencia, CA, USA) according to manufacturer instructions. cDNA was synthesized with the Sensiscript RT kit (Qiagen, Valencia, CA, USA). The reaction mixture contained 10 μL total RNA, 2 μL random primers (3 μg/μL; Invitrogen, Carlsbad, CA, USA), 2 μL 10× RT-PCR buffer, 2 μL dNTP mix, 1 μL RNAse inhibitor (40 U/μL; Invitrogen, Carlsbad, CA, USA), and 1 μL Sensiscript Reverse Transcriptase in a volume of 20 μL. cDNA was synthesized in a Gene Amp® PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA) for 1 h at 37 °C. The samples were stored at −20 °C. 2.8.2. Real time RT-PCR The following primers were used for real time PCR of the FUM1 and FUM19 genes of the F. verticillioides and F. proliferatum isolates: FUM1: PQF1-F (5′-GAGCCGAGTCAGCAAGGATT-3′) and PQF1-R (5′AGGGTTCGTGAGCCAAGGA-3′); FUM19: PQF19-F (5′-ATCAGCATCGGTAACGCTTATGA-3′) and PQF19-R (5′-CGCTTGAAGAGCTCCTGGAT-3′) (López-Errasquín et al., 2007). The amplification conditions were as follows: 50 °C for 2 min, 95 °C for 2 min, 40 cycles at 95 °C for 15 s and 60 °C for 30 s. The Platinum SYBR Green qPCR SuperMix-UDG reagent (Invitrogen, Carlsbad, CA, USA) was used as the reaction mixture. The mixture contained 3 μL sterile Milli-Q water, 2.0 μL of each primer, 5 μL template cDNA, 12.5 μL of the SYBR reagent, and 0.5 μL ROX in a final volume of 25 μL. Each sample was amplified in duplicate. An appropriate negative control containing no template was used in all reactions to exclude possible contaminations. The quantification of mRNA was normalized using TUB (β-tubulin gene) cDNA amplifications run on the same plate. The following primer pairs were used: TUB1-F (5′-CCGGTATGGGTACTCTGCTC-3′) and TUB2-R (5′-CTCAACGACGGTGTCAGAGA-3′), designed from the U27303.1 sequence deposited at NCBI (http://www.ncbi.nlm.nih.gov/) using the Primer 3 program (http://frodo.wi.mit.edu/primer3/). Relative quantification based on ΔΔCt values was the analytical method of choice in this study, since the efficiencies of compatibility tests between the endogenous control (TUB) and the target genes (FUM1 and FUM19) were highly similar, with slopes between −0.1 and 0.1 (Pfaffl, 2001; Schmittgen and Livak, 2008). In this method, a comparison within a sample is made between the gene of interest (FUM1 and FUM19) and the endogenous control gene (TUB). Quantification is relative to the control gene by subtracting the Ct of the endogenous control gene (TUB) from the Ct of the gene of interest (ΔCt). Each ΔCt value (corresponding to each sample) was subtracted by a calibrator value (in this study strain 45BA was chosen, which presented the lowest Ct values for the FUM1 and FUM19 genes) to obtain the corresponding ΔΔCt values. ΔΔCt values were transformed to log2 to generate the relative expression levels (Gizinger, 2002). Relative quantification based on ΔΔCt values is used to compare the gene expression of one sample (or a strain) in relation to another

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(control strain). The control could be an isolate with the lowest level of mRNA (lowest Ct value) of the target gene or a reference strain and even a non treated strain (in the case of susceptibility drugs tests). In this method, it is not necessary to use a calibration curve, if the efficiencies of the endogenous control and target genes are compatible. Moreover, because the time consuming quantification models based on calibration curves and the difficulties in relation to purity and stability of the mRNA standards, relative quantification is an ideal and simple tool for the cDNA quantification (Gizinger, 2002; Pfaffl, 2001). For validation of the method, serial dilutions of the target genes (FUM1 and FUM19) and of the endogenous control gene (TUB) were prepared in triplicate. The primers efficiency was calculated based on the curve generated by the software using the formula: E = 10 (− 1/slope) − 1. According to European Network of GMO Laboratories, the acceptance criterion for the slope of the standard curve must range from −3.1 to −3.6 (ENGL, 2008). 2.8.3. Statistical analysis Pearson's correlation coefficient was used for analysis of the correlation between the relative expression levels of FUM1 and FUM19 and production of FB1 and FB2 by the isolates. The data were analyzed using the Statistical Analysis Software (SAS) program, version 6.0 (Statistical Analysis Software version 6.0). 3. Results 3.1. Identification of isolates by AFLP Ninety-one fragments ranging in size from 40 to 430 bp were amplified using EcoRI AC + MseI CC and EcoRI AC + MseI CT primers for selective amplification. Sixty-seven (74%) of these fragments were polymorphic and 24 (26%) were non-polymorphic. The topology generated by the matrix of the fragments analysis revealed eleven distinct groups (Fig. 1), with the similarity between F. verticillioides strains and the F. verticillioides MATA-1 (MRC 8559) reference strain (South African Medical Research Council) ranging from 70% to 100%. The isolates belonging to clusters that were more distant from the reference strain presented a similarity of about 39% (34SP and 35BA) and 35% (14BA and 36BA). These strains were subsequently identified as Fusarium sp. (34SP), F. sacchari (35BA), and F. proliferatum (14BA and 36BA) by partial sequencing of the TEF-1α gene. Similarity was 75% between strains 34SP and 35BA and 94% between strains 36BA and 14BA. The bootstrap value of 80% supported the separation of the clades of F. verticillioides from F. proliferatum and F. sacchari. The results demonstrated the lack of a relationship between the groups of the phylogenetic tree and the different geographic origins, with most groups containing isolates from the four regions studied. 3.2. Strains identification by TEF-1α partial gene sequencing Identity analysis using the partial sequence of the TEF-1α gene (≈650 bp) was confirmed using GenBank and Fusarium ID v1.0 databases, and both, showed similarity of all strains with F. verticillioides (higher than 99.5%). However, isolates 34SP (Fusarium sp.), 35BA (F. sacchari), and 14BA and 36BA (F. proliferatum) presented similarity of 93.8%, 99.5%, 99.1% and 99.2%, respectively, using Fusarium ID v1.0 database. The phylogenetic dendrogram showed the formation of four groups with F. verticillioides isolates and one group with isolates 36BA

and 14BA (Fusarium sp. and F. sacchari). Isolates 35BA and 34SP and F. oxysporum formed no clusters. The bootstrap value of 76% supported the separation of the clades of F. verticillioides from the other species (Fig. 2). 3.3. Identification of the MAT-1 and MAT-2 alleles Amplification of the idiomorph MAT-1 revealed the presence of a fragment of about 200 bp in 21 (88%) F. verticillioides strains isolated from the region of Nova Odessa — SP, 13 (52%) from the region of Várzea Grande — MT, 15 (60%) from Santa Maria — RS, and 18 (82%) from Recôncavo Baiano — BA (Tables 1 and 2). One fragment of approximately 800 bp corresponding to idiomorph MAT-2 was detected in three (12%) strains isolated from the region of Nova Odessa — SP, 12 (48%) from Várzea Grande — MT, 10 (40%) from Santa Maria — RS, and 4 (18%) from Recôncavo Baiano — BA (Tables 1 and 2). Sixty-seven (70%) of the 96 F. verticillioides isolates carried the MAT-1 allele and 29 (30%) the MAT-2 allele (proportion of 7:3). 3.4. Production of fumonisins B1 and B2 and quantification of the FUM1 and FUM19 genes by real time RT-PCR All 98 isolates identified as F. verticillioides and F. proliferatum were producers of FB1, whereas 89% produced FB1 and FB2 (Tables 1 and 2). Production levels ranged from 0.2 μg/g (16SP and 28RS) to 762 μg/g (39BA) for FB1, and from 0.02 μg/g (01SP, 48SP, 28RS, 13MT and 49MT) to 165 μg/g (39BA) for FB2. The isolates identified as F. sacchari and Fusarium sp. did not produce fumonisins (Tables 1 and 2). The efficiencies of the quantitative real time RT-PCR assays were below 110% for all genes, with slopes of − 3.17 (efficiency = 107%), −3.15 (efficiency = 108%) and − 3.13 (efficiency = 109%) for TUB, FUM1 and FUM19, respectively. The ΔCt values for FUM1 and FUM19 were plotted against the log dilutions and the graphs obtained presented a slope of 0.02 (FUM 1 in relation to TUB) and −0.03 (FUM19 in relation to TUB). Amplification of the FUM1 and FUM19 genes was observed in all isolates identified as F. verticillioides. The relative expression of FUM1 ranged from 0.6 (29RS) to 21.6 (39BA) and the relative expression of FUM19 from 0.6 (27SP, 37SP and 41SP) to 22.2 (39BA) (Tables 1 and 2). The levels of fumonisins (μg/g) were plotted against the expression of the FUM1 and FUM19 genes and the following Pearson's correlation coefficients were obtained: r = 0.95 (P b 0.0001) and r = 0.79 (P b 0.0001) (correlation of FUM1 with FB1 and FB2, respectively), and r = 0.93 (P b 0.0001) and r = 0.78 (P b 0.0001) (correlation of FUM19 with FB1 and FB2, respectively) (Figs. 3 and 4). 4. Discussion Phylogenetic analysis of the isolates obtained from corn grains freshly harvested in different regions of Brazil based on AFLP markers showed a wide intraspecific variability within F. verticillioides, as demonstrated by the formation of different groups and subgroups in the dendrogram. In addition, clusters of strains identified as Fusarium sp. and F. sacchari and of two F. proliferatum isolates were observed, which presented less than 40% similarity to the F. verticillioides MAT-A1 reference strain. According to Leslie et al. (2007), strains of the same species share about 60% to 70% of AFLP fragments. An intermediate degree of similarity is defined when strains share 40% to 60% of fragments and

Fig. 1. AFLP dendrogram inferred by distance analysis using neighbor-joining algorithm for Fusarium strains isolated from corn of different geographic origins. Selective primers (EcoRI AC + MseI CC and EcoRI AC + MseI CT) were used and the AFLP data were analyzed by PAUP 4.0b10 program. Nonparametric bootstrap analysis was performed for 1000 replicates and the results (in percentage) are described at the internodes. F. oxysporum is the outgroup (OG), and FA1, FA11 and FA12 are F. verticillioides reference strains obtained from the South African Medical Research Council (MRC 8559 and MRC 8560). G1 to G11 are the groups inferred by AFLP technique. All strains from G1 to G19 were identified as F. verticillioides. MAT-1 and MAT-2 alleles are described for each strain.

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15

01OG (F. oxysporum)

80

100

100 80 100

0.1

14BA (MAT-1, F. proliferatum) 36BA (MAT-1, F. proliferatum)

G11

46SP (MAT-1) 16SP (MAT-1) 22SP (MAT-1) 06SP (MAT-1) 41SP (MAT-1) 03SP (MAT-2) 41MT (MAT-2) 40MT (MAT-2) 13MT (MAT-1) 35MT (MAT-2) 03MT (MAT-1) G1 03RS (MAT-1) 08RS (MAT-2) 19RS (MAT-2) 35RS (MAT-2) 80 38RS (MAT-2) 46RS (MAT-2) 49RS (MAT-1) 05RS (MAT-1) 98 01RS (MAT-1) 04RS (MAT-1) 02BA (MAT-1) 99 15BA (MAT-1) 40BA (MAT-1) 15MT (MAT-1) 80 33MT (MAT-1) 05MT (MAT-2) 76 36MT (MAT-2) 80 80 08SP (MAT-1) 36SP (MAT-1) 03BA (MAT-1) G2 67 16BA (MAT-1) 37BA (MAT-1) 60 45BA (MAT-1) 26BA (MAT-1) 78 45MT (MAT-2) 48MT (MAT-2) 31MT (MAT-2) 44MT (MAT-2) 20RS (MAT-1) 80 80 11SP (MAT-1) 28SP (MAT-1) 80 24MT (MAT-1) 100 11RS (MAT-2) G3 80 80 23MT (MAT-1) 60 29RS (MAT-1) 42BA (MAT-1) 100 32SP (MAT-1) 100 01SP (MAT-1) 74 26RS (MAT-1) 40RS (MAT-1) 17RS (MAT-2) 02RS (MAT-1) 11MT (MAT-1) 14MT (MAT-1) 80 43MT (MAT-1) G4 48SP (MAT-1) 42SP (MAT-1) 04BA (MAT-1) 79 06MT (MAT-1) 80 09MT (MAT-1) 12BA (MAT-2) 80 18BA (MAT-2) 60 43BA (MAT-2) 50BA (MAT-1) 60 19BA (MAT-1) 60 39BA (MAT-1) 60 13BA (MAT-1) 80 27BA (MAT-1) 47BA (MAT-2) 80 80 29BA (MAT-1) 21RS (MAT-1) 27RS (MAT-2) 33RS (MAT-1) 28RS (MAT-1) 12RS (MAT-1) 30MT (MAT-1) G6 80 37SP (MAT-2) 38SP (MAT-1) 21SP(MAT-1) FA12 (MAT-2) FA1 (MAT-1) 80 FA11 (MAT-1) 36RS (MAT-2) 60 27SP (MAT-1) 100 24SP (MAT-1) 21BA (MAT-1) 80 30SP (MAT-1) G7 80 08MT (MAT-2) 49MT (MAT-2) 60 32MT (MAT-2) 31RS (MAT-2) 96 12MT (MAT-1) 14SP (MAT-1) G8 80 47SP (MAT-1) 25SP (MAT-1) 100 10SP (MAT-2) 47RS (MAT-1) G9 22BA (MAT-1) 34SP (MAT-1, Fusarium sp.) G10 35BA (MAT-1, F. sacchari)

G5

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still undergo speciation, as is the case of mating C (F. fujikuroi) and mating D (F. proliferatum) populations which show about 50% similarity and intercross. Similarity of less than 40% indicates that the isolates do not belong to the same species. AFLP proved to be a valuable tool for the differentiation of Fusarium species associated with corn. According to Parisi et al. (2010), AFLP presents a series of advantages in terms of reproducibility and applicability when compared to other genotyping techniques. In addition, the use of capillary electrophoresis and an automated sequencer permits a high-resolution differentiation of the fragments obtained and rapid analysis of the genotype profiles of different microbial strains. Several studies have shown the importance of AFLP for the determination of inter- and intraspecies genetic variability in Fusarium genus (Chulze et al., 2000; Zeller et al., 2003; Leslie et al., 2005; Leslie et al., 2007; Qu et al., 2008; Lima et al., 2009; Reynoso et al., 2009). However, few studies have used an automated sequencer for analysis as described by Moretti et al. (2004) and Schmidt et al. (2004). In the present investigation, partial sequencing of the TEF-1α gene was performed to test the AFLP data and to confirm the identity of the isolates as proposed by Geiser et al. (2004). This gene is highly useful for phylogenetic differentiation of Fusarium species since it shows a high level of polymorphism when compared to other protein-coding genes such as calmodulin, beta-tubulin and histone H3. Thus, TEF-1α has become the marker of choice for single-locus identification in Fusarium (Geiser et al., 2004). The dendrogram demonstrated the clear separation of F. verticillioides, F. proliferatum, F. sacchari and Fusarium sp. strains, confirming the interspecies clusters generated by AFLP. With respect to intraspecific variability of F. verticillioides, the formation of four distinct groups was observed in the dendrogram of the partial sequencing of TEF-1α gene, whereas the AFLP technique provided nine subgroups with F. verticillioides strains and two more subgroups containing F. proliferatum, F. sacchari and Fusarium sp. This finding might be explained by the fact that AFLP technique includes the whole genome of F. verticillioides, whereas sequencing of the TEF-1α gene involves a part of the fungal genome; therefore, distinct molecular markers are obtained comparing these two techniques. As demonstrated by the present results, AFLP and partial sequencing of the TEF-1α gene present a good alternative for interspecies differentiation, facilitating the identification of F. verticillioides in different regions and hosts. Identification of Fusarium species by conventional methods is time consuming and requires a high level of knowledge (Leslie and Summerell, 2006). To further complicate both taxonomy and the identification of strains in the Gibberella fujikuroi species complex, there are some Fusarium species that morphologically resemble those are included in section Liseola, but produce chlamydospores. Such species are also distinct genetically from those in section Liseola. Undoubtedly, the application of phylogenetic species concept associated with biological concept to species in Fusarium contributes to the identification of species whose status has been questioned (Leslie and Summerell, 2006). Our phylogenetic analysis demonstrated the lack of relationship between strains and geographic origin. This finding is probably due to the easy dispersal of fungi in nature. In addition, F. verticillioides and F. proliferatum are well adapted to different habitats worldwide and the former is widely found in Brazilian corn (Abdala et al., 2000; Gong et al., 2009; Rocha et al., 2009; Almeida et al., 2002). In the present study, F. sacchari was identified as F. subglutinans by morphological analysis, but was subsequently classified as F. sacchari based on the partial sequence of the TEF-1α gene. This species is found in sugar cane in Asia and as a contaminant of sorghum in the Philippines,

Mexico and Brazil, and is occasionally isolated from corn and orchids (Leslie and Summerell, 2006; Desjardins, 2006). Some Fusarium species are adapted to climatic differences, preferring tropical dry and hot or temperate climates, whereas others are considered to be cosmopolitans. Climatic conditions may limit the variety of species found or even influence the relative frequency of species isolation (Vigier et al., 1997; Moschini et al., 2004). The phytopathogenic agent of maize stalk rot in regions of hot and dry climate is F. verticillioides, whereas in cold and humid regions the disease is mainly caused by F. subglutinans (Leslie and Summerell, 2006). There are other species well adapted to different substrates, such as F. subglutinans and F. mangiferae (associated with corn and mango, respectively) and F. verticillioides and F. thapsinum (associated with corn and sorghum, respectively). These species are morphologically similar but are unable to intercross and present distinct genetic profiles (Britz et al., 2002; Leslie et al., 2005). Moretti et al. (2004), studying F. verticillioides isolated from banana and corn, observed that the banana isolates did not produce fumonisins, whereas those isolates from corn produced substantial levels of these toxins. A difference was also noted in the AFLP profile, with the observation of two distinct clusters, one associated with banana and the other with corn. As F. verticillioides is a pathogen on maize and found worldwide wherever maize is cultivated (Leslie and Summerell, 2006), the AFLP dendrogram generated in this investigation showed that the different geographic origins, considering the climatic disparities among the four regions (Rocha et al., 2009) did not influence the genotypic profile of the Brazilian isolates. The distribution of the groups and subgroups in the dendrogram indicates the possibility of sexual reproduction. The similar ratio of mating type alleles 1 and 2, especially at local level (Mato Grosso and Rio Grande do Sul) seemed to be agreeable with this hypothesis. The MAT-1 and MAT-2 alleles are responsible for recombination in teleomorphic and heterothallic Fusarium species. Therefore, these genes can be used to identify Fusarium species by crosses (Leslie and Summerell, 2006). The MATA-1:MATA-2 ratio obtained was 67:29, considering all the isolated strains. Similar results have been reported by Visentin et al. (2009), Cumagun (2007), Chulze et al. (1998) and Mansuetus et al. (1997). Jurado et al. (2010) and Torres et al. (2001) also detected a larger number of MAT-1 allele than MAT-2 for F. proliferatum and F. subglutinans, respectively. An inverse ratio for isolates from the mating A population was reported by Silva et al. (2006), Chulze et al. (2000) and Leslie and Klein (1996). All F. verticillioides strains were able to produce fumonisins. According to Nelson et al. (1991), F. verticillioides strains can produce low (traces to 49 μg/g), intermediate (50–500 μg/g) and high (N500 μg/g) levels of FB1. In the present study, 75 isolates exhibited a potential of FB1 production of less than 50 μg/g, 20 isolates produced intermediate levels, and one isolate exhibited a high level of FB1 production (isolate 39BA, 762 μg/g). Although most strains produced levels of less than 50 μg/g, these values can be increased when climatic conditions are favorable (Hurst, 2001). The levels of FB1 and FB2 production observed here were lower than those reported by Ariño et al. (2007), Sánchez-Rangel et al. (2005), Mirete et al. (2003), Mateo et al. (2001) and Chulze et al. (1998). In this respect, the Food and Drug Administration recommends FB1 levels of 2 to 4 μg/g for products intended for human consumption and of 5 to 100 μg/g for products intended for animal feed (FDA, 2001). We emphasized that, the strain 36BA, identified as F. proliferatum, produced substantial levels of FB1 (139 μg/g). In the present study, the PQF1 and PQF19 primers developed by López-Errasquín et al. (2007) were used for the amplification of FUM1

Fig. 2. Cladogram based on the partial sequencing of the translation elongation factor 1α gene of Fusarium strains isolated from corn of different geographic origins inferred by the maximum likelihood method under an HKY85+G model using PAUP 4.0b10 program. Nonparametric bootstrap analysis was performed for 1000 replicates and the results (in percentage) are described at the internodes. F. oxysporum is the outgroup, F. verticillioides are reference strains obtained from the South African Medical Research Council (MRC 8559). G1 to G5 are the groups inferred by the technique. All strains from G1 to G4 were identified as F. verticillioides. MAT-1 and MAT-2 alleles are described for each strain.

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17

35BA (MAT-1,

F. sacchari) F. oxysporum 34SP (MAT-1, Fusarium sp.) 11RS (MAT-2) 37BA (MAT-1) 04BA (MAT-1) 26BA (MAT-1) 21BA (MAT-1) 33RS (MAT-1) 40RS (MAT-1) 37SP (MAT-2) 36RS (MAT-2) 38RS (MAT-2) 12BA (MAT-2) 28SP (MAT-1) 46RS (MAT-2) 02BA (MAT-1) 30MT (MAT-1) 03BA (MAT-1) 01SP (MAT-1) 12MT (MAT-1) 11SP (MAT-1) 14SP (MAT-1)

76

G1

F. verticillioides (MAT-1) 22BA (MAT-1) 30 SP (MAT-1) 42BA (MAT-1) 15BA (MAT-1) 48SP (MAT-1) 04 RS (MAT-1) 46SP (MAT-1) 32SP (MAT-1) 45BA (MAT-1) 39BA (MAT-1) 16BA (MAT-1) 12RS (MAT-1) 32MT (MAT-2) 45MT (MAT-2) 42SP (MAT-1) 23MT (MAT-1) 11MT (MAT-1) 08MT (MAT-2) 15MT (MAT-1) 05MT (MAT-2) 13BA (MAT-1) 44MT (MAT-2) 19BA (MAT-1) 47BA (MAT-2) 50BA (MAT-1) 17RS (MAT-2) 31RS (MAT-2) 48MT (MAT-2) 06SP (MAT-1)

F. verticillioides (MAT-1)

76 59

71

71

100

18BA (MAT-2) 03SP (MAT-2) 31MT (MAT-2) 36MT (MAT-2) 41SP (MAT-1) 43BA (MAT-2) 09MT (MAT-1) 27SP (MAT-1) 33MT (MAT-1) 24MT (MAT-1) 41MT (MAT-2) 24SP (MAT-1) 40MT (MAT-2) 35RS (MAT-2) 49MT (MAT-2) 16SP (MAT-1) 06MT (MAT-1) 26RS (MAT-1) 49 RS (MAT-1) 35MT (MAT-2) 02RS (MAT-1) 43MT (MAT-1) 27BA (MAT-1) 28RS (MAT-1) 10SP (MAT-2) 19RS (MAT-2) 08RS (MAT-2) 47RS (MAT-1) 08SP (MAT-1) 20RS (MAT-1) 38SP (MAT-1) 29RS (MAT-1) 40BA (MAT-1) 47SP (MAT-1) 25SP (MAT-1) 36SP (MAT-1) 29BA (MAT-1) 21RS (MAT-1) 01RS (MAT-1) 03RS (MAT-1) 27RS (MAT-2) 05RS (MAT-1) 21SP (MAT-1) 13MT (MAT-1) 03MT (MAT-1) 14MT (MAT-1) 22SP (MAT-1)

G2

G3

G4

36BA (MAT-1, F. proliferatum) 14BA (MAT-1, F. proliferatum)

G5

18 L. de Oliveira Rocha et al. / International Journal of Food Microbiology 145 (2011) 9–21 Fig. 3. Correlation among FUM1, FUM19 expression and FB1 production by Fusarium verticillioides isolated strains from freshly harvested corn in different origins in Brazil. Pearson's correlation coefficients were obtained: r = 0.95 (P b 0.0001) and r = 0.93 (P b 0.0001) (correlation of FUM1 with FB1 and FUM19 with FB2, respectively).

L. de Oliveira Rocha et al. / International Journal of Food Microbiology 145 (2011) 9–21 Fig. 4. Correlation among FUM1, FUM19 expression and FB2 production by Fusarium verticillioides isolated strains from freshly harvested corn in different origins in Brazil. Pearson's correlation coefficients were obtained: r = 0.79 (P b 0.0001) and r = 0.78 (P b 0.0001) (correlation of FUM1 with FB2 and FUM19 with FB2, respectively).

19

20

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and FUM19 cDNA, respectively, in F. verticillioides strains isolated from corn grains. The FUM1 gene encodes a polyketide synthase that catalyzes the first step in the biosynthesis of fumonisins. The FUM19 gene encodes a protein that is highly similar to the ATP-binding cassette of multidrug resistance transporters, which act as efflux pumps to reduce cellular concentrations of toxins and thereby confer protection (Desjardins, 2006). In the present study, Pearson's correlation test showed a positive correlation between the expression of the FUM1 and FUM19 genes and the production of FB1 and FB2, in agreement with the results of López-Errasquín et al. (2007). Jurado et al. (2010), analyzing the relationship between FUM1 expression and FB1 production in F. proliferatum, also observed a good correlation. Fumonisin regulation is a complex process governed by environmental conditions and by the interaction with the host plant. Studies involving expression of fungal genes can provide differences on their expression levels under wide variety of conditions and can contribute to the understanding of the fumonisin biosynthesis regulation (Picot et al., 2010). The finding that most isolates were able to produce FB1 and FB2 emphasizes the need for the control of fumonisin contamination in corn-derived products. Molecular characterization of F. verticillioides was found to be a useful tool for the identification of this species. Real time RT-PCR used in this research was shown to be a suitable alternative for the estimation of fumonisin production and can contribute to further investigations considering the influence of abiotic and biotic factors on expression of these genes, aiming to reduce the potential for mycotoxin production in commodities. This is the first report of the expression of fumonisin biosynthetic genes applied in Fusarium strains isolated from freshly harvested Brazilian corn. Acknowledgements This study was supported by the Brazilian funding agency FAPESP (Fundação de Amparo a Pesquisa no Estado de São Paulo). We thank Prof. Dr. Marta Maria Geraldes Teixeira and Prof. Dr. Maria Notomi Sato for granting us access to the automated sequencer and to the real time PCR equipment in their respective laboratories. We also thank the technicians Carmen Sílvia Takata, Jansen Araújo and Mayce Helena Azor for the technical assistance. References Abdala, M.Y., A l-Rokibah, A., Moretti, A., Mulè, G., 2000. Pathogenicity of toxigenic Fusarium proliferatum from date palm in Saudi Arabia. Plant Disease 84, 321–324. Almeida, A.P., Fonseca, H., Fancelli, A.L., Direito, G.M., Ortega, E.M., Corrêa, B., 2002. Mycoflora and fumonisin contamination in Brazilian corn from sowing to harvest. Journal of Agriculture and Food Chemistry 50, 3877–3882. Ariño, A., Juan, T., Estopañan, G., González-Cabo, J.F., 2007. Natural occurrence of Fusarium species, fumonisin production by toxigenic strains, and concentrations of fumonisins B1, and B2 in conventional and organic maize grown in Spain. Journal of Food Protection 70, 151–156. Berjak, P., 1984. Report of seed storage committee working group on the effects of storage fungi on seed viability. Seed Science and Technology 12, 233–253. Bezuidenhout, S.C., Gelderblom, W.C.A., Gorst-Allman, C.P., Horak, R.M., Marasas, W.F.O., Spiteller, G., Vleggaar, R., 1988. Structure elucidation of the fumonisins, mycotoxins from Fusarium moniliforme. Journal of the Chemical Society, Chemical Communications 743–745. Britz, H., Steenkamp, E.T., Coutinho, T.A., Wingfield, B.D., Marasas, W.F.O., Wingfield, M.J., 2002. Two new species of Fusarium section Liseola associated with mango malformation. Mycological Research 94, 722–730. Chulze, S.N., Ramirez, M.L., Pascale, M., Visconti, A., 1998. Fumonisin production by, and mating populations of, Fusarium section Liseola isolates from maize in Argentina. Mycological Research 102, 141–144. Chulze, S.N., Ramirez, M.L., Torres, A., Leslie, J.F., 2000. Genetic variation in Fusarium section Liseola from no till maize in Argentina. Applied and Environmental Microbiology 66, 5312–5315. CONAB, 2009. Companhia Nacional de Abastecimento. Monitoring of the Brazilian Grain Harvest, 2009/2010. Third Survey — December/2009. National Supply Company, Brazilhttp://www.conab.gov.br/conabweb/index.php. Cumagun, C.J., 2007. Female fertility and mating type distribution in a Philippine population of Fusarium verticillioides. Journal of Applied Genetics 48, 123–126.

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