Profiles of mycotoxin production by Fusarium graminearum lineage 7 isolates

ALVAREZ, C.; AZCARATE, M.P.; CABRAL, D.; FERNANDEZ PINTO, V.

Rosario, Argentina

Conferencia; 1st Latin American Conference –ICC (International Conference on Cereals and Cereals Products); 2007

ICC (International Conference on Cereals and Cereals Products).

Title: Profiles of mycotoxin production by Fusarium graminearum lineage 7 isolates. Authors: Alvarez, CLa; Azcárate MPb, Cabral Da and Fernández Pinto Vc   a PHRIDEB-PROPLAME. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Micología N° 69, 4to Piso, Pabellón II. Ciudad Universitaria. C1428 EHA, Buenos Aires, Argentina. b Departamento de Química Orgánica, Laboratorio de Microbiología de Alimentos, 3er Piso, Pabellón II. Ciudad Universitaria. C1428 EHA, Buenos Aires, Argentina. c Estación Experimental Anguil, INTA. C.C.: 11 (6326). Anguil, La Pampa, Argentina.   Keywords: Fusarium graminaerum lineage 7, Deoxynivalenol, Nivalenol, 3- and 15-Acetyl deoxynivalenol   Sesión 4: “Seguridad e inocuidad en productos de cereales”   Fusarium Head Blight (FHB) is an important disease affecting the production of wheat worldwide. In Argentina, 90 % of the pathogens isolated from blighted heads have been Fusarium graminearum [teleomorph Gibberella zeae (Schwein) Petch] (Galich, 1996). O’Donnell et al. (2000) defined seven phylogenetic lineages. In 2002, (Ward et al.) and in 2004  O’Donnell et al. two more lineages were added and the previous lineages were converted to separate phylogenetic species. In Argentina, there is a recent study based on the sequence analysis with translation elongation factor 1-a (TEF) and b-tubulin genes (Alvarez et al., 2006), that showed the predominance of Fusarium graminearum sensu stricto (lineage 7). Isolates of that study were used in the present work in order to determine their pattern of trichotecene production . MATERIALS AND METHODS Thirty three 33 of  fields located in 19 districts of Buenos Aires province were analyzed . At least 0.5kg of wheat was randomly collected from each farm during the 2001-2002 and the 2003-2004 harvests. For the isolation of the internal mycoflora, 100 kernels of each sample were superficially disinfected in a 10 % aqueous solution of commercial sodium hypochlorite for 1 min, rinsed with sterile water, submerged in a 70 % ethyl alcohol solution and dried over a filter paper. The kernels were placed, 10 per plate, on potato dextrose agar (PDA) and incubated for 5 days at 25°C under fluorescent light (12 h photoperiod). The Fusarium species were taken and monosporic cultures were obtained. Preliminary identification was carried out on PDA and Spezieller Nährstoffarmer Agar (SNA) medium according to Nelson et al.(1983). The cultivation on Petri dishes with carnation leaves agar (CLA) was also performed to obtain perithecia. To test for trichothecenes production, Fusarium graminearum isolates were placed in 25g of sterilized rice in 250-ml Erlenmeyer flasks. Fifteen ml of sterile distilled water was added before sterilization by autoclaving at 121°C for 20 minutes. Each flask was inoculated with a piece of agar (diameter of 1cm) from a seven-day-old SNA culture, cultivated at 25°C for 15 days and at 10°C for 10 days and kept at –20 °C until extraction. The extraction of DON, NIV, 15 ADON and 3 ADON was carried out with 125 ml of acetonitrile: ethyl acetate: water (50:41:9) as extraction solvent for 1 hour at 300 rpm; the clean-up was performed with a column packed with charcoal: alumina: celite (0.7:0.5:0.3); and dried in Rotavap®. Trichothecenes were detected and quantified by gas chromatography with 63Ni electron capture detection Shimadzu Model GC17, equipped with split/splitless injector and fitted with RX-5MS capillary column (25mx0, 2mm id). The derivatization was carried out with heptafluorbutyric anhydride with the method reported by Crocteau et al. (1994). The detection limit for DON and its acetyl derivatives was 20 mg kg-1. The limit detection for NIV was 50 mg kg-1. Standarized data were subjected to principal component analysis (PCA), a method to reduce the number of variables and to find the combination of the most important ones. Isolate was the operational taxonomic unit (OTU) ant the variables were the toxins produced. PCA were performed with the Statistica software package (Statsoft Inc 2002). Results and discussion From the 33 wheat samples, 164 F. graminearum lineage 7 were isolated. According to their mycotoxin profile production isolates could be grouped into 4 groups. (Table 1) Four groups with different mycotoxin production profiles could be defined. Group 2, with the highest number of isolates, where all the toxins were produced at intermediate values. Group 4 has the greatest production of DON and NIV, with 15 AcDON also produced. Group 3 with a similar profile than 4 but with lower DON production. Group 1 that has the highest value of 3 AcDON with lower values of all the other toxins. It seems that the more frequent profile is that of group 2 with DON and the repective acetilderivates was produced by almost all the isolates. NIV is produced The  164 strains morphologically identified as Fusarium graminearum sensu stricto (Alvarez et al., 2006). are listed in Table 1. in a minor proportion - Crocteau S, Prelusky D & Trenholm H, 1994. Analysis of trichothecenes mycotoxins by CG with ECD. Journal of  Agriculture and  Food Chemistry 42: 928-933. - McCune B and Mefford J, 1997. Multivariate analysis of Ecological data version 3.0, MjM Software, Gleneden Beach, Oregon, USA.