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Trichothecene genotypes of Fusarium graminearum from wheat in Uruguay
Trichothecene genotypes of Fusarium graminearum from wheat in Uruguay Dinorah Pan, Natalia Calero, Ana Mionetto, Lina Bettucci PII: DOI: Reference:
S0168-1605(13)00012-3 doi: 10.1016/j.ijfoodmicro.2013.01.002 FOOD 6084
To appear in:
International Journal of Food Microbiology
Received date: Revised date: Accepted date:
20 September 2012 8 December 2012 4 January 2013
Please cite this article as: Pan, Dinorah, Calero, Natalia, Mionetto, Ana, Bettucci, Lina, Trichothecene genotypes of Fusarium graminearum from wheat in Uruguay, International Journal of Food Microbiology (2013), doi: 10.1016/j.ijfoodmicro.2013.01.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Trichothecene genotypes of Fusarium graminearum from wheat in Uruguay
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Dinorah Pan *, Natalia Calero, Ana Mionetto and Lina Bettucci.
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* Laboratorio de Micología, Facultad de Ciencias - Facultad de Ingeniería,
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UdelaR, Julio Herrera y Reissig 565, 11200 Montevideo, Uruguay
* Corresponding autor: Dra. Dinorah Pan, Laboratorio de Micología, Facultad de Ciencias - Facultad de Ingeniería, UdelaR, Julio Herrera y Reissig 565, 11200 Montevideo, Uruguay. Phone/Fax: ++ 59827120626; E-mail: [email protected]
ACCEPTED MANUSCRIPT Abstract Gibberella zeae (Scwein.) Petch (anamorph F. graminearum Schwabe) is the primary causal agent of FHB of wheat in Uruguay. In the last decade, F.
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graminearum has produced destructive epidemics on wheat in Uruguay, causing yield losses and price discounts due to reduced seed quality. Strains of
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F. graminearum clade usually express one of three strain-specific profiles of trichothecene metabolites: nivalenol and its acetylated derivatives (NIV
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chemotype), deoxynivalenol and 3-acetyldeoxynivalenol (3-AcDON chemotype), or deoxynivalenol and 15-acetyldeoxynivalenol (15-AcDON chemotype).
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multiplex PCR assay of Tri3, Tri5, and Tri7 was used to determine the trichothecene genotype of 111 strains of F. graminearum collected during 2003
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and 2009 growing season from fields located in the major wheat production area of Uruguay. The result showed that all except one of the isolates were of DON genotype, with the remainder of NIV genotype in years 2003 and 2009. All
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strains with the DON genotype were also of the 15-AcDON genotype in 2003 and nearly all (45/50) in 2009. No DON/ 3-AcDON genotypes were found in
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either growing season. No potential shifts in the populations were found in the trichothecene genotypes between 2003 and the 2009 epidemic FHB harvest season. This study provides the first data on trichothecene genotypes of F. graminearum strains isolated from wheat in Uruguay and add to the current
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regional knowledge of trichothecene genotypes.
Keywords:
Deoxynivalenol,
Nivalenol,
3-acetyldeoxynivalenol,
acetyldeoxynivalenol, Fusarium graminearum, wheat
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ACCEPTED MANUSCRIPT 1.1 Introduction Fusarium head blight (FHB) is one of the most economically important fungal diseases of wheat worldwide (Goswami and Kistler, 2005). Gibberella zeae
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(Scwein.) Petch (anamorph F. graminearum Schwabe) is the primary causal agent of FHB of wheat and barley in Uruguay (Ackerman et al., 2002). It
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manifests as an epiphytic disease that results in a decrease in both germination ability and grain weight. At the same time, this species is able to contaminate
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grains with trichothecene mycotoxins such as deoxynivalenol (DON) and nivalenol (NIV) that constitute a health risks to both humans and domesticated animals (Zeller et al., 2003). Both DON and NIV also are potent phytotoxins and play a role in pathogenesis of F. graminearum (Edues et al., 2001).
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In the last decade, F. graminearum has caused destructive epidemics on wheat in Uruguay, causing yield losses and price discounts due to reduced seed quality. During the harvest seasons of 2001, 2002 and 2009 coincidentally with
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flowering to early stages of grain maturity, a heavy rainfall period occurred and a high rate of FHB was observed, causing levels of DON contamination of 6593
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µg/kg, 5880 µg/kg and 7652 µg/kg, respectively (Pan et al., 2009). Strains of the F. graminearum clade usually express one of three strain-specific profiles of trichothecene metabolites: nivalenol and its acetylated derivatives (NIV chemotype), deoxynivalenol and 3-acetyldeoxynivalenol (3-AcDON chemotype), or deoxynivalenol and 15-acetyldeoxynivalenol (15-AcDON
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chemotype) (Ward et al. 2002). PCR assays have been developed to rapidly assess trichothecene genotypes of F. graminearum. Such assays rely on the amplification of gene portions that code for key enzymes involved in trichothecene biosynthesis (Quarta et al., 2006; Ward et al., 2002). Primers based on the sequences of alleles at Tri3, Tri5 and Tri7 (Quarta et al., 2006) were designed to differentiate three toxin genotypes (3-AcDON, 15-AcDON and NIV) and on Tri7 and Tri13 (Lee et al. 2001, 2002) to distinguish DON and NIV producers. Since wheat represents the major staple food for Uruguayan people, the main aim of this work was to determine and evaluate any potential shift in the trichothecene mycotoxin genotypes (3-AcDON, 15-AcDON and NIV) in F. graminearum isolates obtained from two years’ sampling of wheat grains from Uruguay.
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1.2 Materials and methods
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1.2.1 Sampling
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Fusarium species were isolated from 93 and 26 samples of wheat grains harvest during 2003 and 2009 growing season, respectively, from commercial
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fields located in the major wheat production area of Uruguay (Fig.1). Samples weighting 500 g, obtained after harvesting operations, were stored at -20ºC in
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paper bags in the laboratory.
Figure 1. Sampling sites where wheat grains were obtained.
1.2.2 Fungal isolation and identification
Grains of each sample were surface-disinfected by soaking in 1 % aqueous NaOCl for 1 min, and then rinsed three times with sterile water. One hundred grains
were
plated
(10
grains
per
Petri
dish)
on
peptone
+
pentachloronitrobenzene (PCNB) medium. These plates were incubated at 24ºC for 7 days with 12/12 h photoperiod under cool with and black light fluorescent lamps (Leslie and Summerell, 2006). All Fusarium isolates were
ACCEPTED MANUSCRIPT identified based on conidial morphology according to Leslie and Summerell (2006). The isolates of F. graminearum were confirmed by their ability to produce homothallic perithecia on carrot agar and /or carnation leaf agar and
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also identified as members of F. graminearum lineage 7 by AFLP analyses (Pan et al. 2010). Sixty one isolates of F. graminearum were obtained from locations
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of Soriano (21), San José (5), Río Negro (10), Colonia (15) and Flores (10) in the year 2003 and 50 isolates from locations of Soriano (3), San José (32),
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Colonia (14) and Florida (1) in 2009. The isolates were maintained as spore suspensions in 10% glycerol and frozen at -80ºC. Duplicate subcultures of each isolate are held in the culture collection of the Laboratorio de Micología, Facultad de Ciencias – Facultad de Ingeniería, Universidad de la República,
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Uruguay.
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1.2.3 DNA isolation
Isolates of F. graminearum were grown potato dextrose agar (PDA) plates for 5-
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6 days and genomic DNA was extracted from aerial mycelium using a cetyltrimethylamonium bromide (CTAB) method (Lee and Taylor, 1990).
1.2.4 Trichothecene genotype determination
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Trichothecene genotypes of F. graminearum were identified using a suite of primers directed to portions of genes that are predictive of isolates producing DON, NIV, 3-AcDON and 15-AcDON. For the identification of DON- producing isolates the primer pair 3551H/4056H was selected based on the Tri5 gene sequences. For the identification of 3-AcDON, 15-AcDON and NIV genotype, the primer sets Tri3F971/Tri3R1679, Tri3F1325/Tri3R1679 and Tri7F340/ Tri7R965 directed to portions of Tri3 and Tri7 genes were used. PCR products of 525 bp, 708 bp, 354 bp and 625 bp are produced for DON, 15-AcDON, 3AcDON and NIV, respectively (Quarta et al., 2005, 2006). Multiplex PCR experiments were conducted with 25 ng of fungal DNA in a total volume of 50 mL of 1× reaction buffer containing 1.5 mM MgCl2, 5 U Taq DNA polymerase (Promega), 200 mM dNTPs, 0.2 mM each of the Tri3 primers (Tri3F971, Tri3F1325 and Tri3R1679), and 0.1 mM each of primers Tri7F340,
ACCEPTED MANUSCRIPT Tri7R965, 3551H and 4056H (Quarta et al. 2005, 2006). A negative control, containing all reagents and primers but no fungal DNA was included with every set of reactions. PCR was conducted in a PTC-2000 Thermal Cycler (MJ
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Research Inc., Watertown, MA). The PCR conditions were 94ºC, 3 min then 35 cycles of 94ºC, 30 s, 53ºC, 30 s, 72ºC, 1 min, followed by a final extension step
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of 10 min, 72ºC. PCR products were separated by electrophoresis through 2% agarose gels. Gels were stained with 1 mg/ml ethidium bromide and
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photographed under UV light. DNA from strains with known NIV, 3-AcDON and 15-AcDON chemotypes were used as positive controls.
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1.3 Results
A total of 111 single-spore isolates of F. graminearum were recovered from wheat grain from different localities. Sixty one isolates were obtained in the
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2003 growing season and 50 in 2009. All isolates were identified as members of F. graminearum sensu stricto (lineage 7).
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The multiplex PCR assay demonstrated that all except one isolate were of DON genotype and the remainder belong to the NIV genotype in 2003 and 2009 years. These assays demonstrated that all strains with DON genotype were of the 15-AcDON genotype in 2003 and in nearly all the strains (45/50) in 2009. In this latter year, 4 strains showed DON genotype because they yielded only a
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525 bp product as described by Quarta et al. (2006). No DON/ 3-AcDON genotypes occurred in either growing seasons (Table 1).
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Table 1. Number of trichothecene genotypes of F. graminearum strains collected from kernels of wheat during two growing seasons.
2003
60
DON/ 15-AcDON
1
NIV
45 4
-
+
-
-
+
-
DON/ 15-AcDON
-
+
-
+
DON
-
+
-
-
NIV
-
-
+
-
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+
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1
-
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2009
Amplified fragment (bp) 354 525 625 708
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PCR genotype
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Nº of isolates
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Year
1.4 Discussion
This study provides the first data on trichothecene genotypes of F. graminearum
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strains isolated from wheat in Uruguay. We found that among the populations evaluated in 2003 and 2009 the predominant genotype was DON/15- AcDON. The high DON levels found in samples of wheat grain analyzed (Pan et al., 2009) suggests that the degree of contamination represents a high risk to consumers. For this reason the presence of 15-AcDON in the Uruguay Fusarium
population
is
of
great
health
importance
requiring
further
epidemiological studies to determine conditions favoring production of this mycotoxin and the frequency and distribution of these mycotoxins in food for human and animal consumption. Our results are similar to those found for strains of F. graminearum in South America (Alvarez et al., 2009; Astolfi et al., 2011; Reynoso et al., 2011; Scoz et al., 2008). A recent study in Argentina revealed that DON/ 15-AcDON were the primary trichothecenes produced by F. graminearum in wheat grains (Reynoso
ACCEPTED MANUSCRIPT et al., 2011). In Brazil, the DON/15-AcDON genotype also seems to predominate in isolates from wheat, although a few strains corresponded to NIV genotype (Scoz et al., 2008).
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In Europe there is more variability. In western Russia and Finland, all F. graminearum isolates were found to be DON/3-AcDON (Yli-Mattila et al., 2008),
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whereas in southern Russia, England, the Netherlands and Italy the predominant genotype was DON/15-AcDON (Jennings et al., 2004; Prodi et al.,
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2009; Waalwijk et al., 2003).
In Korea, Japan, China and other parts of Asia, the NIV genotype is the most common and both DON genotypes are rare (Desjardins et al., 2008; Suga et al., 2008). During our research, isolates of the NIV genotype were not frequently
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identified. The low frequency of strains of NIV genotype in 2003 and 2009 is of special interest because NIV is more toxic than DON in most animal systems (Desjardins, 2006). Furthermore, some studies suggest that NIV producers are
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less virulent than DON producers indicating that the production of DON should confer a strong selective advantage to this plant pathogen in wheat (Desjardins Eudes et al., 2000). As the F.
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and Proctor, 2011; Desjardins et al., 2004;
graminearum population is dominated by the DON/15-AcDON genotype, the level of virulence on wheat may affect the population structure of this pathogen in Uruguayan agroecosystems. Most of the strains of F. graminearum evaluated in 2003 and 2009 showed the
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DON/15-AcDON genotype. This finding is important considering that recent data on F. graminearum populations from USA and Canada indicated that the 3-AcDON genotype was more aggressive in susceptible and resistant wheat genotypes and also exhibits a higher in vitro DON production than the 15AcDON isolates (Puri and Zhong, 2010; von der Ohe et al. 2010; Ward et al., 2008) A small percentage of isolates in 2009 showed the DON genotype using multiple PCR as described by Quarta et al. (2006). This DON genotype is unexpected and suggests that these strains can produce only DON. Strains that produce DON and no acetylated derivatives (DON chemotype) had been previously reported for isolates of F. graminearum sensu stricto in Argentina (Alvarez et al., 2009). Further investigation is necessary in order to characterize these strains.
ACCEPTED MANUSCRIPT No potential shifts in the populations of the genotypes of F. graminearum isolates were found between the 2003 and 2009 growing seasons, even though in the latter year an important epidemic of FHB occurred in Uruguay. This fact
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is especially important because it was recently shown that 3-AcDON genotypes seem to be displacing 15-AcDON in the F. graminearum populations from USA,
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Canada, Finland and the Russian Far East (Gilbert et al., 2010; Prodi et al., 2009; Ward et al., 2008; Yli-Mattila et al., 2009).The study reported here
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provides the first data on trichothecene genotypes of F. graminearum strains isolated from wheat in Uruguay during an FHB epidemic year and during a year when there was no FHB epidemic.
Continuous monitoring of Uruguayan F. graminearum isolates is important to
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understand epidemiological factors that may contribute in the future to the distribution of trichothecene genotypes and to detect if potential shifts exist among isolates of acetylated forms of DON and NIV. Moreover, in terms of
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epidemiological studies the detection of NIV genotype was a very important finding, so it is necessary to examine the frequency and distribution of NIV
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producers in Uruguay and the conditions that favour mycotoxin production. Since PCR assays are useful tools for screening large populations of mycotoxigenic fungi in grain or processed foods they may be used as part of routine monitoring programmes for mycotoxins.
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1.5 Acknowledgements
This work was supported by Agencia Nacional de Investigación e Inovación (ANII), project number: INI-X-2010-2-2875. The authors thank to Ing. Agr. Ariel Bogliachini from Plan Nacional de Silos, Ministerio de Ganadería, Agricultura y Pesca for providing wheat grain samples from commercial field.
1.6 References Ackerman, M., Pereyra, S., Stewart, S., Mieres, J., 2002. Fusariosis de la espiga de trigo y cebada. Boletín de difusión del Instituto Nacional de Investigación Agropecuaria (INIA), Nº 79.
ACCEPTED MANUSCRIPT Alvarez, C., Azcarate, M., Fernandez Pinto, V., 2009. Toxigenic potential of Fusarium graminearum sensu stricto isolates from wheat in Argentina.
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Astolfi, P., dos Santos, J., Schneider, L., Gomes, L., Silva, C., Tessman, D., Del
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Ponte, E., 2011. Molecular survey of trichothecene genotypes of Fusarium graminearum species complex from barley in Southern Brazil. International
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Journal of Food Microbiology 148, 197-201.
Desjardins, A., Jarosz, A., Plattner, R., Alexander, J., Brown, D., Jurgenson, J., 2004. Patterns of trichothecene production, genetic variability, and virulence to
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wheat of Fusarium graminearum from smallholder farms in Nepal. Journal of Agricultural and Food Chemistry 52, 6341-6346.
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Desjardins, A., 2006. Fusarium mycotoxins: chemistry, genetics, and biology.
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APS Press, St. Paul, MN.
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Desjardins, A., Proctor, R., 2011. Genetic diversity and trichothecene chemotypes of the Fusarium graminearum clade isolated from maize in Nepal and identification of a putative new lineage. Fungal Biology 115, 38-48.
Eudes, F., Comeau, A., Rioux, S., Collin, J., 2000. Phytotoxicity of eight mycotoxins associated with Fusarium in wheat head blight. Canadian Journal of Plant Pathology 22, 286-292.
Gilbert, J., Clear, R., Ward, T., Gaba, D., Tekauz, A., Turkington, T., Woods, S., Nowicki, T., O`Donnell, K., 2010. Relative aggressiveness and production of 3or 15-acetyl deoxynivalenol by Fusarium graminearum in spring wheat. Canadian Journal of Plant Pathology 32, 146-152.
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Goswami, R. S., Kistler, H. C., 2005. Pathogenicity and in planta mycotoxin accumulation among members of the Fusarium graminearum species complex
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on wheat and rice. Phytopathology 95, 1397-1404.
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Jennings, P., Coates, M. E., Walsh, K., Turner, J. A. Nicholson, P., 2004. Determination of deoxynivalenol and nivalenol producing chemotypes of
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Fusarium graminearum isolated from wheat crops in England and Wales. Plant Pathology 53, 643-652.
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Identification of deoxynivalenol and nivalenol producing chemotypes of Gibberella zeae by using PCR. Applied and Environmental Microbiology 67,
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determine deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae. Applied and Environmental Microbiology 68, 2148-2154.
Lee, S., Taylor, J. 1990. Isolation of DNA from fungal mycelia and single spores. In: PCR protocols: a guide to methods and applications. Innis, M.,
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Gelfand, D., Snindky, J., White, J. eds., Academic Press, San Diego, California 282-287.
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Pan, D., Graneri, J., Bettucci, L. 2009. Correlation of rainfall and levels of deoxynivalenol in wheat from Uruguay, 1997-2003. Food Additives and Contaminants: Part B 2, 162-165. Pan, D., Reynoso, M.M., Torres, A. 2010. Population genetic study of Gibberella zeae (Fusarium graminearum) isolated from wheat in Uruguay. In: Proceedings of 6th Latin American Congress of Mycotoxicology, Mérida, Yucatán, Mexico. June 2010.
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Prodi, A., Tonti, S., Nipoti, P., Pancaldi, D., Pisi, A., 2009. Identification of deoxynivalenol and nivalenol producing chemotypes of Fusarium graminearum
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isolates from durum wheat in a restricted area of northern Italy. Journal of Plant
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Pathology 91, 727-731.
Puri, K.D., Zhong, S., 2010. The 3ADON population of Fusarium graminearum
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found in North Dakota is more aggressive and produces a higher level and DON than the prevalent 15ADON population in spring wheat. Phytopathology 100, 1007-1014.
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Quarta, A., Mita, G., Haidukowski, M., Santino, A., Mule, G., Visconti, A., 2005. Further data on trichothecene chemotypes of European Fusarium culmorum
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Multiplex PCR assay for the identification of nivalenol, 3-, and 15-acetyl deoxynivalenol chemotypes in Fusarium. FEMS Microbiology Letters 259, 7-13.
Reynoso, M. M., Ramirez, M. L., Torres, A., Chulze, S., 2011. Trichothecene genotypes and chemotypes in Fusarium graminearum strains isolated from
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Scoz, L. B., Astolfi, P., Reartes, D. S., Schmale III, D. G., Moraes, M. G., Del Ponte, E. M., 2007. Trichothecene mycotoxin genotypes of Gibberella zeae in Brazilian wheat. Plant Pathology 58, 344–351.
Suga, H., Karugia, G., Ward, T., 2008. Molecular characterization of the Fusarium graminearum species complex in Japan. Phytopathology 98, 159-66.
von der Ohe, C., Gauthier, V., Tamburic-Ilincic, L., Brule-Babel, A., Fernando, W.G.D., Clear, R., Ward, T.J., Miedaner, T., 2010. A comparison of aggressiveness and deoxynivalenol production between Canadian Fusarium
ACCEPTED MANUSCRIPT graminearum isolates with 3-acetyl and 15-acetyldeoxynivalenol chemotypes in field-grown spring wheat. European Journal of Plant Pathology 127, 407-417
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Waalwijk, C., Kastelein, P., De Vries, I., Kerenyi, Z., van der Lee, T., Hesselink, T., Köhl, J.,Kema, G. H. J., 2003. Major changes in Fusarium spp. in wheat in
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Ward, T. J., Bielawski, J. P., Kistler, H. C., Sullivan, E., O’Donnell, K., 2002. Ancestral polymorphism and adaptive evolution in the trichothecene mycotoxin gene cluster of phytopathogenic Fusarium. Proceedings of the National Academy of Sciences (USA) 99, 9278–9283.
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Ward, T., Clear, R., Rooney, A., O’Donnell, K., Gaba, D., Patrick, S., Starkey, D., Gilbert, J., Geiser, D., Nowicki T. 2008. An adaptive evolutionary shift in
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ACCEPTED MANUSCRIPT Highlights We provided the first data on trichothecene genotypes of F. graminearun from Uruguay.
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A multiplex PCR assay was used to analyzed 111 strains isolate during 2003
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and 2009.
Most of the strains with the DON genotype had the 15-AcDON genotype in
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2003 and 2009.
No 3-AcDON genotype were found between 2003 and the 2009 epidemic FHB harvest season.
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No potential shifts were found in the thrichothecene genotypes between 2003 and 2009.
S0168-1605(13)00012-3 doi: 10.1016/j.ijfoodmicro.2013.01.002 FOOD 6084
To appear in:
International Journal of Food Microbiology
Received date: Revised date: Accepted date:
20 September 2012 8 December 2012 4 January 2013
Please cite this article as: Pan, Dinorah, Calero, Natalia, Mionetto, Ana, Bettucci, Lina, Trichothecene genotypes of Fusarium graminearum from wheat in Uruguay, International Journal of Food Microbiology (2013), doi: 10.1016/j.ijfoodmicro.2013.01.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Trichothecene genotypes of Fusarium graminearum from wheat in Uruguay
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Dinorah Pan *, Natalia Calero, Ana Mionetto and Lina Bettucci.
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* Laboratorio de Micología, Facultad de Ciencias - Facultad de Ingeniería,
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MA
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UdelaR, Julio Herrera y Reissig 565, 11200 Montevideo, Uruguay
* Corresponding autor: Dra. Dinorah Pan, Laboratorio de Micología, Facultad de Ciencias - Facultad de Ingeniería, UdelaR, Julio Herrera y Reissig 565, 11200 Montevideo, Uruguay. Phone/Fax: ++ 59827120626; E-mail: [email protected]
ACCEPTED MANUSCRIPT Abstract Gibberella zeae (Scwein.) Petch (anamorph F. graminearum Schwabe) is the primary causal agent of FHB of wheat in Uruguay. In the last decade, F.
PT
graminearum has produced destructive epidemics on wheat in Uruguay, causing yield losses and price discounts due to reduced seed quality. Strains of
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F. graminearum clade usually express one of three strain-specific profiles of trichothecene metabolites: nivalenol and its acetylated derivatives (NIV
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chemotype), deoxynivalenol and 3-acetyldeoxynivalenol (3-AcDON chemotype), or deoxynivalenol and 15-acetyldeoxynivalenol (15-AcDON chemotype).
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multiplex PCR assay of Tri3, Tri5, and Tri7 was used to determine the trichothecene genotype of 111 strains of F. graminearum collected during 2003
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and 2009 growing season from fields located in the major wheat production area of Uruguay. The result showed that all except one of the isolates were of DON genotype, with the remainder of NIV genotype in years 2003 and 2009. All
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strains with the DON genotype were also of the 15-AcDON genotype in 2003 and nearly all (45/50) in 2009. No DON/ 3-AcDON genotypes were found in
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either growing season. No potential shifts in the populations were found in the trichothecene genotypes between 2003 and the 2009 epidemic FHB harvest season. This study provides the first data on trichothecene genotypes of F. graminearum strains isolated from wheat in Uruguay and add to the current
AC
regional knowledge of trichothecene genotypes.
Keywords:
Deoxynivalenol,
Nivalenol,
3-acetyldeoxynivalenol,
acetyldeoxynivalenol, Fusarium graminearum, wheat
15-
ACCEPTED MANUSCRIPT 1.1 Introduction Fusarium head blight (FHB) is one of the most economically important fungal diseases of wheat worldwide (Goswami and Kistler, 2005). Gibberella zeae
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(Scwein.) Petch (anamorph F. graminearum Schwabe) is the primary causal agent of FHB of wheat and barley in Uruguay (Ackerman et al., 2002). It
RI
manifests as an epiphytic disease that results in a decrease in both germination ability and grain weight. At the same time, this species is able to contaminate
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grains with trichothecene mycotoxins such as deoxynivalenol (DON) and nivalenol (NIV) that constitute a health risks to both humans and domesticated animals (Zeller et al., 2003). Both DON and NIV also are potent phytotoxins and play a role in pathogenesis of F. graminearum (Edues et al., 2001).
MA
In the last decade, F. graminearum has caused destructive epidemics on wheat in Uruguay, causing yield losses and price discounts due to reduced seed quality. During the harvest seasons of 2001, 2002 and 2009 coincidentally with
ED
flowering to early stages of grain maturity, a heavy rainfall period occurred and a high rate of FHB was observed, causing levels of DON contamination of 6593
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µg/kg, 5880 µg/kg and 7652 µg/kg, respectively (Pan et al., 2009). Strains of the F. graminearum clade usually express one of three strain-specific profiles of trichothecene metabolites: nivalenol and its acetylated derivatives (NIV chemotype), deoxynivalenol and 3-acetyldeoxynivalenol (3-AcDON chemotype), or deoxynivalenol and 15-acetyldeoxynivalenol (15-AcDON
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chemotype) (Ward et al. 2002). PCR assays have been developed to rapidly assess trichothecene genotypes of F. graminearum. Such assays rely on the amplification of gene portions that code for key enzymes involved in trichothecene biosynthesis (Quarta et al., 2006; Ward et al., 2002). Primers based on the sequences of alleles at Tri3, Tri5 and Tri7 (Quarta et al., 2006) were designed to differentiate three toxin genotypes (3-AcDON, 15-AcDON and NIV) and on Tri7 and Tri13 (Lee et al. 2001, 2002) to distinguish DON and NIV producers. Since wheat represents the major staple food for Uruguayan people, the main aim of this work was to determine and evaluate any potential shift in the trichothecene mycotoxin genotypes (3-AcDON, 15-AcDON and NIV) in F. graminearum isolates obtained from two years’ sampling of wheat grains from Uruguay.
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1.2 Materials and methods
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1.2.1 Sampling
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Fusarium species were isolated from 93 and 26 samples of wheat grains harvest during 2003 and 2009 growing season, respectively, from commercial
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fields located in the major wheat production area of Uruguay (Fig.1). Samples weighting 500 g, obtained after harvesting operations, were stored at -20ºC in
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paper bags in the laboratory.
Figure 1. Sampling sites where wheat grains were obtained.
1.2.2 Fungal isolation and identification
Grains of each sample were surface-disinfected by soaking in 1 % aqueous NaOCl for 1 min, and then rinsed three times with sterile water. One hundred grains
were
plated
(10
grains
per
Petri
dish)
on
peptone
+
pentachloronitrobenzene (PCNB) medium. These plates were incubated at 24ºC for 7 days with 12/12 h photoperiod under cool with and black light fluorescent lamps (Leslie and Summerell, 2006). All Fusarium isolates were
ACCEPTED MANUSCRIPT identified based on conidial morphology according to Leslie and Summerell (2006). The isolates of F. graminearum were confirmed by their ability to produce homothallic perithecia on carrot agar and /or carnation leaf agar and
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also identified as members of F. graminearum lineage 7 by AFLP analyses (Pan et al. 2010). Sixty one isolates of F. graminearum were obtained from locations
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of Soriano (21), San José (5), Río Negro (10), Colonia (15) and Flores (10) in the year 2003 and 50 isolates from locations of Soriano (3), San José (32),
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Colonia (14) and Florida (1) in 2009. The isolates were maintained as spore suspensions in 10% glycerol and frozen at -80ºC. Duplicate subcultures of each isolate are held in the culture collection of the Laboratorio de Micología, Facultad de Ciencias – Facultad de Ingeniería, Universidad de la República,
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Uruguay.
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1.2.3 DNA isolation
Isolates of F. graminearum were grown potato dextrose agar (PDA) plates for 5-
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6 days and genomic DNA was extracted from aerial mycelium using a cetyltrimethylamonium bromide (CTAB) method (Lee and Taylor, 1990).
1.2.4 Trichothecene genotype determination
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Trichothecene genotypes of F. graminearum were identified using a suite of primers directed to portions of genes that are predictive of isolates producing DON, NIV, 3-AcDON and 15-AcDON. For the identification of DON- producing isolates the primer pair 3551H/4056H was selected based on the Tri5 gene sequences. For the identification of 3-AcDON, 15-AcDON and NIV genotype, the primer sets Tri3F971/Tri3R1679, Tri3F1325/Tri3R1679 and Tri7F340/ Tri7R965 directed to portions of Tri3 and Tri7 genes were used. PCR products of 525 bp, 708 bp, 354 bp and 625 bp are produced for DON, 15-AcDON, 3AcDON and NIV, respectively (Quarta et al., 2005, 2006). Multiplex PCR experiments were conducted with 25 ng of fungal DNA in a total volume of 50 mL of 1× reaction buffer containing 1.5 mM MgCl2, 5 U Taq DNA polymerase (Promega), 200 mM dNTPs, 0.2 mM each of the Tri3 primers (Tri3F971, Tri3F1325 and Tri3R1679), and 0.1 mM each of primers Tri7F340,
ACCEPTED MANUSCRIPT Tri7R965, 3551H and 4056H (Quarta et al. 2005, 2006). A negative control, containing all reagents and primers but no fungal DNA was included with every set of reactions. PCR was conducted in a PTC-2000 Thermal Cycler (MJ
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Research Inc., Watertown, MA). The PCR conditions were 94ºC, 3 min then 35 cycles of 94ºC, 30 s, 53ºC, 30 s, 72ºC, 1 min, followed by a final extension step
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of 10 min, 72ºC. PCR products were separated by electrophoresis through 2% agarose gels. Gels were stained with 1 mg/ml ethidium bromide and
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photographed under UV light. DNA from strains with known NIV, 3-AcDON and 15-AcDON chemotypes were used as positive controls.
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1.3 Results
A total of 111 single-spore isolates of F. graminearum were recovered from wheat grain from different localities. Sixty one isolates were obtained in the
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2003 growing season and 50 in 2009. All isolates were identified as members of F. graminearum sensu stricto (lineage 7).
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The multiplex PCR assay demonstrated that all except one isolate were of DON genotype and the remainder belong to the NIV genotype in 2003 and 2009 years. These assays demonstrated that all strains with DON genotype were of the 15-AcDON genotype in 2003 and in nearly all the strains (45/50) in 2009. In this latter year, 4 strains showed DON genotype because they yielded only a
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525 bp product as described by Quarta et al. (2006). No DON/ 3-AcDON genotypes occurred in either growing seasons (Table 1).
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Table 1. Number of trichothecene genotypes of F. graminearum strains collected from kernels of wheat during two growing seasons.
2003
60
DON/ 15-AcDON
1
NIV
45 4
-
+
-
-
+
-
DON/ 15-AcDON
-
+
-
+
DON
-
+
-
-
NIV
-
-
+
-
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+
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1
-
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2009
Amplified fragment (bp) 354 525 625 708
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PCR genotype
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Nº of isolates
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Year
1.4 Discussion
This study provides the first data on trichothecene genotypes of F. graminearum
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strains isolated from wheat in Uruguay. We found that among the populations evaluated in 2003 and 2009 the predominant genotype was DON/15- AcDON. The high DON levels found in samples of wheat grain analyzed (Pan et al., 2009) suggests that the degree of contamination represents a high risk to consumers. For this reason the presence of 15-AcDON in the Uruguay Fusarium
population
is
of
great
health
importance
requiring
further
epidemiological studies to determine conditions favoring production of this mycotoxin and the frequency and distribution of these mycotoxins in food for human and animal consumption. Our results are similar to those found for strains of F. graminearum in South America (Alvarez et al., 2009; Astolfi et al., 2011; Reynoso et al., 2011; Scoz et al., 2008). A recent study in Argentina revealed that DON/ 15-AcDON were the primary trichothecenes produced by F. graminearum in wheat grains (Reynoso
ACCEPTED MANUSCRIPT et al., 2011). In Brazil, the DON/15-AcDON genotype also seems to predominate in isolates from wheat, although a few strains corresponded to NIV genotype (Scoz et al., 2008).
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In Europe there is more variability. In western Russia and Finland, all F. graminearum isolates were found to be DON/3-AcDON (Yli-Mattila et al., 2008),
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whereas in southern Russia, England, the Netherlands and Italy the predominant genotype was DON/15-AcDON (Jennings et al., 2004; Prodi et al.,
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2009; Waalwijk et al., 2003).
In Korea, Japan, China and other parts of Asia, the NIV genotype is the most common and both DON genotypes are rare (Desjardins et al., 2008; Suga et al., 2008). During our research, isolates of the NIV genotype were not frequently
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identified. The low frequency of strains of NIV genotype in 2003 and 2009 is of special interest because NIV is more toxic than DON in most animal systems (Desjardins, 2006). Furthermore, some studies suggest that NIV producers are
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less virulent than DON producers indicating that the production of DON should confer a strong selective advantage to this plant pathogen in wheat (Desjardins Eudes et al., 2000). As the F.
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and Proctor, 2011; Desjardins et al., 2004;
graminearum population is dominated by the DON/15-AcDON genotype, the level of virulence on wheat may affect the population structure of this pathogen in Uruguayan agroecosystems. Most of the strains of F. graminearum evaluated in 2003 and 2009 showed the
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DON/15-AcDON genotype. This finding is important considering that recent data on F. graminearum populations from USA and Canada indicated that the 3-AcDON genotype was more aggressive in susceptible and resistant wheat genotypes and also exhibits a higher in vitro DON production than the 15AcDON isolates (Puri and Zhong, 2010; von der Ohe et al. 2010; Ward et al., 2008) A small percentage of isolates in 2009 showed the DON genotype using multiple PCR as described by Quarta et al. (2006). This DON genotype is unexpected and suggests that these strains can produce only DON. Strains that produce DON and no acetylated derivatives (DON chemotype) had been previously reported for isolates of F. graminearum sensu stricto in Argentina (Alvarez et al., 2009). Further investigation is necessary in order to characterize these strains.
ACCEPTED MANUSCRIPT No potential shifts in the populations of the genotypes of F. graminearum isolates were found between the 2003 and 2009 growing seasons, even though in the latter year an important epidemic of FHB occurred in Uruguay. This fact
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is especially important because it was recently shown that 3-AcDON genotypes seem to be displacing 15-AcDON in the F. graminearum populations from USA,
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Canada, Finland and the Russian Far East (Gilbert et al., 2010; Prodi et al., 2009; Ward et al., 2008; Yli-Mattila et al., 2009).The study reported here
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provides the first data on trichothecene genotypes of F. graminearum strains isolated from wheat in Uruguay during an FHB epidemic year and during a year when there was no FHB epidemic.
Continuous monitoring of Uruguayan F. graminearum isolates is important to
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understand epidemiological factors that may contribute in the future to the distribution of trichothecene genotypes and to detect if potential shifts exist among isolates of acetylated forms of DON and NIV. Moreover, in terms of
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epidemiological studies the detection of NIV genotype was a very important finding, so it is necessary to examine the frequency and distribution of NIV
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producers in Uruguay and the conditions that favour mycotoxin production. Since PCR assays are useful tools for screening large populations of mycotoxigenic fungi in grain or processed foods they may be used as part of routine monitoring programmes for mycotoxins.
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1.5 Acknowledgements
This work was supported by Agencia Nacional de Investigación e Inovación (ANII), project number: INI-X-2010-2-2875. The authors thank to Ing. Agr. Ariel Bogliachini from Plan Nacional de Silos, Ministerio de Ganadería, Agricultura y Pesca for providing wheat grain samples from commercial field.
1.6 References Ackerman, M., Pereyra, S., Stewart, S., Mieres, J., 2002. Fusariosis de la espiga de trigo y cebada. Boletín de difusión del Instituto Nacional de Investigación Agropecuaria (INIA), Nº 79.
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Astolfi, P., dos Santos, J., Schneider, L., Gomes, L., Silva, C., Tessman, D., Del
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Desjardins, A., Jarosz, A., Plattner, R., Alexander, J., Brown, D., Jurgenson, J., 2004. Patterns of trichothecene production, genetic variability, and virulence to
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wheat of Fusarium graminearum from smallholder farms in Nepal. Journal of Agricultural and Food Chemistry 52, 6341-6346.
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Desjardins, A., 2006. Fusarium mycotoxins: chemistry, genetics, and biology.
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Desjardins, A., Busman, M., Manandhar, G., Jarosz, G., Manandhar, H., proctor, R., 2008.Gibberella ear rot of maize (Zea mays) in Nepal: distribution of the mycotoxins nivalenol and deoxynivalenol in naturally and experimentally
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Desjardins, A., Proctor, R., 2011. Genetic diversity and trichothecene chemotypes of the Fusarium graminearum clade isolated from maize in Nepal and identification of a putative new lineage. Fungal Biology 115, 38-48.
Eudes, F., Comeau, A., Rioux, S., Collin, J., 2000. Phytotoxicity of eight mycotoxins associated with Fusarium in wheat head blight. Canadian Journal of Plant Pathology 22, 286-292.
Gilbert, J., Clear, R., Ward, T., Gaba, D., Tekauz, A., Turkington, T., Woods, S., Nowicki, T., O`Donnell, K., 2010. Relative aggressiveness and production of 3or 15-acetyl deoxynivalenol by Fusarium graminearum in spring wheat. Canadian Journal of Plant Pathology 32, 146-152.
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Goswami, R. S., Kistler, H. C., 2005. Pathogenicity and in planta mycotoxin accumulation among members of the Fusarium graminearum species complex
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Jennings, P., Coates, M. E., Walsh, K., Turner, J. A. Nicholson, P., 2004. Determination of deoxynivalenol and nivalenol producing chemotypes of
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Pan, D., Graneri, J., Bettucci, L. 2009. Correlation of rainfall and levels of deoxynivalenol in wheat from Uruguay, 1997-2003. Food Additives and Contaminants: Part B 2, 162-165. Pan, D., Reynoso, M.M., Torres, A. 2010. Population genetic study of Gibberella zeae (Fusarium graminearum) isolated from wheat in Uruguay. In: Proceedings of 6th Latin American Congress of Mycotoxicology, Mérida, Yucatán, Mexico. June 2010.
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Prodi, A., Tonti, S., Nipoti, P., Pancaldi, D., Pisi, A., 2009. Identification of deoxynivalenol and nivalenol producing chemotypes of Fusarium graminearum
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Puri, K.D., Zhong, S., 2010. The 3ADON population of Fusarium graminearum
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Quarta, A., Mita, G., Haidukowski, M., Santino, A., Mule, G., Visconti, A., 2005. Further data on trichothecene chemotypes of European Fusarium culmorum
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Quarta, A., Mita G., Haidukowski, M., Logrieco, A., Mule, G., Visconti, A., 2006.
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Multiplex PCR assay for the identification of nivalenol, 3-, and 15-acetyl deoxynivalenol chemotypes in Fusarium. FEMS Microbiology Letters 259, 7-13.
Reynoso, M. M., Ramirez, M. L., Torres, A., Chulze, S., 2011. Trichothecene genotypes and chemotypes in Fusarium graminearum strains isolated from
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Scoz, L. B., Astolfi, P., Reartes, D. S., Schmale III, D. G., Moraes, M. G., Del Ponte, E. M., 2007. Trichothecene mycotoxin genotypes of Gibberella zeae in Brazilian wheat. Plant Pathology 58, 344–351.
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ACCEPTED MANUSCRIPT graminearum isolates with 3-acetyl and 15-acetyldeoxynivalenol chemotypes in field-grown spring wheat. European Journal of Plant Pathology 127, 407-417
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Waalwijk, C., Kastelein, P., De Vries, I., Kerenyi, Z., van der Lee, T., Hesselink, T., Köhl, J.,Kema, G. H. J., 2003. Major changes in Fusarium spp. in wheat in
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Ward, T. J., Bielawski, J. P., Kistler, H. C., Sullivan, E., O’Donnell, K., 2002. Ancestral polymorphism and adaptive evolution in the trichothecene mycotoxin gene cluster of phytopathogenic Fusarium. Proceedings of the National Academy of Sciences (USA) 99, 9278–9283.
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Ward, T., Clear, R., Rooney, A., O’Donnell, K., Gaba, D., Patrick, S., Starkey, D., Gilbert, J., Geiser, D., Nowicki T. 2008. An adaptive evolutionary shift in
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ACCEPTED MANUSCRIPT Highlights We provided the first data on trichothecene genotypes of F. graminearun from Uruguay.
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A multiplex PCR assay was used to analyzed 111 strains isolate during 2003
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and 2009.
Most of the strains with the DON genotype had the 15-AcDON genotype in
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2003 and 2009.
No 3-AcDON genotype were found between 2003 and the 2009 epidemic FHB harvest season.
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No potential shifts were found in the thrichothecene genotypes between 2003 and 2009.