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Species diversity and toxigenic potential of Fusarium graminearum complex isolates from maize fields in northwest Argentina
International Journal of Food Microbiology 145 (2011) 359–364
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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 ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Short Communication
Species diversity and toxigenic potential of Fusarium graminearum complex isolates from maize fields in northwest Argentina D.A. Sampietro a,⁎, C.G. Díaz b, V. Gonzalez c, M.A. Vattuone a, L.D. Ploper c, C.A.N. Catalan a, T.J. Ward d a
INQUINOA - CONICET, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, España 2903, 4000, San Miguel de Tucumán, Argentina Facultad de Agronomía y Zootecnia, Universidad Nacional de Tucumán, España 2903, 4000, San Miguel de Tucumán, Argentina Sección Fitopatología, Estación Experimental Agroindustrial Obispo colombres, William Cross 3150 (T4101XAC), Las Talitas, Tucumán, Argentina d Bacterial Foodborne Pathogens and Mycology Research Unit, U.S. Department of Agriculture, Agricultural Research Service, 1815 N. University St., Peoria, IL 61604, USA b c
a r t i c l e
i n f o
Article history: Received 19 August 2010 Received in revised form 7 December 2010 Accepted 22 December 2010 Keywords: ASPE Fusarium meridionale Fusarium boothii Multilocus genotyping Trichothecene Population genetics
a b s t r a c t Members of the Fusarium graminearum species complex (Fg complex) are the causal agents of ear rot in maize and Fusarium head blight of wheat and other small grain cereals. The potential of these pathogens to contaminate cereals with trichothecene mycotoxins is a health risk for both humans and animals. A survey of ear rot isolates from maize collected in northwest Argentina recovered 66 isolates belonging to the Fg complex. A multilocus genotyping (MLGT) assay for determination of Fg complex species and trichothecene chemotypes was used to identify 56 of these isolates as F. meridionale and 10 isolates as F. boothii. F. meridionale was fixed for the nivalenol (NIV) chemotype, and all of the F. boothii isolates had the 15acetyldeoxynivalenol (15ADON) chemotype. The results of genetic diversity analysis based on nine variable number tandem repeat (VNTR) loci supported the hypothesis of genetic isolation between F. meridionale and F. boothii, and provided little evidence of geographic substructure among populations of the dominant pathogen species, F. meridionale. This is the first study to indicate that F. meridionale and F. boothii may play a substantial role in the infection and trichothecene contamination of maize in Argentina. In addition, dominance of the NIV chemotype among Fg complex isolates from Argentina is unprecedented, and of significant concern to food safety and animal production. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Members of the Fusarium graminearum species complex (Fg complex) are responsible for diseases of a variety of cereal crops worldwide and are important grain rotting pathogens in the subtropical and temperate regions of Argentina (Chulze et al., 1996). Infection of cereal crops by these fungi significantly lowers grain yield and quality, and can result in the contamination of grain with B-trichothecenes. These mycotoxins are a significant risk to food safety and animal health because they inhibit DNA, RNA and protein synthesis in eukaryotic cells and can modify immune function (Pestka and Smolinski, 2005; Ueno et al., 1973; Rocha et al., 2005). In addition, trichothecenes can be acutely phytotoxic and act as virulence factors on sensitive cereal hosts (Jansen et al., 2005). Until recently, members of the Fg complex were considered a single cosmopolitan species (Booth, 1971; Gerlach and Nirenberg, 1982; Leslie and Summerell, 2006) due to the failure of morphological species recognition to accurately assess species limits for this group. However, studies employing phylogenetic species recognition based ⁎ Corresponding author. Tel.: +54 381 4247752x7082 7007. E-mail addresses: [email protected], [email protected] (D.A. Sampietro). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.12.021
on genealogical concordance (GCPSR) (Taylor et al., 2000) have demonstrated that F. graminearum sensu lato is a complex of at least 13 phylogenetically distinct species with marked biogeographic structure (O'Donnell et al., 2004, 2008; Starkey et al., 2007, YliMattila et al., 2009). Within the Fg complex, individual isolates can produce one of three strain-specific profiles (chemotypes) of Btrichothecene metabolites (Miller et al., 1991; O'Donnell et al., 2004): nivalenol and acetylated derivatives (NIV chemotype), deoxynivalenol and primarily 3-acetyldeoxynivalenol (3ADON chemotype), and deoxynivalenol and primarily 15-acetyldeoxynivalenol (15ADON chemotype). This variation is important to food safety, as differences in toxicity have been reported for different B-trichothecenes (Forsell and Pestka, 1985; Luongo et al., 2008). In addition, B-trichothecene chemotype differences have been maintained by natural selection and appear to have important consequences for pathogen fitness (Ward et al., 2002). Although members of the Fg complex pose a significant threat to cereal production and food safety worldwide, relatively little is known about the prevalence and distribution of Fg complex species and Btrichothecene chemotypes from maize in Argentina. F. graminearum sensu stricto was the only Fg complex species identified in analyses of isolates from wheat fields in the central region of Argentina (Scoz et al., 2009), and a recent study indicated that the 15ADON chemotype is
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predominant across the main wheat growing regions of the country (Alvarez et al., 2009). However, a preliminary analysis based on seven Fg complex isolates from maize in northwestern Argentina suggested additional species and trichothecene chemotype variation (Sampietro et al., 2010). The main objective of this study was to determine species, trichothecene chemotype, and population-level diversity among Fg complex isolates collected from maize in northwest Argentina in order to test the hypothesis that Fg complex diversity in Argentina is greater than previously recognized.
(Sampietro et al., 2010) in order to identify isolates that were morphologically consistent with the Fg complex. The morphology of macroconidia, and chlamydospores was assessed from cultures grown on SNA and CLA. Morphological identification of isolates was based on the criteria of Gerlach and Nirenberg (1982) and Leslie and Summerell (2006).
2. Materials and methods
Morphological variation within the Fg complex provides an insufficient basis for discerning species identity. Therefore, species identification was performed along with trichothecene chemotype determination using a previously published multilocus genotyping (MLGT) assay that is based on allele-specific primer extension (ASPE) with probes targeting species and trichothecene chemotype-specific genetic variation (O'Donnell et al., 2008; Ward et al., 2008; Yli-Mattila et al., 2009). MLGT was performed as described previously (Ward et al., 2008) and consisted of a multiplex amplification of six genomic regions containing species or trichothecene chemotype-specific nucleotide variation. Amplification products served as template for a multiplexed primer extension reaction. Biotinylated extension products from ASPE reactions were sorted by hybridization to a suspension microarray consisting of polystyrene microspheres coated with antitag sequences specific to 5′ sequence tags that were appended to each of the extension probes. Microsphere complexes were detected using a Luminex 100 flow cytometer (Luminex Corporation) and reactions were scored by fluorescence intensity and spectrum.
2.1. Maize plots and collection of fungal isolates Plots of commercial maize hybrids were cultivated in locations provided by Agro-Industrial Experimental Station Obispo Colombres (Tucumán, Argentina) to determine the natural incidence of Fg complex species and trichothecene chemotypes. Fields were located in three distinct zones (Fig. 1): South (La Cocha), East (Viclos, Monte Redondo, La Cruz, La Virginia, El Azul and Monte Quemado) and North (Trancas). Twenty-one maize hybrids were cultivated: AX1013 MG, NK120 TD MAX, NK135 TD MAX, NK138 TD MAX, DK 390 MG, DK 190 MG, AGRI 105, AGRI 103, SPS 1104, MASS636 Hx MG, Pioneer 30T17, DK 910 MG, Avant, 2A120 Hx, 2B710 Hx, 2B688 Hx, NK 3234, XTA8089, XTA5306, XTA80/5 and AGROMEN 31A31. Each hybrid plot consisted of 14 rows of 200 m length, with row spacing of 0.52 m. Plots were sown in the first week of January and harvested in the last week of July, 2007. Grain samples were randomly collected from each maize hybrid and analyzed in the laboratory as described previously
2.2. Species and trichothecene chemotype determination
Fig. 1. Geographical locations of Fusarium isolated from maize ears in Northwest Argentina during 2007. Numbers indicate sampling locations: 1) La Cocha; 2) Viclos; 3) Monte Redondo; 4) La Virginia; 5) La Cruz; 6) El Azul; 7) Monte Quemado; 8) Trancas.
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2.3. Analysis of population structure and diversity Genetic variation at nine variable number tandem repeat (VNTR) loci (Suga et al., 2004) was used to assess population structure and diversity. Forward primers for each VNTR locus were synthesized with fluorescent labels (Applied Biosystems) and amplifications were performed in three multiplex reactions as described previously (Ward et al., 2008). Reaction products were scored relative to a GS500 ROX (Applied Biosystems) internal size standard using an ABI3100 Genetic Analyzer with GENEMAPPER 3.7 software (Applied Biosystems). FST based genetic distance (Michalakis and Excoffier, 1996) values were estimated using the program ARLEQUIN 3.5 (Excoffier et al., 2005). Pairwise FST distance estimates were calculated based on the number of different alleles, and the statistical significance of pairwise FST estimates were assessed using a permutation test with 10,000 permutations. Population subdivision was also analysed using the Bayesian model-based clustering procedure implemented in STRUCTURE 2.3 (Pritchard et al., 2000), which assigns isolates to a user-defined number of population clusters (K) based on their multilocus genotypes. The Bayesian clustering analyses were performed for values of K from 1 to 5, and utilized 100,000 step Markov Chains after a burn-in period of 10,000 iterations. 3. Results 3.1. Ocurrence of Fg complex species and chemotypes in maize grains A total of 66 isolates collected from maize grains in northwest Argentina during 2007 were identified by morphological analysis as members of the Fg species complex (Table 1). A previously published MLGT assay was used to investigate species and trichothecene chemotype diversity among these isolates. Using the MLGT assay, we identified 85% (56/66) of the isolates as F. meridionale, while the remainder (15%, 10/66) were identified as F. boothii. In contrast to expectations based on previous reports, F. graminearum sensu stricto was not represented among the isolates examined. F. meridionale isolates were obtained from all eight sampling locations. However, nine of the 10 F. boothii isolates were obtained from a single location (Trancas) in the North, and the remaining isolate was obtained from one of the six locations (Viclos) sampled in the East. The 56 F. meridionale shared the NIV chemotype, while the 10 F. boothii isolates shared the 15ADON chemotype. 3.2. Analysis of genetic diversity and population structure Genetic variation at nine VNTR loci was analyzed to assess diversity and population structure. Five of the nine VNTR loci were polymorphic within F. meridionale, and four loci were polymorphic within F. boothii (Table 2). Thirteen unique haplotypes were identified among the 56 F. meridionale, and seven unique haplotypes were identified among the 10 F. boothii isolates (Table 2). Only two loci (HK1073 and HK917) were polymorphic in both species, and two loci were monomorphic across all 66 isolates. Substantial genetic divergence was observed between F. meridionale and F. boothii (FST = 0.73, P b 0.001). In addition, 26 of the 29 (89.7%) alleles identified among the seven polymorphic loci were unique to one of the two species. No significant population structure was observed within F. meridionale. Using the Bayesian clustering approach to assess population structure among the 56 F. meridionale isolates, a single population (K = 1) model provided the best fit to the observed data. Setting K N 1 failed to improve likelihood scores and resulted in most individuals being assigned equally to each of the user-defined populations. Similar results were obtained when we specifically tested the hypothesis of geographic structuring after assigning
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F. meridionale isolates into three groups based on geographic origin (North, South, and East; FST b 0.04; P N 0.24). These results indicated that F. meridionale isolates from the sampled regions behave as a single population. Similar analyses for F. boothii were not possible due to the small number of isolates. 4. Discussion The results reported here demonstrate that Fg complex diversity in Argentina is greater than previously recognized. They also indicate for the first time that F. meridionale and F. boothii may play a substantial role in the infection and trichothecene contamination of Argentinian maize. In contrast, previous analyses of Fg complex isolates collected from wheat in the center of Argentina identified all 113 isolates as F. graminearum sensu stricto (Ramirez et al., 2007). F. graminearum sensu stricto was also the dominant species (92.7%) isolated from wheat in southern Brazil; however F. meridionale accounted for six (7.3%) of the 82 isolates examined (Scoz et al., 2009). The dramatic differences in chemotypes and Fg complex species composition observed among wheat in center Argentina and the maize isolates collected in the current work from the northwest is suggestive of a potential difference in host preference within the Fg complex. However, interpretations are limited by the small number of studies published to date that incorporate molecular differentiation of species within the Fg complex. Trichothecene chemotype diversity among the isolates examined was directly tied to species differences. All of the F. meridionale had the NIV chemotype, while all F. boothii isolates had the 15ADON chemotype. These results are consistent with previous analyses of mycotoxin production in these two species (Ward et al., 2002). However, this situation contrasts with recent analyses of F. graminearum sensu stricto and F. asiaticum, in which trichothecene chemotype polymorphism was detected within each species and correlated with population subdivision (Gale et al., 2007; Ward et al., 2008; Yli-Mattila and Gagkaeva, 2010; Zhang et al., 2010). In a previous analysis of trichothecene production by Fg complex isolates collected from maize in Argentina, Molto et al. (1997) reported that all isolates produced DON. Nivalenol was detected in only one of 120 wheat samples collected in the northern Buenos Aires province of Argentina (González et al., 2008), and the NIV chemotype was absent from a recent survey of Fg complex isolates collected from across the major wheat production area in Argentina (Alvarez et al., 2009). The 15ADON chemotype predominated in a recent analysis of species and trichothecene chemotype diversity in southern Brazil, although the six F. meridionale that were identified in that study were found to have the NIV chemotype (Scoz et al., 2009). Interestingly, a previous compilation of global data reported substantially lower levels of NIV contamination in food and feed as compared to DON, but also found that Brazil was a rare exception in that DON and NIV contamination levels were similar (Placinta et al., 1999). Taken together with the data from the current study, these reports suggest that while DON is likely to be the predominant trichothecene contaminate of cereals in South America, the potential exists for substantial NIV contamination. This has significant implications for food safety as NIV appears to be more cytotoxic than DON (Forsell and Pestka, 1985; Luongo et al., 2008). In response to evidence of higher toxicity for NIV, the European Scientific Committee on Food has proposed a lower tolerance limit for NIV than for DON (Schothorst and Van Egmond, 2004). F. meridionale and F. boothii, along with the other members of the Fg complex, were originally described on the basis of genealogical exclusivity evident in phylogenetic trees derived from analyses of the DNA sequences of multiple single-copy nuclear genes (O'Donnell et al., 2000, 2004, 2008; Starkey et al., 2007, Yli-Mattila et al., 2009). These results are indicative of an extended history of genetic isolation consistent with the establishment of species boundaries. However, a
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Table 1 Geographic origin, host maize hybrid, Fg complex species and trichothecene chemotype of isolates collected in Northwest Argentina. Isolate
Geographic origin
Maize hybrid
Species Identity
Trichothecene chemotype
F1 F2 F3 F4 F5 F9 F10 F13 F14 F15 F16 F21 F23 F24 F26 F28 F29 F30 F31 F32 F33 F34 F35 F36 F37 F38 F39 F40 F41 F42 F44 F45 F46 F49 F50 F51 F52 F53 F55 F56 F58 F59 F62 F63 F64 F65 F66 F67 F68 F69 F73 F74 F75 F76 F77 F78 F79 F80 F81 F82 F83 F85 F86 F89 F90 F92
Viclos (east) Viclos (east) Viclos (east) Viclos (east) Viclos (east) Viclos (east) Viclos (east) La Cruz (east) La Virginia (east) La Virginia (east) Monte Redondo (east) Monte Redondo (east) Monte Redondo (east) Monte Redondo (east) Monte Redondo (east) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) Trancas (north) Trancas (north) Trancas (north) Trancas (north) Trancas (north) Trancas (north) Trancas (north) Trancas (north) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) La Cruz (east) Monte Redondo (east) Monte Redondo (east) Monte Quemado (east) Monte Quemado (east) Trancas (north) Trancas (north) Trancas (north) Trancas (north) Trancas (north) El Azul (east) El Azul (east) El Azul (east) Trancas (north) Trancas (north) Monte Redondo (east)
AX 1013 MG AX 1013 MG NK 135 TD MAX NK 135 TD MAX NK 120 TD MAX DK 190 MG NK 138 TD MAX NK 120 TD MAX NK 138 TD MAX NK 138 TD MAX AGRI 105 AGRI 105 NK 120 TD MAX NK 120 TD MAX 2B 710 Hx MASS 636 Hx MG NK 135 TD MAX NK 135 TD MAX NK 120 TD MAX NK 120 TD MAX AX 1013 MG AX 1013 MG PIONEER 30T17 Hx PIONEER 30T17 Hx DK 390 MG AGROMEN 31A31 AGROMEN 31A31 AX 1013 MG AX 1013 MG DK 390 MG DK 910 MG NK 138 TD MAX NK 138 TD MAX NK 120 TD MAX NK 120 TD MAX NK 138 TD MAX NK 138 TD MAX NK 120 TD MAX 2B 710 Hx AGRI 105 2A 120 Hx 2A 120 Hx DK 910 MG DK 910 MG SPS 1104 SPS 1104 PIONEER 30T17 Hx PIONEER 30T17 Hx MASS 636 Hx MG MASS 636 Hx MG 2B 688 AVANT 2B 688 XTA 8089 2B 688 AVANT XTA 5306 NK 3234 2B 688 XTA 80/5 2B 688 NK 3234 AVANT NK 138 TD MAX DK 390 MG NK 120 TD MAX
F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. boothii F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. boothii F. boothii F. boothii F. boothii F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. boothii F. meridionale F. boothii F. meridionale F. boothii F. meridionale F. meridionale F. meridionale F. boothii F. boothii F. meridionale
NIV NIV NIV NIV NIV NIV 15ADON NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV 15ADON 15ADON 15ADON 15ADON NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV 15ADON NIV 15ADON NIV 15ADON NIV NIV NIV 15ADON 15ADON NIV
lack of morphologically diagnostic characters and some evidence of interfertility in the laboratory (Bowden and Leslie, 1999) resulted in questions regarding the permeability of barriers to gene flow between these species. In the present study, F. meridionale and F. boothii were isolated from maize in the same locations within Argentina. Despite their coexistence, these species did not share trichothecene chemo-
types or VNTR haplotypes in common, they had a very high percentage of private alleles (89.7%) at polymorphic loci, and a very high level of genetic divergence (FST = 0.73, P b 0.001). These results are consistent with previous field studies (Lee et al., 2009; Suga et al., 2008) in indicating that there is limited potential for gene flow between Fg complex species in nature. This question is relevant to our
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Table 2 VNTR haplotype frequencies among Fg complex species identified from maize in Northwest Argentina. Haplotypesa
Haplotype frequency
HK913
HK1073
HK977
HK1059
HK630
HK957
HK917
F. meridionale
F. boothii
228 222 222 225 225 228 225 195 195 195 195 195 195 195 195 195 195 195 195 195
168 246 127 209 203 209 173 150 137 150 130 137 137 150 125 137 137 137 137 150
194 194 194 194 194 194 194 188 188 188 188 188 228 188 188 188 188 ? 188 188
218 218 218 218 218 218 218 218 218 218 218 218 ? 218 218 234 218 ? 218 234
218 240 218 218 218 218 218 218 218 218 218 218 218 218 218 218 218 218 218 218
166 166 166 166 166 166 166 186 186 186 186 186 186 166 186 186 186 186 166 166
229 227 227 229 227 227 227 219 223 221 221 221 221 221 221 219 219 221 235 221
0 0 0 0 0 0 0 0.052 0.017 0.293 0.017 0.397 0.035 0.017 0.017 0.035 0.052 0.035 0.017 0.017
0.1 0.2 0.1 0.3 0.1 0.1 0.1 0 0 0 0 0 0 0 0 0 0 0 0 0
a Allele sizes are given in base pairs (bp) for each of the polymorphic markers. All isolates had a 210 bp allele at the HK1043 locus and had a 202 bp allele or were unresolved (?) at the HK967 locus.
understanding of pathogen diversity and to future analyses of pathogen ecology. 5. Conclusions Our results indicated that the diversity of the Fg species complex in Argentina is greater than previously believed. Dominance of the NIV chemotype among isolates collected from maize is of significant concern to food safety and animal production because of the greater toxic potential of NIV relative to DON. Additional studies incorporating molecular species identification and analysis of toxin potential are needed to more fully assess the importance of species other than F. graminearum sensu stricto in infection and contamination of cereals in Argentina, and will also be needed to address hypotheses of species-specific preferences for particular hosts or environmental conditions in different regions. Acknowledgments Dr. Sampietro wants to thank the financial support provided by National Council (CONICET, Argentina) and the Fulbright Commission. This work has been partially supported by grants from CONICET and SECyT. We thank Thomas Usgaard, Nathane Orwig, and Stacy Sink for technical assistance. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentioned. References Alvarez, C.L., Azcarate, M.P., Pinto, V.F., 2009. Toxigenic potential of Fusarium graminearum sensu stricto isolates from wheat in Argentina. International Journal of Food Microbiology 135, 131–135. Booth, C., 1971. The Genus Fusarium. Commonwealth Mycological Institute, Kew, Surrey, England. Bowden, R.L., Leslie, J.F., 1999. Sexual recombination in Gibberella zeae. Phytopathology 89, 182–188. Chulze, S.N., Ramirez, M.L., Farnochi, M., Pascale, M., Visconti, A., March, G., 1996. Fusarium and fumonisins occurrence in Argentinian corn at different ear maturity stages. Journal of Agricultural and Food Chemistry 44, 2797–2801. Excoffier, L., Laval, G., Schneider, S., 2005. Arlequín ver. 3.0: An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online 1, 47–50.
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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 ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Short Communication
Species diversity and toxigenic potential of Fusarium graminearum complex isolates from maize fields in northwest Argentina D.A. Sampietro a,⁎, C.G. Díaz b, V. Gonzalez c, M.A. Vattuone a, L.D. Ploper c, C.A.N. Catalan a, T.J. Ward d a
INQUINOA - CONICET, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, España 2903, 4000, San Miguel de Tucumán, Argentina Facultad de Agronomía y Zootecnia, Universidad Nacional de Tucumán, España 2903, 4000, San Miguel de Tucumán, Argentina Sección Fitopatología, Estación Experimental Agroindustrial Obispo colombres, William Cross 3150 (T4101XAC), Las Talitas, Tucumán, Argentina d Bacterial Foodborne Pathogens and Mycology Research Unit, U.S. Department of Agriculture, Agricultural Research Service, 1815 N. University St., Peoria, IL 61604, USA b c
a r t i c l e
i n f o
Article history: Received 19 August 2010 Received in revised form 7 December 2010 Accepted 22 December 2010 Keywords: ASPE Fusarium meridionale Fusarium boothii Multilocus genotyping Trichothecene Population genetics
a b s t r a c t Members of the Fusarium graminearum species complex (Fg complex) are the causal agents of ear rot in maize and Fusarium head blight of wheat and other small grain cereals. The potential of these pathogens to contaminate cereals with trichothecene mycotoxins is a health risk for both humans and animals. A survey of ear rot isolates from maize collected in northwest Argentina recovered 66 isolates belonging to the Fg complex. A multilocus genotyping (MLGT) assay for determination of Fg complex species and trichothecene chemotypes was used to identify 56 of these isolates as F. meridionale and 10 isolates as F. boothii. F. meridionale was fixed for the nivalenol (NIV) chemotype, and all of the F. boothii isolates had the 15acetyldeoxynivalenol (15ADON) chemotype. The results of genetic diversity analysis based on nine variable number tandem repeat (VNTR) loci supported the hypothesis of genetic isolation between F. meridionale and F. boothii, and provided little evidence of geographic substructure among populations of the dominant pathogen species, F. meridionale. This is the first study to indicate that F. meridionale and F. boothii may play a substantial role in the infection and trichothecene contamination of maize in Argentina. In addition, dominance of the NIV chemotype among Fg complex isolates from Argentina is unprecedented, and of significant concern to food safety and animal production. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Members of the Fusarium graminearum species complex (Fg complex) are responsible for diseases of a variety of cereal crops worldwide and are important grain rotting pathogens in the subtropical and temperate regions of Argentina (Chulze et al., 1996). Infection of cereal crops by these fungi significantly lowers grain yield and quality, and can result in the contamination of grain with B-trichothecenes. These mycotoxins are a significant risk to food safety and animal health because they inhibit DNA, RNA and protein synthesis in eukaryotic cells and can modify immune function (Pestka and Smolinski, 2005; Ueno et al., 1973; Rocha et al., 2005). In addition, trichothecenes can be acutely phytotoxic and act as virulence factors on sensitive cereal hosts (Jansen et al., 2005). Until recently, members of the Fg complex were considered a single cosmopolitan species (Booth, 1971; Gerlach and Nirenberg, 1982; Leslie and Summerell, 2006) due to the failure of morphological species recognition to accurately assess species limits for this group. However, studies employing phylogenetic species recognition based ⁎ Corresponding author. Tel.: +54 381 4247752x7082 7007. E-mail addresses: [email protected], [email protected] (D.A. Sampietro). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.12.021
on genealogical concordance (GCPSR) (Taylor et al., 2000) have demonstrated that F. graminearum sensu lato is a complex of at least 13 phylogenetically distinct species with marked biogeographic structure (O'Donnell et al., 2004, 2008; Starkey et al., 2007, YliMattila et al., 2009). Within the Fg complex, individual isolates can produce one of three strain-specific profiles (chemotypes) of Btrichothecene metabolites (Miller et al., 1991; O'Donnell et al., 2004): nivalenol and acetylated derivatives (NIV chemotype), deoxynivalenol and primarily 3-acetyldeoxynivalenol (3ADON chemotype), and deoxynivalenol and primarily 15-acetyldeoxynivalenol (15ADON chemotype). This variation is important to food safety, as differences in toxicity have been reported for different B-trichothecenes (Forsell and Pestka, 1985; Luongo et al., 2008). In addition, B-trichothecene chemotype differences have been maintained by natural selection and appear to have important consequences for pathogen fitness (Ward et al., 2002). Although members of the Fg complex pose a significant threat to cereal production and food safety worldwide, relatively little is known about the prevalence and distribution of Fg complex species and Btrichothecene chemotypes from maize in Argentina. F. graminearum sensu stricto was the only Fg complex species identified in analyses of isolates from wheat fields in the central region of Argentina (Scoz et al., 2009), and a recent study indicated that the 15ADON chemotype is
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predominant across the main wheat growing regions of the country (Alvarez et al., 2009). However, a preliminary analysis based on seven Fg complex isolates from maize in northwestern Argentina suggested additional species and trichothecene chemotype variation (Sampietro et al., 2010). The main objective of this study was to determine species, trichothecene chemotype, and population-level diversity among Fg complex isolates collected from maize in northwest Argentina in order to test the hypothesis that Fg complex diversity in Argentina is greater than previously recognized.
(Sampietro et al., 2010) in order to identify isolates that were morphologically consistent with the Fg complex. The morphology of macroconidia, and chlamydospores was assessed from cultures grown on SNA and CLA. Morphological identification of isolates was based on the criteria of Gerlach and Nirenberg (1982) and Leslie and Summerell (2006).
2. Materials and methods
Morphological variation within the Fg complex provides an insufficient basis for discerning species identity. Therefore, species identification was performed along with trichothecene chemotype determination using a previously published multilocus genotyping (MLGT) assay that is based on allele-specific primer extension (ASPE) with probes targeting species and trichothecene chemotype-specific genetic variation (O'Donnell et al., 2008; Ward et al., 2008; Yli-Mattila et al., 2009). MLGT was performed as described previously (Ward et al., 2008) and consisted of a multiplex amplification of six genomic regions containing species or trichothecene chemotype-specific nucleotide variation. Amplification products served as template for a multiplexed primer extension reaction. Biotinylated extension products from ASPE reactions were sorted by hybridization to a suspension microarray consisting of polystyrene microspheres coated with antitag sequences specific to 5′ sequence tags that were appended to each of the extension probes. Microsphere complexes were detected using a Luminex 100 flow cytometer (Luminex Corporation) and reactions were scored by fluorescence intensity and spectrum.
2.1. Maize plots and collection of fungal isolates Plots of commercial maize hybrids were cultivated in locations provided by Agro-Industrial Experimental Station Obispo Colombres (Tucumán, Argentina) to determine the natural incidence of Fg complex species and trichothecene chemotypes. Fields were located in three distinct zones (Fig. 1): South (La Cocha), East (Viclos, Monte Redondo, La Cruz, La Virginia, El Azul and Monte Quemado) and North (Trancas). Twenty-one maize hybrids were cultivated: AX1013 MG, NK120 TD MAX, NK135 TD MAX, NK138 TD MAX, DK 390 MG, DK 190 MG, AGRI 105, AGRI 103, SPS 1104, MASS636 Hx MG, Pioneer 30T17, DK 910 MG, Avant, 2A120 Hx, 2B710 Hx, 2B688 Hx, NK 3234, XTA8089, XTA5306, XTA80/5 and AGROMEN 31A31. Each hybrid plot consisted of 14 rows of 200 m length, with row spacing of 0.52 m. Plots were sown in the first week of January and harvested in the last week of July, 2007. Grain samples were randomly collected from each maize hybrid and analyzed in the laboratory as described previously
2.2. Species and trichothecene chemotype determination
Fig. 1. Geographical locations of Fusarium isolated from maize ears in Northwest Argentina during 2007. Numbers indicate sampling locations: 1) La Cocha; 2) Viclos; 3) Monte Redondo; 4) La Virginia; 5) La Cruz; 6) El Azul; 7) Monte Quemado; 8) Trancas.
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2.3. Analysis of population structure and diversity Genetic variation at nine variable number tandem repeat (VNTR) loci (Suga et al., 2004) was used to assess population structure and diversity. Forward primers for each VNTR locus were synthesized with fluorescent labels (Applied Biosystems) and amplifications were performed in three multiplex reactions as described previously (Ward et al., 2008). Reaction products were scored relative to a GS500 ROX (Applied Biosystems) internal size standard using an ABI3100 Genetic Analyzer with GENEMAPPER 3.7 software (Applied Biosystems). FST based genetic distance (Michalakis and Excoffier, 1996) values were estimated using the program ARLEQUIN 3.5 (Excoffier et al., 2005). Pairwise FST distance estimates were calculated based on the number of different alleles, and the statistical significance of pairwise FST estimates were assessed using a permutation test with 10,000 permutations. Population subdivision was also analysed using the Bayesian model-based clustering procedure implemented in STRUCTURE 2.3 (Pritchard et al., 2000), which assigns isolates to a user-defined number of population clusters (K) based on their multilocus genotypes. The Bayesian clustering analyses were performed for values of K from 1 to 5, and utilized 100,000 step Markov Chains after a burn-in period of 10,000 iterations. 3. Results 3.1. Ocurrence of Fg complex species and chemotypes in maize grains A total of 66 isolates collected from maize grains in northwest Argentina during 2007 were identified by morphological analysis as members of the Fg species complex (Table 1). A previously published MLGT assay was used to investigate species and trichothecene chemotype diversity among these isolates. Using the MLGT assay, we identified 85% (56/66) of the isolates as F. meridionale, while the remainder (15%, 10/66) were identified as F. boothii. In contrast to expectations based on previous reports, F. graminearum sensu stricto was not represented among the isolates examined. F. meridionale isolates were obtained from all eight sampling locations. However, nine of the 10 F. boothii isolates were obtained from a single location (Trancas) in the North, and the remaining isolate was obtained from one of the six locations (Viclos) sampled in the East. The 56 F. meridionale shared the NIV chemotype, while the 10 F. boothii isolates shared the 15ADON chemotype. 3.2. Analysis of genetic diversity and population structure Genetic variation at nine VNTR loci was analyzed to assess diversity and population structure. Five of the nine VNTR loci were polymorphic within F. meridionale, and four loci were polymorphic within F. boothii (Table 2). Thirteen unique haplotypes were identified among the 56 F. meridionale, and seven unique haplotypes were identified among the 10 F. boothii isolates (Table 2). Only two loci (HK1073 and HK917) were polymorphic in both species, and two loci were monomorphic across all 66 isolates. Substantial genetic divergence was observed between F. meridionale and F. boothii (FST = 0.73, P b 0.001). In addition, 26 of the 29 (89.7%) alleles identified among the seven polymorphic loci were unique to one of the two species. No significant population structure was observed within F. meridionale. Using the Bayesian clustering approach to assess population structure among the 56 F. meridionale isolates, a single population (K = 1) model provided the best fit to the observed data. Setting K N 1 failed to improve likelihood scores and resulted in most individuals being assigned equally to each of the user-defined populations. Similar results were obtained when we specifically tested the hypothesis of geographic structuring after assigning
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F. meridionale isolates into three groups based on geographic origin (North, South, and East; FST b 0.04; P N 0.24). These results indicated that F. meridionale isolates from the sampled regions behave as a single population. Similar analyses for F. boothii were not possible due to the small number of isolates. 4. Discussion The results reported here demonstrate that Fg complex diversity in Argentina is greater than previously recognized. They also indicate for the first time that F. meridionale and F. boothii may play a substantial role in the infection and trichothecene contamination of Argentinian maize. In contrast, previous analyses of Fg complex isolates collected from wheat in the center of Argentina identified all 113 isolates as F. graminearum sensu stricto (Ramirez et al., 2007). F. graminearum sensu stricto was also the dominant species (92.7%) isolated from wheat in southern Brazil; however F. meridionale accounted for six (7.3%) of the 82 isolates examined (Scoz et al., 2009). The dramatic differences in chemotypes and Fg complex species composition observed among wheat in center Argentina and the maize isolates collected in the current work from the northwest is suggestive of a potential difference in host preference within the Fg complex. However, interpretations are limited by the small number of studies published to date that incorporate molecular differentiation of species within the Fg complex. Trichothecene chemotype diversity among the isolates examined was directly tied to species differences. All of the F. meridionale had the NIV chemotype, while all F. boothii isolates had the 15ADON chemotype. These results are consistent with previous analyses of mycotoxin production in these two species (Ward et al., 2002). However, this situation contrasts with recent analyses of F. graminearum sensu stricto and F. asiaticum, in which trichothecene chemotype polymorphism was detected within each species and correlated with population subdivision (Gale et al., 2007; Ward et al., 2008; Yli-Mattila and Gagkaeva, 2010; Zhang et al., 2010). In a previous analysis of trichothecene production by Fg complex isolates collected from maize in Argentina, Molto et al. (1997) reported that all isolates produced DON. Nivalenol was detected in only one of 120 wheat samples collected in the northern Buenos Aires province of Argentina (González et al., 2008), and the NIV chemotype was absent from a recent survey of Fg complex isolates collected from across the major wheat production area in Argentina (Alvarez et al., 2009). The 15ADON chemotype predominated in a recent analysis of species and trichothecene chemotype diversity in southern Brazil, although the six F. meridionale that were identified in that study were found to have the NIV chemotype (Scoz et al., 2009). Interestingly, a previous compilation of global data reported substantially lower levels of NIV contamination in food and feed as compared to DON, but also found that Brazil was a rare exception in that DON and NIV contamination levels were similar (Placinta et al., 1999). Taken together with the data from the current study, these reports suggest that while DON is likely to be the predominant trichothecene contaminate of cereals in South America, the potential exists for substantial NIV contamination. This has significant implications for food safety as NIV appears to be more cytotoxic than DON (Forsell and Pestka, 1985; Luongo et al., 2008). In response to evidence of higher toxicity for NIV, the European Scientific Committee on Food has proposed a lower tolerance limit for NIV than for DON (Schothorst and Van Egmond, 2004). F. meridionale and F. boothii, along with the other members of the Fg complex, were originally described on the basis of genealogical exclusivity evident in phylogenetic trees derived from analyses of the DNA sequences of multiple single-copy nuclear genes (O'Donnell et al., 2000, 2004, 2008; Starkey et al., 2007, Yli-Mattila et al., 2009). These results are indicative of an extended history of genetic isolation consistent with the establishment of species boundaries. However, a
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Table 1 Geographic origin, host maize hybrid, Fg complex species and trichothecene chemotype of isolates collected in Northwest Argentina. Isolate
Geographic origin
Maize hybrid
Species Identity
Trichothecene chemotype
F1 F2 F3 F4 F5 F9 F10 F13 F14 F15 F16 F21 F23 F24 F26 F28 F29 F30 F31 F32 F33 F34 F35 F36 F37 F38 F39 F40 F41 F42 F44 F45 F46 F49 F50 F51 F52 F53 F55 F56 F58 F59 F62 F63 F64 F65 F66 F67 F68 F69 F73 F74 F75 F76 F77 F78 F79 F80 F81 F82 F83 F85 F86 F89 F90 F92
Viclos (east) Viclos (east) Viclos (east) Viclos (east) Viclos (east) Viclos (east) Viclos (east) La Cruz (east) La Virginia (east) La Virginia (east) Monte Redondo (east) Monte Redondo (east) Monte Redondo (east) Monte Redondo (east) Monte Redondo (east) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) La Cocha (south) Trancas (north) Trancas (north) Trancas (north) Trancas (north) Trancas (north) Trancas (north) Trancas (north) Trancas (north) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) El Azul (east) La Cruz (east) Monte Redondo (east) Monte Redondo (east) Monte Quemado (east) Monte Quemado (east) Trancas (north) Trancas (north) Trancas (north) Trancas (north) Trancas (north) El Azul (east) El Azul (east) El Azul (east) Trancas (north) Trancas (north) Monte Redondo (east)
AX 1013 MG AX 1013 MG NK 135 TD MAX NK 135 TD MAX NK 120 TD MAX DK 190 MG NK 138 TD MAX NK 120 TD MAX NK 138 TD MAX NK 138 TD MAX AGRI 105 AGRI 105 NK 120 TD MAX NK 120 TD MAX 2B 710 Hx MASS 636 Hx MG NK 135 TD MAX NK 135 TD MAX NK 120 TD MAX NK 120 TD MAX AX 1013 MG AX 1013 MG PIONEER 30T17 Hx PIONEER 30T17 Hx DK 390 MG AGROMEN 31A31 AGROMEN 31A31 AX 1013 MG AX 1013 MG DK 390 MG DK 910 MG NK 138 TD MAX NK 138 TD MAX NK 120 TD MAX NK 120 TD MAX NK 138 TD MAX NK 138 TD MAX NK 120 TD MAX 2B 710 Hx AGRI 105 2A 120 Hx 2A 120 Hx DK 910 MG DK 910 MG SPS 1104 SPS 1104 PIONEER 30T17 Hx PIONEER 30T17 Hx MASS 636 Hx MG MASS 636 Hx MG 2B 688 AVANT 2B 688 XTA 8089 2B 688 AVANT XTA 5306 NK 3234 2B 688 XTA 80/5 2B 688 NK 3234 AVANT NK 138 TD MAX DK 390 MG NK 120 TD MAX
F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. boothii F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. boothii F. boothii F. boothii F. boothii F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. meridionale F. boothii F. meridionale F. boothii F. meridionale F. boothii F. meridionale F. meridionale F. meridionale F. boothii F. boothii F. meridionale
NIV NIV NIV NIV NIV NIV 15ADON NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV 15ADON 15ADON 15ADON 15ADON NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV NIV 15ADON NIV 15ADON NIV 15ADON NIV NIV NIV 15ADON 15ADON NIV
lack of morphologically diagnostic characters and some evidence of interfertility in the laboratory (Bowden and Leslie, 1999) resulted in questions regarding the permeability of barriers to gene flow between these species. In the present study, F. meridionale and F. boothii were isolated from maize in the same locations within Argentina. Despite their coexistence, these species did not share trichothecene chemo-
types or VNTR haplotypes in common, they had a very high percentage of private alleles (89.7%) at polymorphic loci, and a very high level of genetic divergence (FST = 0.73, P b 0.001). These results are consistent with previous field studies (Lee et al., 2009; Suga et al., 2008) in indicating that there is limited potential for gene flow between Fg complex species in nature. This question is relevant to our
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Table 2 VNTR haplotype frequencies among Fg complex species identified from maize in Northwest Argentina. Haplotypesa
Haplotype frequency
HK913
HK1073
HK977
HK1059
HK630
HK957
HK917
F. meridionale
F. boothii
228 222 222 225 225 228 225 195 195 195 195 195 195 195 195 195 195 195 195 195
168 246 127 209 203 209 173 150 137 150 130 137 137 150 125 137 137 137 137 150
194 194 194 194 194 194 194 188 188 188 188 188 228 188 188 188 188 ? 188 188
218 218 218 218 218 218 218 218 218 218 218 218 ? 218 218 234 218 ? 218 234
218 240 218 218 218 218 218 218 218 218 218 218 218 218 218 218 218 218 218 218
166 166 166 166 166 166 166 186 186 186 186 186 186 166 186 186 186 186 166 166
229 227 227 229 227 227 227 219 223 221 221 221 221 221 221 219 219 221 235 221
0 0 0 0 0 0 0 0.052 0.017 0.293 0.017 0.397 0.035 0.017 0.017 0.035 0.052 0.035 0.017 0.017
0.1 0.2 0.1 0.3 0.1 0.1 0.1 0 0 0 0 0 0 0 0 0 0 0 0 0
a Allele sizes are given in base pairs (bp) for each of the polymorphic markers. All isolates had a 210 bp allele at the HK1043 locus and had a 202 bp allele or were unresolved (?) at the HK967 locus.
understanding of pathogen diversity and to future analyses of pathogen ecology. 5. Conclusions Our results indicated that the diversity of the Fg species complex in Argentina is greater than previously believed. Dominance of the NIV chemotype among isolates collected from maize is of significant concern to food safety and animal production because of the greater toxic potential of NIV relative to DON. Additional studies incorporating molecular species identification and analysis of toxin potential are needed to more fully assess the importance of species other than F. graminearum sensu stricto in infection and contamination of cereals in Argentina, and will also be needed to address hypotheses of species-specific preferences for particular hosts or environmental conditions in different regions. Acknowledgments Dr. Sampietro wants to thank the financial support provided by National Council (CONICET, Argentina) and the Fulbright Commission. This work has been partially supported by grants from CONICET and SECyT. We thank Thomas Usgaard, Nathane Orwig, and Stacy Sink for technical assistance. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentioned. References Alvarez, C.L., Azcarate, M.P., Pinto, V.F., 2009. Toxigenic potential of Fusarium graminearum sensu stricto isolates from wheat in Argentina. International Journal of Food Microbiology 135, 131–135. Booth, C., 1971. The Genus Fusarium. Commonwealth Mycological Institute, Kew, Surrey, England. Bowden, R.L., Leslie, J.F., 1999. Sexual recombination in Gibberella zeae. Phytopathology 89, 182–188. Chulze, S.N., Ramirez, M.L., Farnochi, M., Pascale, M., Visconti, A., March, G., 1996. Fusarium and fumonisins occurrence in Argentinian corn at different ear maturity stages. Journal of Agricultural and Food Chemistry 44, 2797–2801. Excoffier, L., Laval, G., Schneider, S., 2005. Arlequín ver. 3.0: An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online 1, 47–50.
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