Mycotoxin Res (2017) 33:121–127 DOI 10.1007/s12550-017-0271-4
ORIGINAL ARTICLE
Fusarium verticillioides and fumonisin contamination in Bt and non-Bt maize cultivated in Brazil Vinícius M. Barroso 1 & Liliana O. Rocha 1 & Tatiana A. Reis 1 & Gabriela M. Reis 1 & Aildson P. Duarte 2 & Marcos D. Michelotto 2 & Benedito Correa 1
Received: 13 September 2016 / Revised: 5 February 2017 / Accepted: 13 February 2017 / Published online: 6 March 2017 # Society for Mycotoxin Research and Springer-Verlag Berlin Heidelberg 2017
Abstract Fusarium verticillioides is one of the main pathogens of maize, causing ear and stalk rots. This fungus is also able to produce high levels of fumonisins, which have been linked to various illnesses in humans and animals. Previous studies have shown that maize hybrids genetically modified with the cry genes from the bacterium Bacillus thuringiensis (Bt) presented lower incidence of F. verticillioides and fumonisin levels, presumably through the reduction of insects, which could act as vectors of fungi. The aim of this study was to assess the incidence of F. verticillioides and the concentration of fumonisins in Bt and isogenic non-Bt hybrids (2B710Hx, 30F35YG, 2B710, and 30F35, respectively). The samples of 2B710Hx and 30F35YG presented lower F. verticillioides frequency than 2B710 and 30F35 samples. However, there was no statistical difference between fumonisin contamination when Bt and non-Bt samples were compared (P > 0.05). The results suggest that other environmental parameters could possibly trigger fumonisin production during plant development in the field; consequently, other management strategies should be applied to aid controlling fumonisin contamination in maize. Keywords Fusarium . Mycotoxins . Fungi . Corn . Transgenic crops * Benedito Correa
[email protected] 1
Microbiology Department, Institute of Biomedical Sciences, University of Sao Paulo, Avenue Prof. Lineu Prestes, 1374, Laboratory 249, Sao Paulo, SP 05508-000, Brazil
2
Centre of Grains and Fibres, Agronomic Institute of Campinas, Agro-Business Agency of Sao Paulo (Agência Paulista de Tecnologia dos Agronegócios (APTA)), Avenue Theodureto de Almeida Camargo, 1500, Campinas, SP 13075-630, Brazil
Introduction Maize (Zea mays L.) is an important agricultural crop worldwide, being fundamental in production chains, including poultry, pig, and dairy cattle farming, as it is the main raw component of animal feed. Brazil is the third largest producer, yielding 83.59 million tons during 2015 and 2016 (Conab 2016). Maize production can be affected by environmental conditions, soil type, water stress, presence of weeds, insects, and diseases caused by microorganisms. Fungi are associated with several maize diseases and pose a serious threat to production (Pomeranz 1982). Colonization can occur from sowing to harvest, causing various problems, leading to loss of quality and mycotoxin production (Frisvad and Samson 1991; Pomeranz 1982). Insects cause physical injuries to stalks and ears and, therefore, facilitate fungal infection (Miller et al. 2007). The Fusarium fujikuroi species complex harbors the fumonisinproducing Fusarium species, including Fusarium verticillioides. This fungus is one of the main pathogens of maize, causing ear and stalk rots (Leslie and Summerell 2006). It is also able to produce high levels of fumonisins, important mycotoxins associated with leukoencephalomalacia in horses (Kellerman et al. 1990), pulmonary edema in swine, and possibly esophageal cancer and neural tube defects in humans (Marasas et al. 2004; Missmer et al. 2006). The International Agency for Research on Cancer (IARC) has classified fumonisins as group 2B, a possible human carcinogen (IARC 1993). Fumonisins in grains is a worldwide concern. In consequence, the Commission Regulation (EC) of the European Union set a maximum level of 4 μg/g for FB1 + FB2 in unprocessed maize and 1 μg/g in maize intended for human consumption (ECR 2007). The US Food and Drug Administration (FDA) set a limit of 2–4 μg/g of FB1 + FB2
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+ FB3 contamination in human foods and 5–100 μg/g in animal feeds (FDA 2001). Due to the complications regarding the presence of F. verticillioides in grains, efforts have been made to reduce the contamination of this fungus. Bt hybrids are genetically modified cultivars of maize that express certain cry genes of Bacillus thuringiensis, which encode toxins (cry proteins) that are able to control the larvae of lepidopteran pests (Saxena and Sottzky 2000). Previous studies have demonstrated that the reduction of insects through the use of Bt maize could possibly diminish fungal contamination, therefore decreasing mycotoxin accumulation in grains (Munkvold et al. 1999; Papst et al. 2005). For reasons cited above, the aim of the current study was to evaluate the association between F. verticillioides frequency and fumonisin contamination levels in Bt (2B710Hx/ 30F35YG) and non-Bt maize, in order to determine the efficiency of these Bt technologies to control fungal contamination in the environmental conditions of Brazil.
Materials and methods Maize hybrids and experimental design The study was conducted in the southeast region of Brazil, in the city of Cruzália (22° 44′ 08″ S, 50° 47′ 37″ W) (Fig. 1). This region is mainly characterized by tropical weather, with
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mean minimum/maximum temperature of 12.1/32 °C, respectively, and annual precipitation of 1419.1 mm (http://www. cpa.unicamp.br/outras-informacoes/clima-dos-municipiospaulistas.html). Maize grains were sown in November 2013 and harvested in March 2014. The following hybrids were used in this investigation: 2B710Hx and 30F35YG (Bt maize) and 2B710 and 30F35 (non-Bt maize). Sampling was performed according to the method proposed by Delp et al. (1986). Forty samples of each hybrid were used for mycobiota and fumonisin contamination (total of 160 samples).
Mycobiota of maize The species of fungi were isolated according to the methodology proposed by Berjark (1984). In short, a sub-sample of 30 g of maize grains was disinfected in 0.4% chlorine for 3 min. Thirty-three grains were selected randomly and plated onto petri dishes containing dichloran rose bengal chloramphenicol agar (Oxoid, Basingstoke, HPH, England). The plates were incubated for 5 days at 25 °C, and the results were expressed as percentage of the total number of grains infected with fungi. The water activity (aw) of the grains was determined with an AquaLab device (Decagon, Washington, DC, USA). The Fusarium isolates were identified to species level and maintained in Spezieller Nährstoffarmer agar (Leslie and Summerell 2006; Pitt and Hocking 2009). The EF-1α partial gene sequence was selected in order to confirm the
Fig. 1 Map of Brazil indicating the sampling site (Cruzália, SP, 22° 44′ 08″ S, 50° 47′ 37″ W) from which the Bt and non-Bt maize samples were recovered. SP state of Sao Paulo, Brazil
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morphological identification of the isolates (Geiser et al. 2004). Fusarium strains were grown in yeast extract sucrose (YES) agar (Degola et al. 2007) for 3 days at 25 °C. The DNA was extracted using the Easy-DNA Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. The PCR reaction was performed according to Geiser et al. (2004). Amplicons were purified with ExoSAP-IT (Affymetrix, Santa Clara, CA, USA) and sent to Centre of Human Genome Studies, University of Sao Paulo, Brazil. BioEdit v7.0.9.0 software (www.mbio.ncsu.edu/BioEdit/BioEdit.html) was used to edit the sequences; afterwards, the sequences were blasted against GenBank and Fusarium ID databases (http://blast.ncbi.nlm.nih.gov/Blast.cgi and http://isolate. fusariumdb.org/blast.php). Determination of fumonisins in maize Fumonisins were extracted according to Visconti et al. (2001). The residue was resuspended in 1 mL acetonitrile/water (50:50, v/v). An aliquot of 50 μL of the extract was transferred to a reaction vessel, and 50 μL of OPA reagent was added (Sigma, St. Louis, MO, USA) (40 mg orthophthalaldehyde in 1 mL methanol, diluted in 5 mL of 38.14 g/L sodium tetraborate solution with 50 μL of 2-mercaptoethanol). After 60 s, 20 μL of the solution was injected into a Shimadzu LC10AD liquid chromatography and fumonisins detected by fluorescence (335 and 440 nm for excitation and emission, respectively). The mobile phase was acetonitrile/water/acetic acid (520:480:5, v/v) at a flow rate of 1 mL/min. The following concentrations were used for the calibration curves: 0.039, 0.078, 0.156, 0.3125, 0.625, 0.1.25, 2.5, 5.0, and 10.0 μg/mL, with r2 of 0.99 for both FBs. The limits of quantification (LOQs) were 0.015 μg/g for FB1 and FB2 (Table 1). The retention time for FB1 was 7 min and for FB2 21 min. Statistical analysis Statistical analysis was performed in the software SAS version 9.1, with a significance level of 5%. Spearman correlation was Table 1 Recovery tests for FB1 and FB2 in maize samples
applied to determine the correlation among water activity, incidence of Fusarium, and fumonisin contamination. MannWhitney test was used for comparisons between Bt and nonBt hybrids for (a) F. verticillioides frequency and (b) FB1 and FB2 contamination (Bussab and Morettin 2011).
Results Mycobiota of maize The mycobiota of the 160 freshly harvested maize samples of the Bt and non-Bt hybrids revealed that the most frequent genus was Fusarium, with F. verticillioides the only isolated species. The other genera of isolated fungi are specified in Table 2. Sequencing analysis confirmed that all of the Fusarium isolates were F. verticillioides. Interestingly, the F. verticillioides frequency was lower in the 2B710Hx (P = 0.003) and 30F35YG (P = 0.029) samples, in comparison to their non-Bt isogenic hybrids (Table 2). The mean aw is represented in Table 2. Spearman test demonstrated that there was no correlation between aw and F. verticillioides frequency as well as fumonisin contamination. This may be explained by the high levels of aw in all of the samples and low variability among them (Table 2). Determination of fumonisins in maize The analysis of 160 maize grain samples revealed the presence of FB1 and FB2 in 98 and 50% of the samples, respectively. All of the 30F35YG samples were contaminated with FB1 and 65% with FB2 (Table 3), with levels that ranged from 0.018 to 9.419 μg/g (FB 1 ) and not detected (ND) to 6.157 μg/g (FB2). In the 30F35 hybrid samples, FB1 was detected in all of the samples and FB2 in 47.5%. The concentrations of FB1 and FB2 ranged from 0.035 to 4.308 μg/g and from ND to 2.710 μg/g, respectively (Table 3). Ninety-five percent of the 2B710Hx samples were contaminated with FB1 and 50% with FB2. The concentration of FB1
Spiking concentration (μg/g)
Mean concentration recovered (μg/g)a
Mean recovery (%)a
FB1
FB2
FB1
FB2
0.015 0.025 0.035
0.014 ± 0.14b 0.027 ± 0.07 0.033 ± 0.09
0.013 ± 0.07 0.025 ± 0.05 0.033 ± 0.12
93 109 94
85 100 93
0.085 0.150
77 ± 0.08 146 ± 0.12
84 ± 0.04 148 ± 0.05
91 97
99 98
a
Mean recovery tests of three replicates
b
Standard deviation
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– –
1.74 3.68
Acremonium
– –
– 0.08
– –
0.1 – – – 0.4 – 0.23 1.36 0.45 – 2.8 0.67 1.73 0.07
b
Mean of 40 analysis a
NSF non-sporulating fungi
All of the Fusarium isolates were identified as F. verticillioides
3.2 0.7 2.2 12.6 40.52 66.8 0.90 0.96 2B710 2B710Hx
75.4 54.5
0.08 – 0.15 0.08 1.4 0.2 0.93 0.95 30F35 30F35YG
91.8 84.1
40.7 34.7
10.2 5.2
4.7 2.3
1.4 0.9
– 1.7
– –
Rhizopus NSF Mucor Neurospora Aspergillus Cladosporium Trichoderma Penicillium Fusariumb
awa
Frequency of fungi (%)a
ranged from ND to 5.111 μg/g and the concentration of FB2 from ND to 3.421 μg/g. In the 2B710 hybrid samples, FB1 was detected in 97.5% of the samples and FB2 in 37.5%. The concentrations of FB1 and FB2 ranged from ND to 3.346 μg/g and from ND to 2.083 μg/g, respectively (Table 3). There was no evidence for variation in fumonisin B1 and B2 contamination between Bt and non-Bt maize samples (P = 0.22 for 2B710 and 2B710Hx; P > 0.3 for 30F35 and 30F35YG).
Discussion
Hybrids
Table 2
Mean frequencies (%) of fungal genera and water activity values (aw) in Bt (30F35YG/2B710Hx) and non-Bt (30F35/2B710) hybrids
Alternaria
Curvularia
Yeast
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Maize is susceptible to the contamination of different microorganisms, including fungi. The genera Fusarium, Aspergillus, and Penicillium are the most important mycotoxigenic fungi and are commonly isolated from maize. These species are recovered from several environmental sources, with air and soil as the main dispersal mechanisms (Almeida et al. 2002). Abiotic factors strongly influence fungal growth and the presence of mycotoxins in grains, emphasizing that temperature, soil quality, and precipitation may affect the adaptation of maize hybrids, which can lead to mycotoxin accumulation, and trigger pathogenicity of fungi (Hurst 2001; Miller 2001). F. verticillioides is the main species of fungi associated with maize in tropical areas (Leslie and Summerell 2006), including Brazil, causing ear and stalk rots, as well as producing high levels of fumonisins (Almeida et al. 2005; Hirooka et al. 1996; Mills 1989; Orsi et al. 2000; Pozzi et al. 1995; Rocha et al. 2009). In the current study, there was a high frequency of F. verticillioides, in both Bt and non-Bt maize, even though Bt maize presented a lower incidence of F. verticillioides. Indeed, lower F. verticillioides contamination has been previously demonstrated in Bt maize (Dowd 2000; Munkvold et al. 1999; Papst et al. 2005), and the majority of the studies speculated that Bt maize could possibly diminish Fusarium contamination due to reduced grain damage caused by insect pests (Jouany 2007; Michelotto et al. 2011; Miller 2001; Rocha et al. 2016; Wu et al. 2004; Wu 2006). For this reason, it would also be expected lower fumonisin accumulation in these grains, firstly because insect pests may act as vectors for fungal conidia and secondly because injured grains may facilitate fungal infection as well as mycotoxin production (Duvick 2001; Munkvold 2003; Ostry et al. 2010; Leslie and Logrieco 2014). Our results showed that Bt maize presented higher levels of fumonisins when compared to non-Bt samples, indicating that fumonisin exposure through the consumption of Bt maize may be a concern. Considering that the average consumption of maize in Brazil is about 55 g per day (Ranum et al. 2014), a 70-kg man would have a daily intake of about 0.74 and 1.3 μg/kg body weight fumonisins (FB1 + FB2), if maize
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Frequency of F. verticillioides and fumonisin levels (FB1 and FB2) in Bt (30F35YG/2B710Hx) and non-Bt (30F35/2B710) hybrids
Table 3 Hybrids
30F35a a
30F35YG 2B710a 2B710Hxa
Fusarium FB1 (μg/g) verticillioides (%) Meana Min
Max
90th percentile Median Meana Min Max
90th percentile Median Meana
91.8 84.1 75.4 54.5
4.308 9.419 3.346 5.111
2.669 7.718 2.251 3.974
1.751 3.072 1.322 1.690
0.994 2.698 0.646 1.177
0.035 0.018 NDb ND
FB2 (μg/g)
a
Mean of 40 analyses for each maize hybrid
b
Not detected; limit of quantification 0.015 μg/g for both FB1 and FB2
contains 0.65 and 1.17 μg/g fumonisins (2B710 and 2B710Hx hybrids, respectively) and 0.88 and 2.1 μg/kg body weight fumonisins, if maize contains 0.99 and 2.7 μg/g fumonisins (30F35 and 30F35YB hybrids). This is significant since the provisional maximum daily tolerated intake for fumonisin is 2 μg/kg body weight (JECFA 2012), suggesting that the use of Bt maize only may not be efficient for decreasing fumonisins, at least under the environmental conditions encountered in this experiment. However, it has been shown that several other factors may also influence fumonisin production in the field including environmental conditions, water availability, and the genetic background of contaminating fungi (Bowers et al. 2013). Drought and temperature stress may trigger fumonisin production (FDA 2001; Miller 2001; Shelby et al. 1994). Additionally, hybrids grown outside their area of adaptation may increase their propensity for kernel splitting. This type of injury is generally intensified in drought conditions, favoring Fusarium contamination and fumonisin production (Doko et al. 1995; Visconti 1996). Perhaps, this may explain the patterns of fumonisin contamination in Bt and non-Bt hybrids observed in the current and other studies (Clements et al. 2003). It is relevant to emphasize that the region where the hybrids were sown is considered tropical. Nevertheless, temperature and drought have increased over the last 5 years in the state of Sao Paulo (FAO 2016), Brazil, which could possibly affect mycotoxin accumulation in crops during their development in the field. This hypothesis has previously been raised by Bock and Cotty (1999) who observed higher aflatoxin contamination in Bt cottonseed resulted from warmer and more humid conditions in Arizona, USA. Another study conducted in Argentina and the Philippines also determined that weather explained 47% of the variability in fumonisin contamination, whereas insect damage and Bt hybrids explained only 17 and 11%, respectively (Schaafsma and Hooker 2007). In addition, Marasas et al. (1979) and Shephard et al. (1996) indicated that higher levels of fumonisins may occur under warm and dry conditions, highlighting that a specific environmental ecosystem has its own features and crop plants may be affected by these differences in each part of the world (Fandohan et al. 2005).
0.256 0.570 0.989 1.570
0.507 1.029 0.292 0.482
ND ND ND ND
FB1 + FB2 (μg/g)
2.710 6.157 2.083 3.421
0 0.147 0 0.015
1.501 3.727 0.938 1.659
Conclusion The current study displays important information on the use of Bt maize with the purpose of restricting F. verticillioides and fumonisin contamination. Despite the use of Bt crops being undoubtedly beneficial for controlling insect pests, it is essential to evaluate other field parameters that could affect plant culture and influence mycotoxin production. Considering that Brazil is the third largest maize exporter in the world, strict measures for selecting more resistant hybrids are necessary to aid in the management of fumonisin contamination.
Compliance with ethical standards Source of funding This investigation was funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil. Conflict of interest The authors confirm absence of any conflict of interest.
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