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Fusarium poses food and feed safety problems because most species produce mycotoxins. To understand the epidemiology of the Fusarium disease, efforts must focus more precisely on how environmental variables affect disease presence. The objectives of the present study were to monitor the occurrence of Fusarium species in maize kernels in northwestern Spain to determine the risk of mycotoxin contamination and to identify environmental traits affecting the composition of the Fusarium species identified. A combination of 24 environments was evaluated. The percentage of kernels infected by F. verticillioides ranged from 33 to 99%, supporting the idea that fumonisin contamination is the main maize-based feed and food safety concern in this area. In this region, temperature and humidity primarily affected Fusarium spp. occurrence. Warmer temperatures during the later stages of kernel development and during kernel drying increased the frequency of F. verticillioides in maize kernels, while the presence of F. subglutinans was increased by higher relative humidity during the silking stage and cooler temperatures during kernel drying.
Moulds belonging to the genus Fusarium (Ascomycota: Nectriaceae) are widely found infecting maize kernels in temperate regions. Fusarium poses food and feed safety problems because most species produce mycotoxins (Logrieco et al., 2003). Symptoms of mycotoxicosis depend on the type of mycotoxin, concentration, length of exposure and characteristics of the exposed person (e.g. age and health), but the liver, kidneys and immune, endocrine and/or nervous systems are especially vulnerable (Bennett & Klich, 2003). Mycotoxins can be mutagenic and carcinogenic; potential carcinogenic risk for some mycotoxins has been rated by the International Agency for Research on Cancer (IARC, 1993). Therefore, legislation to limit the levels of some mycotoxins in food has been implemented in many parts of the world (FAO, 2004) to minimize risks to human health.
Climatic conditions determine the predominance of a particular species or group of Fusarium species that cause different types of maize ear rot. In cooler temperate regions, gibberella ear rot predominates and is mainly caused by F. graminearum and related species such as F. culmorum, F. cerealis and F. avenaceum (Bottalico, 1998; Logrieco et al., 2002; Munkvold, 2003). In warmer regions, fusarium ear rot is prevalent; it results from kernel infection by F. verticillioides and other species of the Gibberella fujikuroi complex such as F. proliferatum and F. subglutinans. All these species are mycotoxigenic and, depending on the species, can produce trichothecenes, fumonisins and/or zearalenone, as well as other less-important mycotoxins such as moniliformin, beauvericin, fusaproliferin, fusaric acid and/or enniatins (Logrieco et al., 2002; Jestoi, 2008).
In Spain, maize kernels are predominantly infected by F. verticillioides and, to a lesser extent, by F. proliferatum. Both produce fumonisin (Butrón et al., 2006; Jurado et al., 2006; Arino et al., 2007). Sanchís et al. (1995) noted the potential fumonisin contamination in many Spanish corn-based products containing both Fusarium species, while Butrón et al. (2006) previously reported fumonisin contamination of maize flours above the levels established in the European Regulations. The incidence of Fusarium spp. infection of maize kernels varies significantly among years and locations in many geographical areas (Bottalico, 1998; Goertz et al., 2010; Covarelli et al., 2011; Boutigny et al., 2012). Bakan et al. (2002) found that F. proliferatum was more abundant in kernels in northeastern Spain. In northwestern Spain, climatic characteristics during kernel filling are very different from those in northeastern Spain, which could be responsible for differences in the Fusarium species identified in the area (Marín et al., 1996; Butrón et al., 2006).
Although yearly and geographical variation in the diversity of Fusarium in maize kernels has been noted, there is no information on the environmental traits affecting biodiversity. Therefore, the objectives of the present study were: (i) to monitor the occurrence of Fusarium species in maize kernels in coastal and inland locations of northwestern Spain to determine the potential risk of contamination by each of several mycotoxins; and (ii) to identify environmental traits associated with changes in the Fusarium mycoflora in the area.
Materials and methods
Six maize hybrids derived from crosses among inbred lines EP39, CM151, EP42 and EP47 were used to monitor the prevalence of Fusarium spp. on naturally infected maize kernels. Because corn borer attack is associated with increased kernel infection by fungi (Smith & White, 1988), two inbred lines (EP39 and CM151) were selected that were resistant to the Mediterranean corn borer (Sesamia nonagrioides) attack, while EP42 and EP47 were susceptible (Santiago et al., 2003).
Hybrids were sown both early (end of April) and late (middle of May) in 2007 and 2008 at three locations in northwestern Spain and were harvested both early and late. Early sowing and harvest avoiding wet weather are usually suggested as good agronomic practices. Pontevedra (42°24′N, 8°38′W, 50 m a.s.l.) and Barrantes (42°30′N, 8°46′W, 50m a.s.l.) were near the coast, while Valongo (42°26′N, 8°27′W, 500 m a.s.l.) was situated inland. Therefore, each hybrid was evaluated in a total of 24 environments (2 years × 3 locations × 2 sowing dates × 2 harvest dates). A split-plot design with three replications was used for each environment; hybrids were assigned to main plots and harvest times to subplots. Main plots consisted of two rows (0·80 m apart) with 13 two-kernel hills (0·21 m apart) per row. After thinning, the final density was around 60 000 plants ha−1. Within each plot, ears from one row (subplot) were harvested at the beginning of October (early harvest) and from the other row 1 month later (late harvest). Harvested ears were shelled and kernels were dried at 35°C for 1 week and maintained at 4°C and 50% humidity until analyses were performed.
A meteorological station was installed at each location to record climatic data every 12 min and used to compute climatic parameters: average daily temperatures (°C); mean daily maximum temperature (°C); mean daily minimum temperature (°C); mean daily relative humidity (%); rainfall (mm); number of days with minimum temperature ≤15°C; number of days with maximum temperature ≥30°C; number of days with mean temperature 10–15°C (≥10 and <15°C), 15–20°C, 20–25°C and 25–30°C; and number of days with rainfall ≥2 mm. These climatic parameters were selected according to previous reports on the influence of climate on mould development in wheat and maize (Marin et al., 2004; de la Campa et al., 2005; Schaafsma & Hooker, 2007; Maiorano et al., 2009). Climatic parameters were calculated for the following periods: the entire growing period (from sowing to harvest); the vegetative period (sowing to silking); the reproductive period (silking to harvest); the flowering period (15 days before silking to 15 days after silking); critical period 1 (C1; between 10 and 4 days before silking); critical period 2 (C2; between 4 days before and 2 days after silking); critical period 3 (C3, 2–8 days after silking); critical period 4 (C4; 8–14 days after silking); the milk-dough kernel stage (16–30 days after silking); the dent kernel stage (31–45 days after silking); the kernel developing period (silking to physiological maturity); and the kernel drying period (physiological maturity to harvest).
Other environmental parameters recorded at harvest were: maize husk coverage, evaluated by a visual scale from 0 (loose husks with visible cob) to 5 (tight husks) (Wiseman & Isenhour, 1992); kernel damage by corn borers on a visual scale from 1 (100% of ear damaged by borers) to 9 (no damage); tunnel length; maize stem damage by borers (in cm); kernel humidity (%); kernel damage by Sitotroga cerealella; percentage of kernels with damaged pericarp; and thickness of pericarp (in μm).
Identification of Fusarium species
Fifty kernels from each subplot were used to identify the presence of each Fusarium species in both 2007 and 2008. Maize kernels were grown on Komada medium, which is selective for Fusarium spp. (Komada, 1975). Monosporic isolates were obtained and grown on potato dextrose agar, Spezieller Nährstoffarmer agar and carnation leaf agar media to determine the specific characteristics of each isolate (Leslie & Summerell, 2006).
The Fusarium species were also identified using molecular techniques. Fungal DNA was directly extracted from mycelia of monosporic cultures grown on plates using the commercial kit E.Z.N.A. Fungal DNA Mini (Omega Bio-tek). All monosporic isolates were tested by PCR using primers ITS1 and ITS4 (White et al., 1990) to amplify the internal transcribed spacer (ITS) region of rDNA and primers EF1 and EF2 (O'Donnell et al., 2000) for the elongation factor 1α gene (EF-1α). ITS PCRs contained one PuReTaq Ready-To-Go PCR Bead (GE Healthcare), 1 μL genomic DNA, 0·3 μL each primer (10 μm) and sterile water to a final volume of 25 μL. The EF-1α PCRs contained 1 μL genomic DNA, 25 pmol each primer, 2·5 μL dNTPs (2 mm), 1 U Green Taq DNA polymerase (GenScript), 1× standard PCR buffer and sterile water to a final volume of 25 μL.
Both DNA amplification reactions were carried out in a Thermocycler Biometra T3000 under the following conditions: 5 min at 94°C; 35 cycles at 94°C for 30 s, 55°C (for ITS1/ITS4) or 53°C (for EF1/EF2) for 30 s, and 72°C for 1 min; and a final elongation at 72°C for 10 min. PCR products were electrophoresed on a 2% agarose gel, then stained with ethidium bromide and visualized with a UV transilluminator. Product size was estimated by comparison with a 100 bp standard ladder (Marker XIV; Roche Diagnostics). Amplified products were sequenced with the same primers used for PCR in an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). Sequences were analysed with the NCBI blast program and compared with those deposited in GenBank (National Center for Biotechnology Information, 2012; http://www.ncbi.nlm.nih.gov/). The molecular identification of a species was accepted when the sequence identity was above 98%.
The averaged occurrence of each Fusarium species in each of the 24 environments (2 years × 3 locations × 2 sowing dates × 2 harvest dates) was computed as the mean of individual percentages in 18 subplots (six different maize hybrids replicated three times). Combined analyses of variance for Fusarium spp. occurrence were computed with the general linear model (glm) procedure of SAS following a split-plot design (SAS Institute). All sources of variation were considered as fixed factors. Comparisons of means among years, locations, sowing dates and harvest dates were made by Fisher's protected least significant difference test. In addition, Pearson correlations between Fusarium spp. were calculated.
To examine the relationships between the environmental variables and the Fusarium species in the kernels, a redundancy analysis (RDA) was performed using canoco (Ter Braak & Smilauer, 1997). Previously, a detrended correspondence analysis had been performed to determine whether the data fitted a linear ordination model as RDA or not, following the recommendations of Lepš & Šmilauer (2003). Analyses were applied to the averaged percentage of presence of each Fusarium species in maize kernels in each environment. RDA computations were performed on centered and standardized data and run with a forward selection of the environmental variables procedure and the associated Monte Carlo permutation test (499 unrestricted permutations) to exclude environmental variables that did not contribute significantly (P > 0·05) to the variation in the Fusarium species.
Nine different Fusarium species were isolated from maize kernel samples (Table 1). Five species were found in all locations: F. verticillioides, complex F. subglutinans sensu lato, F. proliferatum, F. poae and F. oxysporum. The prevalent species in all 24 environments was F. verticillioides; the environmental average of F. verticillioides presence ranged from 33 to 99%. Second most abundant was the complex F. subglutinans sensu lato, which was present in all environments at percentages varying from 1 to 27%. The species in this complex were F. begoniae and F. sterilihyphosum. The remaining Fusarium species (F. proliferatum, F. poae, F. oxysporum, F. cerealis, F. equiseti, F. solani and F. culmorum) were present sporadically across environments and never exceeded a kernel presence of 4% (data not shown).
Table 1. Averaged percentages of kernels containing Fusarium spp. isolates in 2007 and 2008 at three locations in northwestern Spain. The numbers of positive samples are in parentheses
F. subglutinans sensu lato
Total % of positive kernels
Total % of negative kernels
There were no differences in occurrence between years, sowing dates or harvest dates, or among locations for the diverse Fusarium species, with the exception of F. verticillioides which was more common in coastal locations (Pontevedra and Barrantes) than in the inland one (Valongo). In addition, it occurred more frequently in the early sowing (86·19 versus 74·55% in the late sowing) and late harvest (80·94 versus 73·52% in the early harvest). No significant differences between years were observed for F. verticillioides presence.
There was a simple positive correlation between abundances for F. oxysporum and F. solani (r = 0·67, P ≤ 0·001), F. cerealis and F. poae (r = 0·56, P ≤ 0·01), F. equiseti and F. culmorum (r = 0·77, P ≤ 0·001), F. equiseti and F. subglutinans sensu lato (r = 0·59, P ≤ 0·01), and F. culmorum and F. subglutinans sensu lato (r = 0·70, P ≤ 0·001). These correlations are based on very low percentages of presence for those species.
The RDA was performed using significant non-categorical environmental factors as explicative variables. The results of the Monte Carlo permutation tests revealed the statistical significance (P ≤ 0·05) of the effects of three environmental variables on Fusarium species composition: number of days with mean temperature of 15–20°C during the drying kernel period, averaged relative humidity at C3, and number of days with minimum temperature ≤ 15°C during the dent kernel stage (Table 2). The first two axes of the RDA using these three environmental parameters as explicative variables explained 71·2% of the variability in Fusarium species occurrence (Fig. 1), 75·0% of the variability for F. verticillioides and 49·0% of the variability for F. subglutinans sensu lato (Table 3). Days with mean temperature of 15–20°C during the drying kernel period and days with minimum temperature ≤15°C during the dent kernel stage contributed substantially to the gradient for the first axis, which explained 75% of the variability for F. verticillioides. The averaged relative humidity during C3 and days with mean temperature of 15–20°C during the drying kernel period strongly affected the second axis. Both axes explained 49% of the variability for F. subglutinans sensu lato and between 6 and 21% of variability for F. poae, F. proliferatum, F. oxysporum, F. cerealis, F. equiseti, F. solani and F. culmorum (Table 3). More days with mean temperatures of 15–20°C during the drying kernel period and fewer days with minimum temperature ≤15°C during the dent kernel stage favoured the occurrence of F. verticillioides in maize kernels, while the presence of F. subglutinans was augmented with increased relative humidity during C3 and fewer days with mean temperatures of 15–20°C during kernel drying (Fig. 1).
Table 2. Statistics of the environmental variables retained after the Monte Carlo permutation test and included in the redundancy analyses for Fusarium species composition in maize kernels cultivated in 24 environments (2 years × 3 locations × 2 sowing dates × 2 harvest dates) in northwestern Spain
Tm15-20S: number of days with mean temperature ≥15°C and <20°C during the kernel drying period; HumC3: relative humidity during critical period C3 (2–8 days after maize silking); Tmin15D: number of days with minimum temperature ≤15°C during the maize kernel dent stage.
Table 3. Accumulated variability for each Fusarium species abundance in 24 environments (2 years × 3 locations × 2 sowing dates × 2 harvest dates) in northwestern Spain explained by three selected significant variables: days with mean temperature ≥15°C and <20°C during the kernel drying period; relative humidity during critical period C3 (2–8 days after maize silking), and days with minimum temperature ≤15°C during the maize kernel dent stage
F. subglutinans sensu lato
All Fusarium species isolated from maize kernel samples were previously found in maize grown in Europe (Logrieco et al., 2002; Dorn et al., 2009; Goertz et al., 2010). These species are, in general, mycotoxigenic and produce fumonisins, trichothecenes, zearalenone, moniliformin, beauvericin, enniatins, and fusaric acid (Logrieco et al., 2003; Leslie & Summerell, 2006; Jestoi, 2008). The results here confirmed that F. verticillioides is the prevalent species in northwestern Spain (Munoz et al., 1990; Butrón et al., 2006).
Fusarium verticillioides is the species most frequently isolated from maize pink ear rot, which occurs commonly from southern to central Europe, while the main species causing maize red ear rot is F. graminearum, which is increasingly distributed from central to northern European regions (Logrieco et al., 2002). In warm southern European areas, F. verticillioides is associated with F. proliferatum, while displacement toward central Europe increases the presence of F. subglutinans to the detriment of F. proliferatum. In this study, F. proliferatum was scarce, and F. graminearum was absent, while F. verticillioides was highly predominant, and F. subglutinans sensu lato was the most abundant group. These observations agree with those of surveys performed in the past 10 years in maize growing areas around the world, in which F. verticillioides in association with F. subglutinans are becoming the dominant species (Bottalico, 1998). The absence of F. graminearum could be a consequence of the early establishment of F. subglutinans, which may act as a biological control mechanism against invasion by F. graminearum (Cooney et al., 2001) and/or possible competition with F. verticillioides (Reid et al., 1999; Marin et al., 2004). Environmental conditions in northwestern Spain, mild temperatures throughout the year, and moderate risk of ear damage by corn borers can affect Fusarium species distribution. Corn borer damage is associated with increased infection rates by F. subglutinans and F. verticillioides and less infection by F. graminearum (Lew et al., 1991). In addition, more extreme temperatures would favour F. graminearum (colder) or F. proliferatum (warmer) (Logrieco et al., 2002).
Fusarium verticillioides is a fumonisin producer, and F. subglutinans produces a range of mycotoxins including moniliformin, fusaproliferin, beauvericin and fumonisin (Jestoi, 2008). The fumonisin-producing capacity of the F. verticillioides isolates in the area has been noted (Cao, 2013). In addition, previous studies showed the risk of fumonisin occurrence in maize kernels in northwestern Spain (Butrón et al., 2006; Cao et al., 2013). The higher presence of F. verticillioides in a wide range of natural environments supports the idea that fumonisin contamination is the main maize-based feed and food safety concern in this area, although emerging mycotoxins such as moniliformin, fusaproliferin and beauvericin should also be taken into account.
The influence of the geographical location on the variability of occurrence of F. verticillioides is important when climatic conditions vary across locations (Boutigny et al., 2012). Fusarium verticillioides presence was higher in coastal locations than the inland one, as expected, because the coastal climate is more temperate. Variation between years was not significant; in southern Europe, minor differences among years for Fusarium variability have been reported (Dorn et al., 2009; Covarelli et al., 2011), while substantial changes in annual Fusarium spp. composition have been found in northern Europe (Dorn et al., 2009; Goertz et al., 2010). With respect to sowing and harvest dates, this study recommends the role of agronomic practices in regulating the occurrence of F. verticillioides (Blandino et al., 2009), although only slight effects on Fusarium presence were noted in this particular study; there was probably no effect on fumonisin contamination. The positive correlation among abundance for F. subglutinans sensu lato, F. equiseti and F. culmorum, as well as between F. cerealis and F. poae, corroborate that these species are adapted to similar environmental conditions that occur in central and northern Europe (Logrieco et al., 2002).
Work must be done to understand the epidemiology of Fusarium infection by focusing more precisely on the relationship between environmental variables and the disease cycle. Temperature and humidity must be considered as factors that influence spore production under field conditions (Indira & Muthusubramanian, 2004). In the same way, mycotoxin contamination is affected by climatic factors such as temperature and relative humidity both pre- and post-harvest (Paterson & Lima, 2010). The two main abiotic factors associated with the life cycle of F. verticillioides are temperature and water activity (Marin et al., 2004; Samapundo et al., 2005). They were considered the main factors in modelling fungal development and fumonisin synthesis (de la Campa et al., 2005; Maiorano et al., 2009). Likewise, it was noted how temperature and humidity affected Fusarium spp. occurrence in northwestern Spain. It is concluded that warmer temperatures during the later stages of kernel development and kernel drying favoured the presence of F. verticillioides in maize kernels, while the presence of F. subglutinans sensu lato was augmented by higher relative humidity when fresh silks were exposed and by cooler temperatures during kernel drying. These results agreed with the idea that F. subglutinans prefers a cooler temperature and more humid conditions (Logrieco et al., 2002; Goertz et al., 2010; Boutigny et al., 2012) than F. proliferatum and F. verticillioides.
This research was supported by the National Plan for Research and Development of Spain (AGL2009-12770), the Autonomous Government of Galicia (PGIDIT06TAL40301PR) and the Deputación de Pontevedra. A. C. acknowledges funding from the JAE Program of the Spanish Council of Research. R. S. acknowledges postdoctoral contract ‘Isidro Parga Pondal’ supported by the Autonomous Government of Galicia and the European Social Fund.