Assessing pigmented pericarp of maize kernels as possible source of resistance to fusarium ear rot, Fusarium spp. infection and fumonisin accumulation

International Journal of Food Microbiology 227 (2016) 56–62

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Assessing pigmented pericarp of maize kernels as possible source of resistance to fusarium ear rot, Fusarium spp. infection and fumonisin accumulation Giovanni Venturini ⁎, Laleh Babazadeh, Paola Casati, Roberto Pilu, Daiana Salomoni, Silvia L. Toffolatti Dipartimento di Scienze Agrarie e Ambientali — Produzione, Territorio, Agroenergia, Università degli Studi di Milano, via G. Celoria 2, 20133 Milano, Italy

a r t i c l e

i n f o

Article history: Received 10 December 2015 Received in revised form 3 March 2016 Accepted 21 March 2016 Available online 4 April 2016 Keywords: Fumonisins Fusarium verticillioides Kernel pericarp Zea mays

a b s t r a c t One of the purposes of maize genetic improvement is the research of genotypes resistant to fusarium ear rot (FER) and fumonisin accumulation. Flavonoids in the pericarp of the kernels are considered particularly able to reduce the fumonisin accumulation (FUM). The aim of this field study was to assess the effect of flavonoids, associated with anti-insect protection and Fusarium verticillioides inoculation, on FER symptoms and fumonisin contamination in maize kernels. Two isogenic hybrids, one having pigmentation in the pericarp (P1-rr) and the other without it (P1-wr), were compared. P1-rr showed lower values of FER symptoms and FUM contamination than P1-wr only if the anti-insect protection and the F. verticillioides inoculations were applied in combination. Fusarium spp. kernel infection was not influenced by the presence of flavonoids in the pericarp. Artificial F. verticillioides inoculation was more effective than anti-insect protection in enhancing the inhibition activity of flavonoids toward FUM contamination. The interactions between FUM contamination levels and FER ratings were better modeled in the pigmented hybrid than in the unpigmented one. The variable role that the pigment played in kernel defense against FER and FUM indicates that flavonoids alone may not be completely effective in the resistance of fumonisin contamination in maize. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Fusarium ear rot (FER) is one of the most widespread diseases affecting maize (Zea mays L.) ears in temperate regions and it can result in high yield losses due to a decrease of grain matter content, kernel density and total grain yield (Presello et al., 2008). Climatic conditions during the growing season are determinant factors for FER incidence and severity (Venturini et al., 2015). FER epidemics are particularly favored at flowering and kernel drying in presence of warm and dry conditions (Cao et al., 2014; Miller, 2001). Beside climatic conditions, many other factors, such as cultural practices, host susceptibility, kernel damage by insects and pest management strategy, contribute to FER (De Curtis et al., 2011; Parson and Munkvold, 2012). In most maize-growing areas of southern Europe, such as northern Italy, FER is caused by several Fusarium species belonging to the Fusarium fujikuroi species complex (FFSC) (Aguín et al., 2014; Shala-Mayrhofer et al., 2013; Venturini et al., 2011). The most important FFSC species responsible of FER are

Abbreviations: (AIP), anti-insect protection; (FER), fusarium ear rot; (FERi), fusarium ear rot incidence; (FERs), fusarium ear rot severity; (FFC), Fusarium fujikuroi species complex incidence; (FUM), fumonisin contamination; (FVI), Fusarium verticillioides inoculation; (IDi), insect damage incidence; (IDs), insect damage severity. ⁎ Corresponding author. E-mail address: [email protected] (G. Venturini).

http://dx.doi.org/10.1016/j.ijfoodmicro.2016.03.022 0168-1605/© 2016 Elsevier B.V. All rights reserved.

the conidial anamorphs of the biological species F. verticillioides (Sacc.) Nirenberg followed by F. proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg and F. subglutinans (Wollenw. & Reinking) P.E. Nelson, Toussoun & Marasas (White, 1999). FFSC strains may occur on maize as seedborne endophytes or infect the plant at various developmental stages without inducing visible disease symptoms (Munkvold et al., 1997; Venturini et al., 2011). Insect wounds and exposed silks constitute the most important pathways for FFSC conidia to colonize kernels (Alma et al., 2005; Duncan and Howard, 2010). In addition, systemic movement of FFSC strains from infected seed and stalk to developing maize kernels has also been reported as an infection pathway (Munkvold, 2009). In northern Italy one of the most relevant pests of maize implicated with FER incidence and severity are corn borers, in particular the European corn borer (ECB) Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) (Mazzoni et al., 2011), although other insects are likely to facilitate FER as well (Cao et al., 2014; Parson and Munkvold, 2012). Strains belonging to FFSC, mainly F. verticillioides and F. proliferatum, are mycotoxin producing fungi. Thus, FFSC infections of maize can result in accumulation of mycotoxins such as fumonisins (FUM) in maize kernels. Fumonisin B1 (FB1) contamination in maize grain is of concern because of its causal role in equine leukoencephalomalacia, porcine pulmonary oedema, liver and renal carcinogenicity in laboratory rodents and possibly even human carcinogenicity (Shephard et al., 2013).

G. Venturini et al. / International Journal of Food Microbiology 227 (2016) 56–62

Northern Italian grown maize undergoes variable levels of fumonisin contamination depending on several factors such as climatic variables, maize maturity class, and growing weeks (Maiorano et al., 2009; Torelli et al., 2012; Venturini et al., 2015). The application of cultural practices to reduce fumonisin levels is not always effective and environmentally safe methods to control FER and FUM levels in maize (De Curtis et al., 2011; Folcher et al., 2009). Maize genetic improvement seems to be the most sustainable approach to control FER and minimize fumonisin accumulation. However, difficulties might be encountered in the search for resistant maize genotype, because maize resistance to both FER and FUM is under polygenic control and it is quantitative in nature. No source of complete resistance has been identified for either FER or FUM (Mesterházy et al., 2012). Focus of many researches, FER and FUM resistance has brought maize breeders to consider diverse approaches that are currently under investigation (Atanasova-Penichon et al., 2014; Picot et al., 2013). Among them, the development of maize genotypes with kernel pericarp enriched with phenolic compounds seems particularly promising in the reduction of mycotoxin accumulation (Pilu et al., 2011; Sampietro et al., 2013). In addition, phenolic pigments accumulated in the maize seeds are associated with antioxidant power and thought to be highly beneficial for human health (Rodrıguez et al., 2013). Phenolic compounds in kernel pericarp such as flavonoids, especially those regulated by the p1 gene (Grotewold et al., 1994), reduced fumonisin accumulation (Pilu et al., 2011). Flavonoids are thought to act as a physical barrier against fungal infection by hardening maize kernel pericarp and therefore reducing the mycelial progress from infected to intact kernels. Moreover, the antifungal activity of flavonoids is probably exerted by complexing irreversibly with nucleophilic amino acids in fungal proteins leading to inactivation of the proteins and loss of their functions (Treutter, 2006). Inhibitory effect of flavonoids on fumonisin biosynthesis has not yet been fully elucidated, but it can be hypothesized that flavonoids may inhibit redox enzymes encoded by the FUM6 gene blocking fumonisin production (Kim et al., 2006). In a previous field study FER and fumonisin levels of two isogenic maize hybrids, one able to accumulate flavonoids and one colourless, were compared. That research showed that the effects of flavonoids in maize pericarp varied depending on the experimental site and was not stable across the years (Venturini et al., 2015). The objective of the current study was to investigate the stability in the field of pigmented maize resistance to FER and FUM contamination in response to artificial inoculation of the ears with F. verticillioides strains and/or to the apposition of physical barrier against insects. The effect of flavonoids on frequency and severity of insect damage and FER, FFSC kernel contamination and FUM accumulation was evaluated.

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was required, F. verticillioides strains were grown on PDA slants at 25 °C under combined visible (white) and ultraviolet (black) light (12 h per day) for 7 days. Conidial suspensions were prepared the day before the field inoculation assays by adding 6 mL of sterile distilled water to the each PDA slant and gentle shaking. The conidia were harvested by filtering the suspensions through two layers of cheesecloth and then enumerated and adjusted to 1.0 × 106 conidia/mL with a haemocytometer (Kova®, Hycor Biomedical Co., Indianapolis). To obtain a mixed inoculum, the conidial suspensions of each F. verticillioides strain were mixed together and stocked overnight at 4 °C. 2.2. Experimental design and inoculation procedure Field experiments were carried out in 2013 in Landriano (PV) at 45°190N, 9°160E, 88 m a.s.l. The field was in a maize–maize rotation and standard soil fertilization, irrigation and weed management practices were applied. The experimental design was a randomized complete block arranged as a split plot with three replicates. Two isogenic maize hybrids, one with P1-rr allele providing pigmentation in pericarp, due to accumulation of phlobaphene pigments, and the other carrying P1-wr allele without phlobaphene accumulation in pericarp (Pilu et al., 2011), were sown in late April. Hybrids were applied to main plots representing P1-wr and P1-rr genotypes, respectively. Each plot consisted in four 70-m rows, 0.7 m apart, and sown at a density of 7 seeds m−1. The growth stages were monitored weekly following the BBCH (Biologische Bundesanstalt, Bundessortenamt and Chemical industry) scale (Lancashire et al., 1991). Treatments were used as sub-plots and were randomized within the two main plots. Treatments included: anti-insect protection (AIP) and F. verticillioides inoculation (FVI). Experimental units were single rows consisting of 60 plants (20 plants per replicate). Anti-insect protection was realized by covering all primary ears in the AIP experimental units, at silking (BBCH 61, silking was assigned when silk emergence could be observed in 50% of maize ears), with a bag (400 × 200 mm) made by transparent insect-proof polyethylene net, mesh size 0.22 mm (Retificio Padano s.r.l., Ospitaletto, BS, Italy), sealed at the basal part of the ear (Castellano et al., 2008). The primary ears of plants within the FVI units were inoculated 7 days after flowering when silks were still green but started to dry out from the tips (Miedaner et al., 2010). Silk channel inoculation was performed by injection of 1 ml inoculum into the silk channel of the primary ear of each plant. Negative controls consisted of experimental units of not covered ears (nAIP) or sterile water-inoculated ears (nFVI). Meteorological data were recorded by an automated weather station placed next to the field, with an hourly step. 2.3. Insect damage and FER incidence and severity

2. Materials and methods 2.1. Fungal inoculum Eight F. verticillioides strains (Fv2003, Fv2010, Fv2170, Fv2198, Fv2221, Fv2232, Fv2233 and Fv2306) were used in this study. These strains were isolated from naturally infected maize kernels in 2011, identified following morphological and biological species concepts (Leslie and Summerell, 2006), and belong to the fungal culture collection of the Mycology Laboratory at the Department of Agricultural and Environmental Sciences — Production, Land, Agrienergy (DiSAA-PTA, University of Milan, Milan, Italy). The isolates were maintained on potato dextrose agar (PDA, Difco®, Becton & Dickinson Co., Sparks) at 4 °C and stored as conidial suspensions in 20% glycerol at −80 °C. Preliminary results performed in the laboratory indicated that all the F. verticillioides isolates showed high levels of pathogenicity on maize seeds following the method described by Venturini et al. (2013). The F. verticillioides isolates were also tested for the fumonisin production in vitro as described by Glenn et al. (2008) (data not shown). They produced from 174.4 to 373.4 μg/g of fumonisin in vitro. When inoculum

Insect damage (ID) and FER incidence and severity were assessed at physiological maturity on 20 ears per replicate. Incidence of ID (IDi) was calculated as the percentage of ears with visible damage due to larval activity. Incidence of FER (FERi) was calculated as the percentage of ears with symptoms per replicate. ID severity (IDs) was calculated as the percentage of kernels per ear with visible damage due to insect activity. A scale of 0–6 was used in which each numerical value corresponds to a percentage interval of surfaces exhibiting visible kernel injuries due to larval activity according to the modified version of a 1– 7 scale suggested by Blandino et al. (2008): 0 no injuries, 1 = 1–5%, 2 = 6–10%; 3 = 11–20%, 4 = 21–35%, 5 = 35–60% and 6 N 60%. FER severity (FERs) was calculated as the percentage of surface with symptoms per ear. A modified version of a 1–7 scale proposed by Reid and Zhu (2005) was adopted, in which each numerical value corresponds to a percentage interval of surfaces exhibiting visible symptoms of the disease such as rot, mycelial growth and kernels with ‘starburst’ streaks within the pericarp, according to the following scale: 0 = no symptoms, 1 = 1–3%, 2 = 4–10%; 3 = 11–25%, 4 = 26–50%, 5 = 51–75% and 6 = 76–100%. ID and FER severity indexes expressed as

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percentages were calculated according to the modified Townsend– Heuberger formula: IDs or FERs = Σ [(n × c)/6 N] × 100, where n is the number of ears per class, c is the numerical value of each class, and N is the total number of ears examined per replicate (Townsend and Heuberger, 1943). 2.4. Mycological analysis After visual assessment, 20 ears per replicate were dehusked and dried with forced air (30–40°C) to c. 13 % grain moisture. The kernels of the ears of each experimental unit were obtained by an electric sheller, pooled and thoroughly mixed. A 1 kg sample was taken from each experimental unit for subsequent mycological analysis. One hundred asymptomatic kernels per replicate, randomly chosen, were surface treated with NaOCl (7%) for 5 min, and rinsed three times in sterile deionized water (Venturini et al., 2015). After drying on sterile paper, the kernels were placed on Petri dishes containing half-strength potato dextrose agar (PDA; Difco) amended with dichloran (0.002 g/L) and broad-spectrum antibiotics (chloramphenicol, 0.1 g/L; streptomycin sulphate, 0.2 g/L; neomycin sulphate, 0.12 g/L). The plates were incubated at 25 °C for 5 days and microscopically examined (×20 magnification). All the developed colonies belonging to the genus Fusarium, identified at genus level through morphological criteria (Pitt and Hocking, 2009), were transferred to PDA and purified by monoconidial isolation. All the monoconidial Fusarium strains were then inoculated on Spezieller Nährstoffarmer agar plates (SNA) (Leslie and Summerell, 2006) and then incubated at 25°C for 7 days under combined visible (white) and ultraviolet (black) light (12 h per day). Morphological identification of FFSC strains was carried out following the criteria recommended by Leslie and Summerell (2006). Data on fungal incidence were recorded as the percentage of kernels infected by FFSC strains (FFC). The identification at species level within FFSC was not relevant to the aim of the present study. 2.5. Fumonisin quantification Concentration of fumonisins in grain was assessed by ELISA (Ridascreen® Fumonisin, R-Biopharm Rhône Ltd). About 100 g of grain samples from each replicate was ground for 1.5 min in a laboratory mill (Foss Cyclotec Sample Mill 1093; Foss Italia S.r.l.) set at the finest setting. Milled samples were thoroughly mixed prior to fumonisin extraction. Fumonisins were extracted blending 5 g of sample and 25 ml of 70% methanol and stirring vigorously for 3 min on orbital shaker. The extracts were filtered through filter Whatman no. 1 (GE Healthcare Ltd) and diluted 1:14 with sterile distilled water. Diluted extracts and six standards, at concentrations of 0.000, 0.025, 0.074, 0.222, 0.666 and 2.000 μg/g of fumonisins, were subjected to ELISA. Absorbance was measured at 450 nm with a microplate reader in a Sunrise® Absorbance Reader (Tecan Group Ltd). Concentration of fumonisins of the samples was estimated on the base of a semilogarithmic function between fumonisin concentration and relative absorbance of the standards using RIDA® SOFT Win software (R-Biopharm). The limits of detection of the method (LOD, signal-to-noise ratio 3:1) and quantification (LOQ, signal-to-noise ratio 10:1) were 0.025 and 0.075 μg/g, respectively. The recovery rates in spiked maize meal samples, corresponding to the standard fumonisin (0.050 and 0.500 μg/g), was approximately 60 %; with a mean coefficient of variation of approximately 8 %. 2.6. Data analysis The SPSS statistical package for Windows, v. 22.0 (SPSS Inc.), was used for all statistical analyses. Normal distribution and homogeneity of variances were verified using the Shapiro–Wilk test and the Levene’s test, respectively. A Student’s t-test was used to compare P1-rr with P1wr hybrid, AIP with nAIP parcels and FVI with nFVI parcels for IDi, IDs, FERi, FERs, FFC and FUM. All the percentage values (p) were converted

with the equation y = arcsin (√ p/100) and FUM data were 4th-root transformed to normalize distribution of residuals and remove heterogeneity of error variances prior to t-test. The effects of hybrid, AIP and FVI on IDi, IDs, FERi, FERs, FFC and FUM were tested by the general linear model (GLM). Finally, data were subjected to regression analyses using the CURVEFIT procedure in order to calculate the regression statistics among measured variables. 3. Results Environmental conditions (mean temperature, relative humidity and rainfall) recorded in 2013 during the maize growing season at the experimental field are reported in Fig. 1. During the flowering stages the climatic conditions in the field were quite uniform, with mean temperatures around 26 °C, relative humidity around 75 % and rare precipitation events above 1 mm (only one day with 2,6 mm). After the FVI treatment, during the grain filling stages, temperatures were extremely variable. Particularly during the second part of August, high maximum temperatures quickly dropped and raised again. A similar trend was followed by relative humidity values. During the ripening stages, several rainfall events above 1 mm occurred particularly during the third decade of August. In general, both treatments AIP and FVI showed significant differences between experimental units. All the AIP units showed significantly lower values of IDi, IDs FERi, FERs and FUM compared to the nAIP units. FFC infection levels were similar among AIP and nAIP experimental units (Table 1). As far as FVI treatment is concerned, in the FVI experimental units significantly higher values of FERi, FERs, FFC and FUM, compared to values obtained in the nFVI parcels. For IDi and IDs, the inoculation treatment failed to show differences between FVI and nFVI parcels as t-tests did not report any significant differences for P b 0.05 (Table 1). Overall, the two hybrids exhibited significant differences for IDi, IDs, FERi, FERs and FUM but not for FFC. Larvae damage was more frequent and severe in P1-wr parcels than in P1-rr experimental units. A similar trend was observed also for FERi, FERs and FUM. Finally, FFC was not different between hybrids (Table 1). Significant differences between the two hybrids were also found when the treatments, AIP and FVI, were applied separately or combined. In the AIP–FVI experimental units for FER and FUM levels it was possible to find significant differences between the two hybrids. FER incidence and severity were lower in P1-rr than in P1-wr (FERi: t = − 6.859, d.f. = 4, P = 0.035; FERs: t = − 7.263, d.f. = 4, P = 0.002) (Table 2). FUM average was higher in P1-wr than in P1-rr (t = −7.273, d.f. = 4, P b 0.001) (Table 2). In the control parcels, nAIP–nFVI, hybrids did not exhibit any significant difference for all the measured variables. In the AIP–nFVI experimental units the P1-rr hybrid showed lower FER incidence and severity than P1-wr (FERi: t = −6.181, d.f. = 4, P = 0.002; FERs: t = − 8.602, d.f. = 4, P b 0.001) (Table 2). Insect damages, FFC and FUM levels did not differ among hybrids. As far as nAIP-FVI parcels are considered, it was possible to find significant differences between hybrids for ID incidence, in P1-wr IDi mean was higher than in P1-rr was (t = −7.196, d.f. = 4, P b 0.001) (Table 2). For ID severity, in P1rr mean was lower than in P1-wr (t = − 6.073, d.f. = 4, P = 0.008), and finally for FUM with P1-wr displaying FUM average higher than P1-rr (t = −7.277, d.f. = 4, P b 0.001) (Table 2). In general the effects of the treatments were significant individually for all measured parameters, except for FFC that was significantly influenced only by FVI (P = 0.038). Two- and triple-way interactions between the source of variations of hybrid (H), AIP and FVI were never found to be significant (Table 3). Because H, AIP and FVI were independently significant for the majority of the measured parameters but their interactions were not, it was possible to combine data across treatments and to analyze the influence of each source of variation on the interactions between variables. Taking into account the ln regression analyses of FUM with IDi, IDs, FERi, FERs and FFC, H played an important role in modeling the interactions

G. Venturini et al. / International Journal of Food Microbiology 227 (2016) 56–62

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Fig. 1. Climatic conditions in the experimental maize field during the 2013 growing season. Temperature (°C), relative humidity (%) and rainfall (mm) recorded by the automated weather station.

between variables since the goodness of fit of the models were clearly different between P1-rr and P1-wr parcels (Table 4). Good and significant models were registered for all the influencers on FUM in P1-rr parcels, only FFC reported a weak but significant influence on FUM in P1-rr samples. On the contrary, in P1-wr only models concerning FERi and FERs were good and significant (Table 4). AIP treatment shaped the interactions between FUM and other variables in a lesser extent than H since only for FERi and FERs good models were reported. Anti-insect protection application was significant in defining the relationship between FUM and FFC since only for nAIP parcels a quite good and significant model was computed (Table 4). In addition, in nAIP ears quite good and significant models were registered for FFC with IDi and FERi, with R2 = 0.574 (y = − 124.946 + 49.683 ln x) and R2 = 0.457 (y = −98.885 + 43.591 ln x) respectively. In AIP parcels, however, ln regression analyses of FFC with the other variables were neither good nor significant (data not shown). The F. verticillioides inoculation treatment (FVI) did not markedly shape the regression analyses for FUM and the other measured variables. Only slight differences were reported between R2 values of FVI and nFVI parcels (Table 4). Interesting and relevant differences were registered between significant quadratic models computed in FVI and nFVI parcels, for FERs with FERi, IDi and IDs. Taking into consideration FVI parcels, models for FERs with FERi, IDi and IDs were R 2 = 0.980 (y = 4.345 − 0.094 x + 0.005 x2), R2 = 0.934 (y = 8.054 − 0.259 x + 0.006 x 2) and Table 1 Insect damage incidence (IDi) and severity (IDs), fusarium ear rot frequency (FERi) and severity (FERs), Fusarium fujikuroi species complex incidence (FFC) and fumonisin contamination (FUM) of maize samples belonging to different parcels of P1-rr and P1-wr hybrids.

Measured variables

IDi (%) IDs (%) FERi (%) FERs (%) FFC (%) FUM (μg/g)

Fusarium verticillioides inoculation (FVI)

Anti-insect protection (AIP)

Maize hybrid

P1-rr

P1-wr

Pa

AIPb

nAIPb

Pa

FVIb

nFVIb

Pa

46.7 12.4 53.7 18.1 83.5 1.6

68.3 23.7 76.7 32.9 91.5 2.4

0.036 0.031 0.041 0.048 0.437 0.039

38.3 8.9 52.9 16.3 86.0 1.7

76.7 27.2 77.5 34.7 89.0 2.5

b0.001 b0.001 0.037 0.018 0.770 0.028

65.0 21.1 83.3 37.1 98.8 2.6

50.0 15.1 47.1 13.9 76.2 1.5

0.158 0.261 b 0.001 0.002 0.027 0.001

a P value for a two tailed t test comparing the means obtained from ears and kernels belonging to different parcels of P1-rr and P1-wr maize hybrids. b Treatments applied: anti-insect protection (AIP), not anti-insect protection (nAIP), F. verticillioides inoculation (FVI), not F. verticillioides inoculation (nFVI).

R2 = 0.950 (y = 3.306 + 0.319 x + 0.018 x 2). In the nFVI parcels models for FERs with FERi, IDi and IDs were R2 = 0.704 (y = 46.687 − 1.589 x + 0.017 x 2 ), R2 = 0.777 (y = 28.811 − 0.471 x + 0.008 x2) and R2 = 0.789 (y = 7.262 + 1.816 x − 0.013 x2). 4. Discussion This research was aimed at assessing the influence of pigmented maize pericarp on FER and FUM levels under field conditions. The presence of flavonoids in maize kernel pericarp is linked to the accumulation of maysin, a flavone defence compound able to reduce the corn earworm larvae development (Byrne et al., 1996; Sharma et al., 2012). The well-demonstrated positive relationship between insect lesions, FER and fumonisin accumulation led to the hypothesis that flavonoids in maize pericarp could decrease FER and fumonisin levels (Pilu et al., 2011). A previous two-year field study compared two isogenic maize hybrids, a genotype able to accumulate flavonoids (P1-rr) with colourless one (P1-wr), concerning FER and fumonisin levels, showed that the effects of flavonoids in maize pericarp varied depending on the experimental site and was not stable across the years (Venturini et al., 2015). In order to give a more comprehensive evaluation of the role of pigmented genotype in the resistance of maize against FER and FUM, in this study artificial inoculations of F. verticillioides strains, responsible of FER and FUM accumulation, were performed. Artificial inoculation of F. verticillioides strains is a suitable strategy for testing maize genotypes against FER and FUM as reported by several previous Authors (Balconi et al., 2014; Butrón et al., 2015; Mesterházy et al., 2012; Santiago et al., 2013). Additionally, a physical barrier against insects, consisting in an insect-proof polyethylene net to mitigate the attacks of pests, was applied in the present study to enhance the differences between the hybrids. This approach was preferred because it could provide information on influence of pigmented maize pericarp on FER and FUM levels under different pest attack (AIP treatment) and FER pressure (FVI treatment). The current study evidenced that the anti-insect method was effective in reducing larvae damage incidence (IDi) and severity (IDs). AIP parcels also exhibited lower FER and FUM levels confirming the significant and positive correlation between insect wounds and FER and FUM well demonstrated in earlier studies (De Curtis et al., 2011; Mazzoni et al., 2011; Parson and Munkvold, 2012; Santiago et al., 2015). However, the present work reveals the lack of a positive and significant correlation between insect damage and Fusarium spp. symptomless infection, because AIP parcels registered similar level of FFC contamination to

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Table 2 Insect damage incidence (IDi) and severity (IDs), fusarium ear rot incidence (FERi) and severity (FERs), F. fujikuroi species complex incidence (FFC) and fumonisin contamination (FUM) of maize samples belonging to different parcels of P1-rr and P1-wr hybrids. AIP-FVIa

AIP-nFVIa

Measured variables IDi (%) IDs (%) FERi (%) FERs (%) FFC (%) FUM (μg/g)

nAIP-FVIa

nAIP-nFVIa

P1-rr

P1-wr

Pb

P1-rr

P1-wr

Pb

P1-rr

P1-wr

Pb

P1-rr

P1-wr

Pb

40.0 8.3 63.3 20.0 99.3 2.0

50.0 12.2 86.7 30.6 100.0 2.7

0.423 0.390 0.035 0.002 0.374 b0.001

20.0 4.2 15.0 4.2 64.0 0.4

43.3 11.1 45.7 10.6 80.7 1.4

0.124 0.130 0.002 b0.001 0.630 0.055

70.0 21.1 86.7 36.7 96.0 2.6

100.0 42.7 96.7 61.1 100.0 2.9

b0.001 0.008 0.265 0.375 0.680 b0.001

56.7 16.1 50.0 11.7 74.7 1.6

80.0 28.9 76.7 29.4 85.3 2.5

0.245 0.148 0.116 0.429 0.456 0.062

a Treatments applied in the present study and their combinations: anti-insect protection (AIP), not anti-insect protection (nAIP), F. verticillioides inoculation (FVI), not F. verticillioides inoculation (nFVI). b P value for a two tailed t test comparing the means obtained from ears and kernels belonging to different parcels of P1-rr and P1-wr maize hybrids.

nAIP replicates. Probably a deeper insight into the composition of species within FFSC may better explain the association between insect wounds and Fusarium spp. contamination (De Curtis et al., 2011). Fusarium verticillioides silk-channel inoculations (FVI) resulted in higher visible FER symptoms, FFSC infection levels and FUM contamination values than not inoculated parcels (nFVI). These results clearly demonstrated that silk-channel technique is suitable for evaluating maize hybrids leading to more uniform disease incidence and severity as opposed to previous studies that used a kernel inoculation technique (Balconi et al., 2014; Butrón et al., 2015; Santiago et al., 2013). Under natural conditions, FER levels could be highly variable from site to site since in some locations natural infections cause moderate to low FER epidemics creating not uniform FER ratings for the selection of disease resistant genotypes (Mesterházy et al., 2012). Meteorological conditions, specifically mean temperatures and rainfall, registered at the experimental field around silking stages were not so favorable to initiate natural FER epidemics on the base of previous models developed in past studies (Cao et al., 2014; Parson and Munkvold, 2012). Although climatic parameters reported during ripening were apparently not so conducive for FER, uniform FER levels were registered in FVI parcels showing that the F. verticillioides isolates used could reliably infect maize even if environmental conditions did not favor the disease development. In addition, FVI treatment influenced both ID incidence and severity reporting higher ID levels in FVI parcels than not FVI ones. These results could be explained by the emission of volatile organic compounds, produced by FFSC during the colonization of maize ears, which are attractive to insects (Bartelt and Wicklow, 1999; Cardwell et al., 2000). The effects of flavonoids in maize pericarp against FER symptoms, FFSC contamination and FUM assessed in this study varied depending on the treatment applied. The results obtained in the present research showed that if AIP and FVI treatments were applied in combination Table 3 F-values, obtained with GLM regression, showing the effect of the main factors and their interactions on insect damage incidence (IDi), severity (IDs), Fusarium ear rot incidence (FERi), severity (FERs), Fusarium fujikuroi species complex incidence (FFC) and fumonisin contamination (FUM) of maize samples to different parcels of P1-rr and P1-wr hybrids. Source of variationa

dfb

IDi (%)

IDs (%)

FERi (%)

FERs (%)

H AIP FVI H × AIP H × FVI AIP × FVI H × AIP × FVI

1 1 1 1 1 1 1

23.31⁎⁎⁎ 24.87⁎⁎⁎ 20.58⁎⁎⁎ 13.57⁎⁎ 72.97⁎⁎⁎ 64.78⁎⁎⁎ 23.68⁎⁎⁎ 20.98⁎⁎⁎ 2.17⁎ 3.06⁎ 51.50⁎⁎⁎ 33.18⁎⁎⁎ 1.24 2.80 0.82 2.47 0.14 0.41 1.53 0.45 0.14 2.25 2.56 1.68 1.24 1.73 0.17 0.25

FFC (%)

FUM (μg/g)

0.63 24.24⁎⁎⁎ 0.89 17.13⁎⁎ 5.18⁎ 53.42⁎⁎⁎ 0.41 0.58 0.72 3.38 0.62 3.72 0.54 0.41

⁎ Denotes significance at the 0.05 probability level. ⁎⁎ Denotes significance at the 0.01 probability level. ⁎⁎⁎ Denotes significance at the 0.001 probability level. a Source of variation. H: Hybrid; AIP: Anti-insect protection; FVI: F. verticillioides inoculation. b d.f.: degrees of freedom.

the pigmented pericarp exerted an inhibition activity towards FER and FUM levels than the unpigmented one. Such inhibition activity, exalted by low insect damages and high disease pressure, although this mechanism has not been yet elucidated, could be explained both physically and biochemically. In particular, in the AIP and FVI plots, FER level and FUM biosynthesis could be reduced by the presence of phenolic compounds in the pigmented pericarp (eg phlobaphenes), because flavonoids impede the fungal infection making the maize kernel pericarp harder (Treutter, 2006). Moreover, the antifungal activity of flavonoids is probably exerted by complexing irreversibly with fungal proteins leading to inactivation of the proteins and loss of their functions (Kim et al., 2006). Phlobaphenes have never been characterized for their in vitro efficiency to inhibit fumonisin as other phenolic compounds (Beekrum et al., 2003; Dambolena et al., 2008; Samapundo et al.,

Table 4 Logarithmic regression analyses of insect damage incidence (IDi), severity (IDs), Fusarium ear rot incidence (FERi), severity (FERs), F. fujikuroi species complex incidence (FFC) as influencers of fumonisin contamination (FUM) of maize samples belonging to different parcels of P1-rr and P1-wr hybrids. Maize hybrida P1-rr Influencers IDi (%) IDs (%) FERi (%) FERs (%) FFC (%)

P1-wr

R2c

SEc

Fc

Pc

R2

SE

F

P

0.730 0.562 0.748 0.763 0.375

0.498 0.634 0.483 0.466 0.768

27.02 12.81 29.35 32.24 5.54

b0.001 0.005 b0.001 b0.001 0.04

0.277 0.298 0.623 0.696 0.132

0.609 0.600 0.440 0.395 0.666

3.83 4.24 16.51 22.86 1.575

0.079 0.067 0.002 0.001 0.238

Anti-insect protectionb AIP Influencers IDi (%) IDs (%) FERi (%) FERs (%) FFC (%)

nAIP

R2

SE

F

P

R2

SE

F

P

0.511 0.373 0.652 0.755 0.281

0.717 0.811 0.605 0.507 0.869

10.43 5.96 18.75 30.81 3.91

0.009 0.035 0.001 b0.001 0.076

0.706 0.649 0.796 0.755 0.400

0.317 0.346 0.264 0.289 0.452

23.99 18.48 38.98 30.78 6.68

0.001 0.002 b0.001 b0.001 0.027

Fusarium verticillioides inoculationb FVI Influencers R IDi (%) IDs (%) FERi (%) FERs (%) FFC (%) a

2

0.582 0.596 0.693 0.795 0.001

nFVI SE

F

P

R2

SE

F

P

0.226 0.222 0.194 0.191 0.350

13.91 14.72 22.51 23.65 0.015

0.004 0.003 0.001 0.001 0.906

0.693 0.721 0.576 0.702 0.229

0.531 0.506 0.624 0.523 0.842

22.61 25.88 13.61 23.61 2.96

0.001 b0.001 0.004 0.001 0.116

Main plots used in the present study: P1-rr and P1-wr maize hybrids. Treatments applied in the present study: anti-insect protection (AIP), not anti-insect protection (nAIP), Fusarium verticillioides inoculation (FVI), not F. verticillioides inoculation (nFVI). c Regression statistics: R2, adjusted R2; SE, standard error of the estimate; F, F-value of the model; P, level of significance. b

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2007). Lower FUM averages in the pigmented inoculated parcels than unpigmented inoculated ones lead to infer that such difference was due to fumonisin inhibition activity by pigment in pericarp as reported in previous field studies (Pilu et al., 2011; Sampietro et al., 2013; Venturini et al., 2015). Further metabolomic researches could be carried out in order to analyze the inhibitory effects, against FUM biosynthesis by FFSC, exerted by phenolic compounds in the pigmented pericarp as reported for other maize metabolites (Campos-Bermudez et al., 2013; Atanasova-Penichon et al., 2014). Hybrid reacted to ear rot in a different manner when the AIP treatment was applied: P1-rr hybrid had lower FER incidence and severity than P1-wr. It is possible to argue that in the absence of additional biotic stress, such as insect damages, flavonoids in the pericarp limited Fusarium mycelial growth both in the inoculated parcels and in not-inoculated ones. However, neither hybrids nor anti-insect protection ever affected the levels of FFSC infection in kernels. These results might be explained by the dual behavior of FFSC strains colonizing maize kernels as either endophytes or pathogens (Bacon et al., 2008). FFSC could colonize maize kernel without showing any symptom or mycotoxin production (Duncan and Howard, 2010). The similar FFC level between hybrids was the result of FFSC internal contamination of kernel and was not influenced by pericarp composition as mycelial growth and fumonisin production. In this regard it is important to mention that FFC was never found to be influenced by any source of variation except FVI treatment. It was not surprisingly that inoculated parcels showed higher levels of FFSC contamination than not inoculated ones since F. verticillioides isolates could replace other Fusarium spp. as previously reported (Miedaner et al., 2010). The presence of pigment in the pericarp shaped the interactions between the measured variables, in particular between FUM and FER ratings. Regression analyses showed better goodness of fit of the models computed in P1-rr than in P1-wr hybrid. This suggests that the interactions of fumonisin contamination levels with visual ratings of disease and insect damages are better modeled in the pigmented hybrid. Differences among maize genotypes in regression analyses of fumonisin contaminations with FER were reported in previous studies (Löffler et al., 2011; Santiago et al., 2013). The most interesting differences between models computed for the two hybrids were found between FUM and ID. Only in the pigmented hybrid models for fumonisin levels and insect damages were good and significant aligning with previous reports (Mazzoni et al., 2011; Parson and Munkvold, 2012). These results could be explained by the fact that only with reduced fumonisin levels the association with decreased insect damages is good and significant. This reinforces the evidence that flavonoids in the pigment of the maize pericarp might provide resistance to insect damages, fusarium ear rot and fumonisin contamination. It is possible to infer that p1 locus could represent one of the QTLs associated with resistance to FER and FUM accumulation. In fact in a QTL meta-analysis study a QTL position, associated with FER resistance, was compatible with the p1 map position (chromosome 1, bin 1.03–1.04) (Xiang et al., 2012). If this hypothesis is correct, p1 gene represents the first mendelized QTL involved in FER and thus it might be useful in marker-assisted selection programs. Finally, very good and significant models were observed for fumonisin levels and disease severity. This evidence agrees with past reports supporting the selection of resistant maize genotypes with indirect selection of fumonisin levels using ear rot ratings (Balconi et al., 2014; Butrón et al., 2015; Santiago et al., 2013). It is possible to argue that fumonisin could act as virulence factor involved in FER development although fumonisin in the FFSC infection process may play multiple and different roles depending on the mode of FFSC infection (Desjardins et al., 2002). In summary, the results presented here suggest the potential role of flavonoid pigments in maize pericarp in the reduction of FER and fumonisin accumulation, considering that such contribution is related to the cultivation practices such as the protection against insects. Possible interactions of seasonal and geographical variables with the treatments applied in the present study (AIP and FVI) could be analyzed in

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additional field tests with a similar approach to the one reported in a previous study (Venturini et al., 2015). The role that the pigment plays in kernel defense against FER and FUM is not so consistent across the parcels, indicating that flavonoids alone may not be a unique component in the resistance of fumonisin contamination in maize. Further data on a better definition of flavonoid composition in the pericarp of pigmented maize kernels might provide more information on the involvement of flavonoids in resistance to FER and fumonisin contamination. Other factors should be included in breeding programs of pigmented maize such as silk, cob and pericarp composition regarding the antioxidant fraction, pericarp thickness, husk tightness and coverage. Dedication This work is dedicated to the memory of Professor Annamaria Vercesi. Her pursuit of scientific knowledge in plant pathology and mycology was exceptional. The homage to her is being paid in these pages. Acknowledgements The work presented here was supported, in part, by Regione Lombardia, “Fondo per la promozione di Accordi Istituzionali, project BIOGESTECA 15083/RCC". References Aguín, O., Cao, A., Pintos, C., Santiago, R., Mansilla, P., Butrón, A., 2014. Occurrence of Fusarium species in maize kernels grown in northwestern Spain. Plant Pathol. 63, 946–951. Alma, A., Lessio, F., Reyneri, A., Blandino, M., 2005. Relationships between Ostrinia nubilalis Hübner (Lepidoptera Crambidae), crop technique and mycotoxin contamination of corn kernel in northwestern Italy. Int. J. Pest Manag. 51, 165–173. Atanasova-Penichon, V., Bernillon, S., Marchegay, G., Lornac, A., Pinson-Gadais, L., Pons, N., Zehraoui, E., Barreau, C., Richard-Forget, F., 2014. Bioguided isolation, characterization, and biotransformation by Fusarium verticillioides of maize kernel compounds that inhibit fumonisin production. Mol. Plant-Microbe Interact. 27, 1148–1158. Bacon, C.W., Glenn, A.E., Yates, I.E., 2008. Fusarium verticillioides: managing the endophytic association with maize for reduced fumonisins accumulation. Toxin Rev. 27, 411–446. Balconi, C., Berardo, N., Locatelli, S., Lanzanova, C., Torri, A., Redaelli, R., 2014. Evaluation of ear rot (Fusarium verticillioides) resistance and fumonisin accumulation in Italian maize inbred lines. Phytopathol. Mediterr. 53, 14–26. Bartelt, R.J., Wicklow, D.T., 1999. Volatiles from Fusarium verticillioides (Sacc.) Nirenb. and their attractiveness to Nitidulid beetles. J. Agric. Food Chem. 47 (6), 2447–2454. Beekrum, S., Govinden, R., Padayachee, T., Odhav, B., 2003. Naturally occurring phenols: a detoxification strategy for fumonisin B1. Food Addit. Contam. 20, 490–493. Blandino, M., Reyneri, A., Vanara, F., Pascale, M., Haidukowski, M., Saporiti, M., 2008. Effect of sowing date and insecticide application against European corn borer (Lepidoptera: Crambidae) on fumonisin contamination in maize kernels. Crop. Prot. 27, 1432–1436. Butrón, A., Reid, L.M., Santiago, R., Cao, A., Malvar, R.A., 2015. Inheritance of maize resistance to gibberella and fusarium ear rots and kernel contamination with deoxynivalenol and fumonisins. Plant Pathol. 64, 1053–1060. Byrne, P.F., McMullen, M.D., Snook, M.E., Musket, T.A., Theuri, J.M., Widstrom, N.W., Wiseman, B.R., Coe, E.H., 1996. Quantitative trait loci and metabolic pathways: genetic control of the concentration of maysin, a corn earworm resistance factor, in maize silks. Proc. Natl. Acad. Sci. 93, 8820–8825. Campos-Bermudez, V.A., Fauguel, C.M., Tronconi, M.A., Casati, P., Presello, D.A., Andreo, C.S., 2013. Transcriptional and metabolic changes associated to the infection by Fusarium verticillioides in maize inbreds with contrasting ear rot resistance. PLoS One 8, e61580. Cao, A., Santiago, R., Ramos, A.J., Souto, X.C., Aguín, O., Malvar, R.A., Butrón, A., 2014. Critical environmental and genotypic factors for Fusarium verticillioides infection, fungal growth and fumonisin contamination in maize grown in northwestern Spain. Int. J. Food Microbiol. 177, 63–71. Cardwell, K.F., Kling, J.G., Maziya-Dixon, B., Bosque-Pérez, N.A., 2000. Interactions between Fusarium verticillioides, Aspergillus flavus, and insect infestation in four maize genotypes in lowland Africa. Phytopathology 90, 276–284. Castellano, S., Scarascia Mugnozza, G., Russo, G., Briassoulis, D., Mistriotis, A., Hemming, S., Waaijenberg, D., 2008. Plastic nets in agriculture: a general review of types and applications. Appl. Eng. Agric. 24, 799–808. Dambolena, J.S., Lopez, A.G., Canepa, M.C., Theumer, M.G., Zygadlo, J.A., Rubinstein, H.R., 2008. Inhibitory effect of cyclic terpenes (limonene, menthol, menthone and thymol) on Fusarium verticillioides MRC 826 growth and fumonisin B1 biosynthesis. Toxicon 51, 37–44. De Curtis, F., De Cicco, V., Haidukowski, M., Pascale, M., Somma, S., Moretti, A., 2011. Effects of agrochemical treatments on the occurrence of Fusarium ear rot and fumonisin contamination of maize in Southern Italy. Field Crop Res. 123, 161–169.

62

G. Venturini et al. / International Journal of Food Microbiology 227 (2016) 56–62

Desjardins, A.E., Munkvold, G.P., Plattner, R.D., Proctor, R.H., 2002. FUM1 — a gene required for fumonisin biosynthesis but not for maize ear rot and ear infection by Gibberella moniliformis in field tests. Mol. Plant-Microbe Interact. 15, 1157–1164. Duncan, K.E., Howard, R.J., 2010. Biology of maize kernel infection by Fusarium verticillioides. Mol. Plant-Microbe Interact. 23, 6–16. Folcher, L., Jarry, M., Weissenberger, A., Gérault, F., Eychenne, N., Delos, M., RegnaultRoger, C., 2009. Comparative activity of agrochemical treatments on mycotoxins levels with regard to corn borers and Fusarium mycoflora in maize (Zea mays L.) fields. Crop. Prot. 28, 302–308. Glenn, A.E., Zitomer, N.C., Zimeri, A.M., Williams, L.D., Riley, R.T., Proctor, R.H., 2008. Transformation-mediated complementation of a FUM gene cluster deletion in Fusarium verticillioides restores both fumonisin production and pathogenicity on maize seedlings. Mol. Plant-Microbe Interact. 21, 87–97. Grotewold, E., Drummond, B.J., Bowen, B., Peterson, T., 1994. The MYB-homologous pgene controls phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset. Cell 76, 543–553. Kim, J.H., Mahoney, N., Chan, K.L., Molyneux, R.J., Campbell, B.C., 2006. Controlling foodcontaminating fungi by targeting antioxidant stress-response system with natural phenolic compounds. Appl. Microbiol. Biotechnol. 70, 735–739. Lancashire, P.D., Bleiholder, H., Langelüddecke, R., Stauss, T., Van Den Boomn, E., Weber, T., Witzen–Berger, A., 1991. A uniformdecimal code for growth stages of crops and weeds. Ann. Appl. Biol. 119, 561–601. Leslie, J.F., Summerell, B.A., 2006. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, Iowa, USA. Löffler, M., Kessel, B., Ouzunova, M., Miedaner, T., 2011. Covariation between line and testcross performance for reduced mycotoxin concentrations in European maize after silk channel inoculation of two Fusarium species. Theor. Appl. Genet. 122, 925–934. Maiorano, A., Reyneri, A., Sacco, D., Magni, A., Ramponi, C., 2009. A dynamic risk assessment model (FUMAgrain) of fumonisin synthesis by Fusarium verticillioides in maize grain in Italy. Crop. Prot. 28, 243–256. Mazzoni, E., Scandolara, A., Giorni, P., Pietri, A., Battilani, P., 2011. Field control of Fusarium ear rot, Ostrinia nubilalis (Hübner), and fumonisins in maize kernels. Pest Manag. Sci. 67, 458–465. Mesterházy, Á., Lemmens, M., Reid, L.M., 2012. Breeding for resistance to ear rots caused by Fusarium spp. in maize—a review. Plant Breed. 131, 1–19. Miedaner, T., Bolduan, C., Melchinger, A.E., 2010. Aggressiveness and mycotoxin production of eight isolates each of Fusarium graminearum and Fusarium verticillioides for ear rot on susceptible and resistant early maize inbred lines. Eur. J. Plant Pathol. 127, 113–123. Miller, J.D., 2001. Factors that affect the occurrence of fumonisin. Environ. Health Perspect. 109, 321–3244. Munkvold, G.P., 2009. Seed pathology progress in academia and industry. Annu. Rev. Phytopathol. 47, 285–311. Munkvold, G.P., McGee, D.C., Carlton, W.M., 1997. Importance of different pathways for maize kernel infection by Fusarium moniliforme. Phytopathology 87, 209–217. Parson, M.W., Munkvold, G.P., 2012. Effects of planting date and environmental factors on fusarium ear rot symptoms and fumonisin B1 accumulation in maize grown in six North American locations. Plant Pathol. 61, 1130–1142. Picot, A., Atanasova-Pénichon, V., Pons, S., Marchegay, G., Barreau, C., Pinson-Gadais, L., Roucolle, J., Daveau, F., Caron, D., Richard-Forget, F., 2013. Maize kernel antioxidants and their potential involvement in Fusarium ear rot resistance. J. Agric. Food Chem. 61, 3389–3395. Pilu, R., Cassani, E., Sirizzotti, A., Petroni, K., Tonelli, C., 2011. Effect of flavonoid pigments on the accumulation of fumonisin B1 in the maize kernel. J. Appl. Genet. 52, 145–152.

Pitt, J.I., Hocking, A.D., 2009. Fungi And Food Spoilage. Springer, Dordrecht, Netherlands. Presello, D.A., Botta, G., Iglesias, J., Eyherabide, G.H., 2008. Effect of disease severity on yield and grain fumonisin concentration of maize hybrids inoculated with Fusarium verticillioides. Crop. Prot. 27, 572–576. Reid, L.M., Zhu, X., 2005. Screening corn for resistance to common diseases in Canada. Technical Bulletin Cat. No.A42-103/2005E. Agriculture and Agri-Food Canada, Ottawa, Canada. Rodrıguez, V.M., Soengas, P., Landa, A., Ordas, A., Revilla, P., 2013. Effects of selection for color intensity on antioxidant capacity in maize (Zea mays L.). Euphytica 193, 339–345. Samapundo, S., De Meulenaer, B., Osei-Nimoh, D., Lamboni, Y., Debevere, J., Devlieghere, F., 2007. Can phenolic compounds be used for the protection of corn from fungal invasion and mycotoxin contamination during storage? Food Microbiol. 24, 465–473. Sampietro, D.A., Fauguel, C.M., Vattuone, M.A., Presello, D.A., Catalán, C.A.N., 2013. Phenylpropanoids from maize pericarp: resistance factors to kernel infection and fumonisin accumulation by Fusarium verticillioides. Eur. J. Plant Pathol. 135, 105–113. Santiago, R., Cao, A., Malvar, R.A., Reid, L.M., Butrón, A., 2013. Assessment of corn resistance to fumonisin accumulation in a broad collection of inbred lines. Field Crop Res. 149, 193–202. Santiago, R., Cao, A., Butrón, A., 2015. Genetic factors involved in fumonisin accumulation in maize kernels and their implications in maize agronomic management and breeding. Toxins 7, 3267–3296. Shala-Mayrhofer, V., Varga, E., Marjakaj, R., Berthiller, F., Musolli, A., Berisha, D., Kelmendi, B., Lemmens, M., 2013. Investigations on Fusarium spp. and their mycotoxins causing Fusarium ear rot of maize in Kosovo. Food Addit. Contam. Part B 6, 237–243. Sharma, M., Chai, C., Morohashi, K., Grotewold, E., Snook, M.E., Chopra, S., 2012. Expression of flavonoid 3′-hydroxylase is controlled by P1, the regulator of 3deoxyflavonoid biosynthesis in maize. BMC Plant Biol. 12, 196. Shephard, G.S., Kimanya, M.E., Kpodo, K.A., Gnonlonfin, G.J.B., Gelderblom, W.C.A., 2013. The risk management dilemma for fumonisin mycotoxins. Food Control 34, 596–600. Torelli, E., Firrao, G., Bianchi, G., Saccardo, F., Locci, R., 2012. The influence of local factors on the prediction of fumonisin contamination in maize. J. Sci. Food Agric. 92, 1808–1814. Townsend, G.R., Heuberger, J.W., 1943. Methods for estimating losses caused by diseases in fungicide experiments. Plant Dis. Rep. 27, 340–343. Treutter, D., 2006. Significance of flavonoids in plant resistance: a review. Environ. Chem. Lett. 4, 147–157. Venturini, G., Assante, G., Vercesi, A., 2011. Fusarium verticillioides contamination patterns in Northern Italian maize during the growing season. Phytopathol. Mediterr. 50, 110–120. Venturini, G., Assante, G., Toffolatti, S.L., Vercesi, A., 2013. Pathogenicity variation in Fusarium verticillioides populations isolated from maize in northern Italy. Mycoscience 54, 285–290. Venturini, G., Toffolatti, S.L., Assante, G., Babazadeh, L., Campia, P., Fasoli, E., Salomoni, D., Vercesi, A., 2015. The influence of flavonoids in maize pericarp on fusarium ear rot symptoms and fumonisin accumulation under field conditions. Plant Pathol. 64, 671–679. White, D.G., 1999. Fusarium kernel and ear rot. In: White, D.G. (Ed.), Compendium of Corn Diseases, third ed. The American Phytopathological Society, Saint Paul, USA, pp. 45–46. Xiang, K., Reid, L.M., Zhang, Z.M., Zhu, X.Y., Pan, G.T., 2012. Characterization of correlation between grain moisture and ear rot resistance in maize by QTL meta-analysis. Euphytica 183, 185–195.