Effects of agrochemical treatments on the occurrence of Fusarium ear rot and fumonisin contamination of maize in Southern Italy

Field Crops Research 123 (2011) 161–169

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Effects of agrochemical treatments on the occurrence of Fusarium ear rot and fumonisin contamination of maize in Southern Italy Filippo De Curtis a,∗ , Vincenzo De Cicco a , Miriam Haidukowski b , Michelangelo Pascale b , Stefania Somma b , Antonio Moretti b a b

Department of Animal, Plant and Environmental Sciences, University of Molise, Via De Sanctis, 86100 Campobasso, Italy Institute of Sciences of Food Production (ISPA), CNR Via Amendola, 122/O Bari, Italy

a r t i c l e

i n f o

Article history: Received 26 November 2010 Received in revised form 1 April 2011 Accepted 12 May 2011 Keywords: Maize Fusarium proliferatum Fusarium verticillioides Fumonisin B1 Fumonisin B2 Fumonisin B3 Fungicides

a b s t r a c t The efficacy of agrochemical treatments, based on three different fungicides combined with an insecticide, was tested in southern Italy for two years on three maize hybrids to control Fusarium ear rot of maize and the accumulation in the maize kernels of the carcinogenic mycotoxins fumonisins. Insect damage incidence and severity, disease incidence and severity, identification of Fusarium species and levels of fumonisin contamination in kernels were determined. Field trials showed in both years that natural colonization of maize kernels by the fumonisin producing species Fusarium proliferatum and F. verticillioides (up to 81.5 and 26.5%, respectively) and total fumonisin contamination (up to 68.2 ␮g g−1 ) were highly severe. For all hybrids and in both years, the treatment with the insecticide applied alone reduced the insect damage severity consistently and the content of fumonisins in the kernel only in half of the cases, whereas fungicide treatments applied in combination with the insecticide showed a further significant reduction of fumonisin contamination in the three hybrids and in both years. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Fusarium ear rot is a severe and worldwide disease of maize (Zea mays L.) that causes significant yield and economic losses. Among the Fusarium species involved in the disease, F. proliferatum (teleomorph Gibberella intermedia) and F. verticillioides (teleomorph G. moniliformis), two species belonging to the G. fujikuroi complex (Leslie and Summerel, 2006), are frequently isolated (Logrieco et al., 1995; Munkvold et al., 1998). Both species produce the carcinogenic mycotoxins fumonisins (IARC, 2002), in particular fumonisin B1 (FB1 ), B2 (FB2 ) and B3 (FB3 ) that can accumulate in maize kernels, generally, at a level higher for FB1 compared to FB2 and/or FB3 (Pascale et al., 2002; Desjardins, 2006). Fumonisins are related to a wide range of animal and human health problems, being epidemiologically associated with human esophageal cancer and neural tube defect, and causing leukoencephalomalacia in equine, pulmonary edema in swine, nephrotoxicity and hepatotoxicity in several animals (Desjardins, 2006). Although the occurrence of fumonisins in maize in Italy has been mainly documented in the northern regions (Ritieni et al., 1997; Battilani et al., 2008), little information is available on the maize cultivated in central-southern Italy. This area is characterized

∗ Corresponding author. Tel.: +39 0874404687; fax: +39 0874404855. E-mail address: [email protected] (F. De Curtis). 0378-4290/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2011.05.012

by warm, dry weather and drought stress during the grain-filling period, conditions that represent key factors for both Fusarium ear rot and fumonisin contamination (Miller, 2001; Rossi et al., 2009). Besides climate parameters, several factors may affect Fusarium ear rot of maize and fumonisin contamination and must be considered, such as host genotype, insect injuries to kernels, cultural practices and pest management strategy (Miller, 2001; Munkvold, 2003; Folcher et al., 2009). Both F. proliferatum and F. verticillioides can infect all parts of the maize plant (root, stalk, silks, cob, kernels) during the whole cropping season and can survive on maize crop residues for several years as a primary inoculum source for the infections on the subsequent maize crop (Cotten and Munkvold, 1998; Munkvold, 2003; Naef and Défago, 2006; Rossi et al., 2009). These pathogens can infect maize ears mainly through three different ways: seeds, silks and wounds caused by insects and birds (Munkvold et al., 1997). Furthermore, although the role and importance of the pathways of pathogen penetration in maize kernels is not yet clear, recent research has further elucidated the mechanism of penetration of the pathogen through the silks (Duncan and Howard, 2010) and has also shown a significant production of airborne conidia of F. verticillioides from maize stalks during the silk emission in open field (Rossi et al., 2009). Therefore, inappropriate management of maize crop residues, especially in areas where maize is grown in short rotation or as continuous crop, can cause a high production of airborne spores as potential inoculum for silk

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infection (Rossi et al., 2009; Duncan and Howard, 2010). In addition, however, a high correlation has also been found between the level of insect damage and Fusarium ear rot severity (Munkvold, 2003). In this regard, significant mycotoxin reductions were obtained by a suitable agrochemical insect control strategy combined with other field practices such as early sowing date, appropriate hybrid selection for each specific area, proper fertilization, and long crop rotation (Munkvold, 2003; Folcher et al., 2009). On the other hand, our recent investigations carried out in southern Italy on different maize hybrids and under natural infection, recorded variable and, in some cases, high Fusarium ear rot severity and fumonisin kernel contamination also in fields treated with insecticide (De Curtis et al., 2008). The aim of this study was to evaluate the efficacy of three fungicides combined with an insecticide in reducing Fusarium ear rot severity and fumonisin kernel contamination under natural conditions. 2. Materials and methods

Fig. 1. Symptoms of Fusarium ear root on maize kernels without any evident insect damage.

2.1. Field trials Field experiments were carried out under natural conditions, during two consecutive growing seasons 2005 and 2006, at the Experimental Farm of the Agricultural Educational Institute “San Pardo” of Larino, CB, Italy (159 m, altitude; 41◦ 50 N, 14◦ 57 E). The experiments were conducted on the same field in both years, where, in the previous two years, 2003 and 2004, winter wheat and maize have been cultivated, respectively. Three hybrids typically cultivated in this area were selected: two belong to the FAO maturity class 300 [NK Surtep (105 days) and NK Cisko (110 days)] and one to the maturity class 400 [NK Stella (115 days)] – Syngenta seeds (Cremona, Italy). The chemical applications were performed in four treatments and based on commercial synthetic fungicides combined with an insecticide treatment as follows: (1) fungicide Folicur WG® [active ingredient (a.i.): 25% tebuconazole] manufactured by Bayer CropScience (Milan, Italy), at the rate of 1.0 kg ha−1 , combined to the insecticide Karate® (a.i.: 2.5% lambda-cyhalothrin) manufactured by Syngenta Crop Protection (Milan, Italy); (2) fungicide Eminent 40 EW® (a.i.: 3.85% tetraconazole) manufactured by Isagro Italia (Milan, Italy), at the rate of 3.0 L ha−1 and combined to the insecticide Karate® ; (3) fungicide Tiptor S® (a.i.: 32.3 and 4.3% prochloraz + cyproconazole, respectively) manufactured by Syngenta Crop Protection, at the rate of 1.25 L ha−1 , combined to the insecticide Karate® . (4) insecticide Karate® , at the rate of 0.5 kg ha−1 . The four treatments were compared to the untreated control. The experimental design was a completely randomized block with three replicates (plots) for each treatment and each maize hybrid. Each experimental plot was 20 m × 6 m, with a total of approximately 800 plants for each plot. Two rows of maize plants were used as a border, along the whole plot. All the soil and plant management practices, as well as weed control and irrigation, were conducted according to the Good Agricultural Practices applied in Italy. Fungicides were applied both to soil and foliage two times: 3 days after complete silk emission (stage 65 of the BBCH scale) and at early dough (stage 83 of the BBCH scale) growth stages; the growth stages were monitored daily according to the BBCH scale (Weber and Bleiholder, 1990). Fungicides were distributed to the soil by means of the drip irrigation system as reported by

De Curtis et al. (2010). Treatments to the foliage were carried out by spraying 600 L ha−1 of the fungicide suspension using a self-propelled sprayer (mod. IRIS, BARGAM® S.P.A., Imola, Italy) equipped with 24 fan nozzles (model, Turbo TwinJet® AITTJ60) at a pressure of 300 kPa and an operation speed of 5 km h−1 . The insecticide was applied only to the foliage and aimed against the corn stalk borer (CSB) Sesamia nonagrioides Lefèbvre (Lepidoptera, Noctuidae), the main maize insect pest in the experimental area; timing of applications was chosen by monitoring insect flight activity by means of oil traps (Germinara et al., 2007) baited with rubber septum dispensers each containing 1 mg of sex attractants: (Z)11-hexadecenyl-acetate, (Z)-hexacedenol, (Z)-11-hexadecenal and dodecanyl-acetate in the ratio of 9:1:1:1 (Novapher, San Donato Milanese, Milan, Italy) (Germinara et al., 2005). The dispensers were replaced every 30 days. Adults captured were collected and counted every 3 days. The insecticide applications were carried out after 5 days of the CSB flight peak spraying 600 L ha−1 of the insecticide suspension by means of a self-propelled sprayer equipped as mentioned above.

2.2. Disease ratings The Fusarium ear rot incidence and severity was evaluated on all the ears of fifty maize plants randomly collected by handpicking at the centre of each plot, at the harvest phenological stage (99 of the BBCH scale). The Fusarium ear rot incidence was calculated as the percentage of ears with clear symptoms of the disease, while the Fusarium ear rot severity was calculated as the percentage of kernels per ear with symptoms of the disease. To this end, the ears were visually evaluated for Fusarium ear rot by using a seven-class disease severity rating scale, proposed by Reid et al. (1999), based on the estimation of ear rot as the percentage of kernels visibly damaged as follows: 1 = 0%, 2 = 1–3%, 3 = 4–10%, 4 = 11–25%, 5 = 26–50%, 6 = 51–75%, and 7 > 75% of kernels with visible symptoms of infection such as rot, discolored kernels and mycelia growth (Fig. 1), as reported by Reid et al. (1999). The Fusarium ear rot severity was calculated according to the McKinney formula (19): Fusarium ear rot severity = [(n × c)/N × C] × 100, where n is the number of ears per class, c the number of classes, N is the total number of ears assessed per plot and C is the number of highest class (=7). The Fusarium ear rot incidence was calculated by evaluating the percentage of the ears visibly showing the symptoms of Fusarium ear rot (discolored kernels, mycelia growth, etc.) on the total number of ears sampled.

F. De Curtis et al. / Field Crops Research 123 (2011) 161–169

2.3. Insect damage ratings The insect damage incidence (IDI) and severity (IDS) were calculated on all ears of fifty maize plants randomly collected at the centre of each plot as above mentioned. The IDI was calculated as the percentage of ears visibly showing the injuries due to insect larvae activity, on the total number of ears sampled. The IDS was calculated as the percentage of kernels per ear with visible damage due to larvae activity. For this purpose, a seven-class rating scale was used in which, 1 = 0%, 2 = 1–3%, 3 = 4–10%, 4 = 11–25%, 5 = 26–50%, 6 = 51–75%, and 7 > 75% of kernels with visible injuries due to larvae activity. 2.4. Fungal isolation and identification For each growing season, the kernels of the fifty ears of each plot were removed by electric sheller, pooled, and 100 kernels from each sample were randomly selected and plated on Petri dishes containing Potato Dextrose Agar (PDA, Oxoid-Unipath Ltd., Basingstoke, England) plus 1% of pentacloronitrobenzen (PCNB code P-3395, Sigma, St Louis, MO, USA), neomycin (1 mg L−1 ) and streptomycin (1 mg L−1 ). Fungal isolation was performed as previously reported (Logrieco et al., 1995). A preliminary morphological identification was performed, according to Leslie and Summerel (2006). 2.5. Molecular identification For strains morphologically identified as F. proliferatum and F. verticillioides, the identity was confirmed by using molecular identification since these two species are morphologically closely related. DNA extraction was performed according to Somma et al. (2010). As a first step, the identity of representative strains of each species was confirmed by sequencing a portion of calmodulin gene, a translation elongation factor (TEF; Somma et al., 2010). Genomic DNA was used as a template for amplified calmodulin gene by using primer pairs CL1/CL2 and protocol from O’Donnell and Cigelnik (1997); for TEF sequences, the protocol of Somma et al. (2010) was followed. The identification of the whole set of strains was successively confirmed by using DNA species specific primers as reported in Mulè et al. (2004). 2.6. Fumonisin analysis Fumonisin B1 (FB1 ), B2 (FB2 ) and B3 (FB3 ) were analyzed according to the AOAC Official Method No. 995.15, with minor modifications. 20 g maize samples, previously ground in a laboratory mill, were extracted with 100 mL acetonitrile–water (1:1, v/v) by shaking for 1 h. After filtration through Whatman No. 4 filter paper, the pH of the extract was measured and adjusted to 5.8–6.5 with 0.1 M NaOH or 0.1 M HCl, if necessary. Extract (10 mL) was cleaned up at a flow rate of 2 mL min−1 through a strong anion-exchange (500 mg, SAX) cartridge, previously conditioned by the successive passage of methanol (5 mL) and methanol/water (3:1, 5 mL). The cartridge was then washed with methanol/water (3:1, 8 mL) followed by methanol (3 mL), and fumonisins were eluted with 1% acetic acid in methanol (10 mL). The eluate was evaporated to dryness at 40 ◦ C under a gentle stream of nitrogen and the residue was redissolved in 500 ␮L of acetonitrile–water (30:70, v/v). 110 ␮L of the purified extract were added with 110 ␮L of o-phtaldialdehyde (OPA) solution (obtained by adding 5 mL of 0.1 M sodium tetraborate and 50 ␮L of 2-mercaptoethanol to 1 mL of methanol containing 40 mg of OPA) by using HPLC autosampler (Varian Inc., Palo Alto, CA, USA) and mixed for 30 s. 50 ␮L of the derivatized solution were injected into the HPLC/fluorescence detection system (Varian Inc.) by full loop at exactly 3 min after adding the OPA reagent. Fluorometric detec-

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tor was set at wavelengths of 335 nm (excitation) and 440 nm (emission). The analytical column was a SymmetryShield RP18 (15 cm × 4.6 mm, 5 ␮m, Waters, MA, USA) preceded by a guard column inlet filter (0.5 ␮m × 3 mm diameter, Rheodyne Inc., CA, USA). The mobile phase consisted of a binary gradient that was applied as follows: the initial composition of the mobile phase, 60% of (A) acetonitrile–water–acetic acid (30/69/1, v/v/v)/40% of (B) acetonitrile–water–acetic acid (60/39/1, v/v/v), was kept constant for 5 min, then (B) solvent was linearly increased to 88% in 21 min and kept constant for 4 min. Finally, to clean the column the amount of acetonitrile was increased to 100% and kept constant for 4 min. The column was thermostated at 30 ◦ C. The flow rate of the mobile phase was 1.0 mL min−1 . The limit of detection of the analytical method was 0.01 ␮g g−1 for each fumonisin. FB1 and FB2 were quantified by measuring peak areas and comparing them with the relevant calibration curves obtained with standard solutions. FB3 was quantified on the basis of the FB2 calibration curve. 2.7. Meteorological data During both years, daily meteorological data (temperature, relative humidity and rainfall) were recorded by the weather station of CO.RE.DI.MO. (Consorzio Regionale di Difesa Molisano – Molise Region – Italy) located fifty meters away from the experimental field location. Meteorological data are shown in Fig. 2. 2.8. Statistical analysis The effects of the agrochemical treatments on ear rot incidence and severity, IDI, IDS and the occurrence of fumonisins on kernels were tested by the analysis of variance (ANOVA) using a completely randomized block design. Means were compared using Tukey’s test and all statistical tests were conducted at a probability level of P < 0.05. All the percentage values of ear rot incidence and severity, IDI, IDS and Fusarium mycoflora were previously converted √ into Bliss angular values (arcsine %) prior to ANOVA (Gomez and Gomez, 1984). Correlation analysis was performed to correlate both Fusarium ear rot severity and IDS with fumonisin kernel occurrence by using the Pearson Index. The SPSS version 18.0 for Windows statistical package (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. 3. Results 3.1. Fusarium ear rot incidence and severity The results on Fusarium ear rot incidence and severity recorded in the 2 years of field experiments (Tables 1-2) resulted significantly higher in 2005 than 2006. The Fusarium ear rot incidence ranged from 65.6% (2006) to 78.3% (2005), while the Fusarium ear rot severity ranged from 15.2% (2006) to 17.9% (2005). In 2005, the untreated control (75% for incidence; 17.9% for severity) and the treatment of the insecticide alone (78.3% for incidence; 17.6% for severity) showed the highest mean values of Fusarium ear rot. On the other hand, both Fusarium ear rot incidence and severity were significantly reduced (up to 100% of reduction in 2005) by all the fungicide treatments performed in combination with the insecticide (Table 1). In the 2006 experiment, all the fungicide treatments performed in combination with the insecticide significantly (P < 0.05) reduced Fusarium ear rot incidence and severity (up to 82% and 86.5%, respectively), although the reduction degree was lower than 2005 (Table 2). It is also interesting to highlight that a Fusarium ear rot incidence significantly higher than other treatments in 2006 occurred in the untreated control of the hybrid NK Stella.

164 Table 1 Effect of agrochemical treatments on insect damage incidence (IDI) and severity (IDS), Fusarium ear rot (FER) incidence and severity, Fusarium proliferatum and F. verticillioides occurrence and fumonisin (FB) contamination on maize in 2005. Maize hybrid

Treatment

IDI (%)a

IDS(%)b

FER incidencec

FER severityd

F. proliferatum

F. verticillioides

NK Surtep

Tebuconazole + insecticide Tetraconazole + insecticide Prochloraz/cyproconazole + insecticide Insecticide alone Untreated control

10.0 bcf 6.7 bc 11.7 b 16.7 ab 21.7 a

2.8 bcf 2.2 bc 3.5 bc 4.1 b 7.5 a

0.0 bf 0.0 b 0.0 b 78.3 a 65.0 a

0.0 bf 0.0 b 0.0 b 17.6 a 11.9 a

12.0 bcf 13.5 bc 9.5 c 22.5 b 44.0 a

3.0 cf 1.0 c 2.0 c 16.0 b 26.5 a

0.0 cf 0.0 c 0.0 c 28.2 b 68.2 a

NK Cisko

Tebuconazole + insecticide Tetraconazole + insecticide Prochloraz/cyproconazole + insecticide Insecticide alone Untreated control

6.7 c 10.0 b 8.3 bc 11.7 ab 20.0 a

2.3 bc 2.5 bc 2.9 bc 3.5 b 5.3 a

0.0 d 25.0 b 13.3 bc 75.0 a 68.3 a

0.0 d 4.5 b 1.9 bc 16.2 a 17.9 a

57.0 ab 4.0 c 24.5 bc 81.5 a 73.0 a

0.0 b 0.5 a 11.5 a 9.0 a 16.5 a

1.3 b 7.6 b 0.2 b 44.1 a 26.5 a

NK Stella

Tebuconazole + insecticide Tetraconazole + insecticide Prochloraz/cyproconazole + insecticide Insecticide alone Untreated control

10.0 b 13.3 b 11.7 b 11.7 b 25.0 a

2.3 bc 3.0 b 2.9 b 3.0 b 7.1 a

18.3 b 0.0 c 18.3 b 65.0 a 75.0 a

2.6 b 0.0 c 3.6 b 12.9 a 16.2 a

25.5 ab 37.0 ab 40.0 a 37.5 a 20.5 b

9.5 a 10.0 a 15.0 a 10.0 a 9.0 a

8.0 b 0.5 c 1.7 c 37.7 a 52.1 a

Fumonisins (␮g g−1 ) (FB1 + FB2 + FB3 ) e

The insect damage incidence (IDI) (%) was calculated as the percentage of ears per plot with injuries due to larvae insect activity on fifty ears per plot. Insect damage severity (IDS) (%) based on a 1–7 insect damage severity rating scale used for the estimation of the percentage of kernels damaged due the larvae as follows: 1 = 0%, 2 = 1–3%, 3 = 4–10%, 4 = 11–25%, 5 = 26–50%, 6 = 51–75%, and 7 > 75% of kernels with visible injuries. c Fusarium ear rot (FER) incidence (%) was calculated as the percentage of ears per plot with visible symptoms of the disease on fifty ears per plot. d Fusarium ear rot (FER) severity (IMcK) based on a 1–7 class disease severity rating scale used for the estimation of the percentage of kernels visibly damaged or discolored as follows: 1 = 0%, 2 = 1–3%, 3 = 4–10%, 4 = 11–25%, 5 = 26–50%, 6 = 51–75%, and 7 > 75% of kernels with visible symptoms of infection such as rot and mycelia growth. e Kernel fumonisin contamination (sum of FB1 , FB2 and FB3 ), expressed as ␮g of mycotoxins per gram of kernels. f Means in each column and for each maize hybrid, followed by the same letter, are not significantly different at a probability level of 0.05 according to Tukey’s test. The reported IDI,IDS and FER incidence and severity, means were transformed into Bliss angular values before their use in statistical analysis. b

F. De Curtis et al. / Field Crops Research 123 (2011) 161–169

a

F. proliferatum/F. verticillioides isolated from kernels (%)

Table 2 Effect of agrochemical treatments on Insect damage incidence (IDI) and severity (IDS), Fusarium ear rot (FER) incidence and severity, Fusarium proliferatum and F. verticillioides occurrence and fumonisin (FB) contamination on maize in 2006. Treatment

IDI (%)a

IDS(%)b

FER incidencec

NK Surtep

Tebuconazole + insecticide Tetraconazole + insecticide Prochloraz/cyproconazole + insecticide Insecticide alone Untreated control

5.0 cdf 8.3 bc 3.3 cd 11.7 ab 18.3 a

2.2 bf 2.7 b 1.1 bc 3.1 b 5.1 a

32.1 abf 23.3 b 11.7 c 28.3 ab 30.6 ab

NK Cisko

Tebuconazole + insecticide Tetraconazole + insecticide Prochloraz/cyproconazole + insecticide Insecticide alone Untreated control

8.3 b 6.7 b 8.3 b 8.3 b 20.0 a

2.3 bc 2.1 bc 2.9 b 3.1 b 7.5 a

NK Stella

Tebuconazole + insecticide Tetraconazole + insecticide Prochloraz/cyproconazole + insecticide Insecticide alone Untreated control

6.7 b 5.0 bc 8.3 b 8.3 b 20.0 a

2.6 b 2.0 bc 2.6 b 2.4 b 6.1 a

a

FER severityd

F. proliferatum/F. verticillioides isolated from kernels (%)

Fumonisins (␮g g−1 ) (FB1 + FB2 + FB3 )e

F. proliferatum

F. verticillioides

5.3 abf 3.6 bc 3.1 bc 5.0 ab 5.4 ab

4.3 af 23.3 a 7.0 a 19.0 a 24.7 a

2.3 af 22.7 a 3.3 a 13.3 a 15.7 a

24.0 b 11.7 c 20.0 b 41.7 a 65.0 a

3.4 cd 1.6 d 3.1 d 9.0 ab 11.9 a

4.3 a 20.7 a 6.0 a 53.0 a 23.3 a

0.3 b 15.3 ab 3.3 ab 24.0 a 9.3 ab

1.7 e 8.8 c 3.8 d 11.5 b 19.8 a

20.0 b 25.0 b 25.0 b 30.0 ab 65.6 a

2.9 d 3.8 cd 4.5 cd 9.2 ab 15.2 a

56.7 a 33.4 a 12.3 a 46.7 a 36.3 a

13.3 a 14.3 a 9.0 a 17.0 a 13.0 a

0.2 c 0.9 c 5.6 b 7.2 b 11.9 a

4.8 bf 5.3 b 0.9 c 8.5 a 7.9 a

The insect damage incidence (IDI) (%) was calculated as the percentage of ears per plot with injuries due to larvae insect activity on fifty ears per plot. Insect damage severity (IDS) (%) based on a 1–7 insect damage severity rating scale used for the estimation of the percentage of kernels damaged due the larvae as follows: 1 = 0%, 2 = 1–3%, 3 = 4–10%, 4 = 11–25%, 5 = 26–50%, 6 = 51–75%, and 7 > 75% of kernels with visible injuries. c Fusarium ear rot (FER) incidence (%) was calculated as the percentage of ears per plot with visible symptoms of the disease on fifty ears per plot. d Fusarium ear rot (FER) severity (IMcK) based on a 1–7 class disease severity rating scale used for the estimation of the percentage of kernels visibly damaged or discolored as follows: 1 = 0%, 2 = 1–3%, 3 = 4–10%, 4 = 11–25%, 5 = 26–50%, 6 = 51–75%, and 7 > 75% of kernels with visible symptoms of infection such as rot and mycelia growth. e Kernel fumonisin contamination (sum of FB1 , FB2 and FB3 ), expressed as ␮g of mycotoxins per gram of kernels. f Means in each column and for each maize hybrid, followed by the same letter, are not significantly different at a probability level of 0.05 according to Tukey’s test. The reported FER incidence and severity, IDI and IDS means were transformed into Bliss angular values before their use in statistical analysis. b

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Maize hybrid

165

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Fig. 2. Climatic conditions in maize field during the two years. Temperature (◦ C), relative humidity (%) and rain (mm) recorded at the weather station of CO.RE.DI.MO. (Consorzio Regionale di Difesa Molisano – Molise Region – Italy (Cb)).

3.2. Insect damage incidence and severity

3.3. Fungal identification

In both years the IDI and IDS were significantly reduced by the insecticide treatment in almost all cases. When the insecticide treatment was applied alone, the IDI was significantly reduced in only three of the six-year hybrid combinations, while the treatment always reduced the IDS compared to the untreated control. In particular, in 2005, the IDI values ranged from 6.7 to 16.7% for the treatments that include the insecticide, and were up to 25% for the untreated control; while the IDS values ranged from 2.2 to 3.5% for the treatments that include the insecticide, and were up to 7.5% for the untreated control (Table 1). In 2006, the IDI values ranged from 3.3 to 11.7, for the treatment that includes the insecticide, and were up to 20% for the untreated control; while the IDS values ranged from 2.0 to 3.1 for the treatment that includes the insecticide, and were up to 7.5% for the untreated control (Table 2).

The kernels randomly sampled in both years from all three hybrids and from all the agrochemical treatments produced abundant colonies belonging to Fusarium species on Petri plates. Data showed a very high variability between treatments, with differences that were significant only in few combinations of hybrid/treatment for both years (Tables 1 and 2). Throughout the two-year trial, the two dominant Fusarium species isolated from kernels under natural conditions were F. proliferatum and F. verticillioides. The total amount of F. proliferatum and F. verticillioides colonies isolated from the maize kernels ranged from 4.5 to 90.5% in 2005 (the total amount of other Fusarium species ranging from 2 to 7%), and from 4.6 to 77% in 2006 (the total amount of other Fusarium species ranging from 1 to 11%, being most of samples free of other Fusarium species). In particular, in 2005 the frequency of

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F. proliferatum ranged from 4 to 81.5% and that of F. verticillioides ranged from 0.5 to 26.5%. In 2006, the frequency of F. proliferatum ranged from 4.3 to 56.7%, while F. verticillioides ranged from 0.3 to 24%. 3.4. Fumonisin contamination There was great variability in fumonisin occurrence between the two years since both in control and in insecticide plots alone; it was significantly higher in 2005 than in 2006. In 2005, the mean values of fumonisin contamination in kernels ranged from 0 to 68.2 ␮g g−1 depending on the treatment and on the maize hybrid (Tables 1 and 2). The treatments with the three fungicides, applied in combination with the insecticide, significantly reduced in all three maize hybrids the fumonisin occurrence with respect to the treatment with the insecticide alone and control. In particular, the higher degree of fumonisin reduction was observed in maize hybrid NK Surtep. For this latest hybrid, in 2005, there was no contamination of the kernels (100% of reduction compared to control), while in 2006, the reduction of the fumonisin contamination in the kernels was lower although significant (Table 2). For the other two maize hybrids, a reduction of the contamination of fumonisin was also recorded although at a lower level. In particular, the treatment with tetraconazole caused a significant fumonisin reduction for the hybrid NK Cisko. On the other hand, the treatment with tebuconazole was effective for fumonisin reduction in the kernels for the hybrid NK Stella. Finally, for all hybrids and in both years, the treatment with insecticide alone significantly reduced fumonisins in only three of the six hybrid-year combinations (Tables 1 and 2). In particular, in 2005 for hybrid NK Surtep fumonisin contamination was significantly reduced to 28.2 ␮g g−1 by the treatment of the insecticide alone compared to the control with 68.2 ␮g g−1 (Table 1); whereas, in 2006 for hybrids NK Cisko and NK Stella fumonisin contamination was significantly reduced by the insecticide alone compared to the untreated control (Table 2); in 2006, only for the two hybrids NK Cisko and NK Stella, the fumonisin contaminations were significantly reduced to 11.5 and 7.2 ␮g g−1 , respectively, by the insecticide alone compared with the untreated control with a fumonisins content of 19.8 and 11.9 ␮g g−1 , respectively (Table 2). Finally, in both years, statistical analysis showed a significant (P < 0.001) degree of correlation between the Fusarium ear rot severity and the level of fumonisin kernel contaminations (R = 0.80 in 2005 and R = 0.77 in 2006) and between IDS and fumonisin kernel content (R = 0.76 in 2005 and R = 0.79 in 2006). 3.5. Meteorological data Meteorological data recorded in two years showed a significant difference in rainfall. In fact, during the period of silk emission, the climate was more rainy in 2005 than in 2006 (Fig. 2). 4. Discussion The current study evidenced that Fusarium ear rot of maize can be a serious problem also in southern Italy. The results showed that Fusarium ear rot incidence (up to 78.3%) and fumonisin contamination (up to 68.2 ␮g g−1 ) were quite severe, being F. proliferatum and F. verticillioides the main Fusarium species isolated from the maize kernels. The treatment with the three fungicides under natural infection and applied in combination with the insecticide treatment yielded a significant reduction of both Fusarium ear rot incidence and fumonisin contamination. On the other hand, the treatment with insecticide alone, significantly (P < 0.05) reduced the IDS in all cases, the kernel fumonisin content was significantly lower only for one hybrid (NK Surtep) in 2005 and two hybrids (NK Cisko and

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NK Stella) in 2006 and, did not reduce the Fusarium ear rot severity in any hybrid in both years. Further, Fusarium ear rot severity, fumonisin contamination, IDI and occurrence and prevalence of Fusarium species varied substantially between the two consecutive seasons (2005–2006) among the three maize hybrids, as well as among the different agrochemical field strategies applied in the research. F. proliferatum and F. verticillioides, as observed in previous investigations (Logrieco et al., 1995; Bottalico, 1998; Munkvold, 2003), were the largely dominant species isolated in both years with a relevant high level in the first year for both species. Indeed, Fusarium ear rot incidence and fumonisin contamination, recorded in 2005 in either control plots and in plots treated with insecticide alone, were significantly higher than those recorded in the same thesis in 2006. According to previous studies (Dorn et al., 2009), these differences can be due to the different environmental factors between the two years at the critical phenological phase (Fig. 2) and the cultural practices adopted in the previous years in the same field and/or in the nearby field crops. However, the possibility that the level of inoculum could have been decreased in 2006 due to the fungicide treatment of the previous year might also be taken into account. It is interesting to underline the high level of F. proliferatum occurrence on maize kernels in both years also in comparison with F. verticillioides. F. proliferatum has rarely been reported in Italy on maize (Logrieco et al., 1995), being largely a species of secondary importance with respect to F. verticillioides. On the other hand, the high level of contamination of two different species both capable to produce fumonisins makes the program of control of fumonisin contamination of maize more difficult since there is the need to face two different biological entities even if phylogenetically closely related. Several epidemiological studies have reported that the amount of airborne spores of F. verticillioides as a potential inoculum available for the main way of infection, the silks at flowering (Rossi et al., 2009; Duncan and Howard, 2010), is significantly related to environmental and cultural practices (temperature, humidity, rainfall, crop rotation, etc.), level of total mycoflora and the genetic variability of Fusarium strains occurring in the field (Munkvold, 2003; Dorn et al., 2009; Rossi et al., 2009). In this respect, Bush et al. (2004) and Fandohan et al. (2005) reported an increase of fumonisin levels associated to the moisture available and rainfall during the period from flowering to maturity that appeared to have had a key role in kernel infection and fumonisin content. Therefore, environmental conditions functional to the development of the disease and/or an improper management of maize crop residues can cause a high production of airborne spores especially in areas where maize is grown in short rotation and/or as continuous crop. Our preliminary epidemiological investigation, carried out in different climatic locations of southern Italy on a variety of maize hybrids in fields with different crop rotation, showed a high variability of Fusarium ear rot incidence and fumonisin contamination (De Curtis et al., 2008), confirming the importance of appropriate crop rotation in the control of Fusarium ear rot of maize. Fusarium ear rot incidence and fumonisin content recorded in this study can be influenced also by the different maize hybrids utilized in 2005 and 2006, as reported by other authors (Melcion et al., 1997; Dorn et al., 2009). The fumonisin content values showed a significant variability even in the same year experiment, depending on the year, hybrid and agrochemical treatment applied. Moreover, a high fumonisin content was registered also in kernel samples harvested from plot in which a low Fusarium ear rot incidence was recorded. On the other hand, not always samples with a high incidence of Fusarium species resulted highly contaminated by fumonisin. In 2006, the statistical analysis showed a significant degree of corre-

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lation (R = 0.84) between Fusarium ear rot severity and fumonisin contamination. Furthermore, we recorded a high fumonisin content also in samples from asymptomatic ears and/or ears with a very low Fusarium ear rot incidence. These results are in agreement with several previous reports on the singular and complex pathosystem and on behaviour of F. proliferatum and F. verticillioides in causing maize ear rot disease with symptomless infection of kernels (Nelson, 1992; Bacon and Hilton, 1996; Munkvold et al., 1997). In this regard, although the kernel infection by these fungi can also be a result of the infection of seeds and kernels (Munkvold et al., 1997), the silk penetration seems to be the most important pathway for the symptomless kernel infection with a high variability of disease incidence and severity (Munkvold et al., 1997; Duncan and Howard, 2010). The insecticide treatment significantly reduced the IDS (P < 0.05) in the two years, as was also reported in recent studies (Folcher et al., 2009; Mazzoni et al., 2011), but had reduced the content of fumonisin only in half of the hybrid–year combinations; when the insecticide was applied in combination with fungicides the agrochemical strategy significantly and consistently reduced the content of fumonisins compared to the untreated control. Indeed, the statistical analysis between IDS and fumonisin kernel content in two years showed a significant (P < 0.001) degree of correlation (R = 0.61, R = 0.65 in 2005 and 2006, respectively), but lower than the degree of correlation between Fusarium ear rot severity and fumonisin kernel content (R = 0.80, R = 0.84 in 2005 and 2006, respectively). These data could demonstrate the additive effect of the treatments with fungicides when applied in combination with insecticides on the contamination by fumonisins, as was also observed in a recent study (Mazzoni et al., 2011); this aspect could explain the importance of the penetration via silks of airborne spores of F. proliferatum and F. verticillioides for both symptomless kernel infection and fumonisin contamination on spring-sown maize in southern Italy. The importance of treatment with fungicides carried out at the flowering of maize (the critical phenological phase) has been clearly shown in this study. These results on the use of synthetic fungicides applied at silk emission and at the milky-waxy ripening phase to the foliage and to root, in combination with insecticide treatment, showed a significant reduction of the Fusarium ear rot severity and fumonisin kernel contamination as also observed in other studies (Folcher et al., 2009; Mazzoni et al., 2011). The effect of the fungicides applied to the soil is confined to decrease pathogen activity that can attack the roots and the first part of the stalk (Munkvold et al., 1997; Ronchi et al., 1997; Duncan and Howard, 2010). Indeed, the foliage treatment carried out in a crucial phenological phase could play a key role in reducing the insidious airborne microconidia as an inoculum for the silks infection and the subsequent fumonisin contamination. In this regard, recent research performed by Duncan and Howard (2010) on the biology of maize kernel infection by F. verticillioides clearly demonstrated the silk penetration by fungus and could more effectively elucidate the spray treatment effect with some fungicides in reducing the kernel infections in our experiments. In earlier research, preventive treatments with some fungicides significantly reduced the Fusarium head blight (FHB), the deoxynivalenol and trichothecene content in some cultivars of soft and durum wheat under field conditions and the maize Fusarium ear rots (Kuck et al., 1995; Heatherington and Meredith, 1988; Edwards et al., 2001; Haidukowski et al., 2005). In this regard, Ronchi et al., 1997 observed that the fungicide tetraconazole affects the phenylpropanoid-flavonoid biosynthesis and the hydroxyprolinerich glycoprotein gene in maize plant, increasing the maize plant defense responses to abiotic and biotic stresses as drought and plant pathogens.

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