M. W. Parsons a and G. P. Munkvold b* Introduction

Transcription

1 Doi: /j x Effects of planting date and environmental factors on fusarium ear rot symptoms and fumonisin B 1 accumulation in maize grown in six North American locations M. W. Parsons a and G. P. Munkvold b* a BASF Corp., 2320 Claranita Ave., Los Osos, CA 93402; and b Iowa State University Department of Plant Pathology and Microbiology, 160 Seed Science Center, Ames, IA 50011, USA Fusarium ear rot, primarily caused by Fusarium verticillioides, causes losses in grain yield and quality and can result in contamination of grain by mycotoxins, primarily fumonisin B 1. Disease severity and fumonisin B 1 contamination vary considerably among maize-growing regions and from year to year. A 2 year field study was conducted in six locations in the USA, to evaluate the roles of planting date, maize hybrid, rainfall, temperature and insect pests in the variation in fusarium ear rot symptoms and fumonisin B 1 contamination. Grain samples were inspected to determine percentage of kernels with fusarium ear rot symptoms, categorized as moulded or starburst ; grain was also analysed by ELISA for fumonisin B 1.Hybridand planting date frequently had significant effects (P 0Æ05) on fusarium ear rot and fumonisin B 1 contamination. Earlier planting consistently resulted in lower ear rot severity, fumonisin B 1 levels and insect damage. Mould symptoms were highly correlated with thrips populations (Frankliniella occidentalis) (r =0Æ78) and with fumonisin B 1 concentration (r =0Æ89). The starburst symptom was not as closely correlated with thrips (r =0Æ33) or fumonisin B 1 (r =0Æ18). A multiple linear regression model identified highly significant effects on fumonisin B 1 for thrips, lower average daily precipitation after flowering, and location. These results strengthen the evidence that locations with high populations of ear-infesting thrips and dry conditions after pollination have an increased risk of fumonisin contamination, and suggest that high fumonisin levels are much more likely in visibly moulded kernels compared to those with starburst symptoms. Keywords: fungal ecology, Fusarium spp., mycotoxins, quantitative epidemiology, thrips Introduction The primary causal organism of fusarium ear rot, Fusarium verticillioides, is worldwide in distribution, and it is the most prevalent pathogen associated with maize (Munkvold & Desjardins, 1997). Ear rot symptoms usually develop on ear tips or as scattered areas of infection over the ear surface. Severely infected ears are covered with white to pink fungal mycelium, which can colonize all the kernels (Farrar & Davis, 1991). Another symptom type is the starburst symptom, which consists of white streaks beneath the pericarp, radiating from the stylar canal (Duncan & Howard, 2010). Fusarium ear rot results in a net decrease in grain dry matter content, kernel density and total grain yield (Presello et al., 2007a). Fusarium verticillioides can also be isolated from a large proportion of symptomless kernels, as well as host root, stalk and leaf tissues (Munkvold et al., 1997). The importance of this symptomless systemic infection is still * munkvold@iastate.edu Published online 20 January 2012 unclear; it is only after a wound occurs (insect feeding, silk-cut, kernel-pop, etc.) that visible disease symptoms are evident on the host ear tissues (Munkvold, 2003b). Fusarium verticillioides produces a group of toxic metabolites known as the fumonisins, of which fumonisin B 1 is the most important (Proctor et al., 2006). The accumulation of fumonisins in maize kernels is highly correlated with fungal biomass in the kernels (Waalwijk et al., 2008). Fumonisin B 1 causes leukoencephalomalacia (LEM) in horses, pulmonary oedema (PEM) in swine, and has been implicated in liver cancer of rats (Marasas, 1996). Fumonisins are also associated with oesophageal cancer and neural tube birth defects in humans (Marasas, 1996; Marasas et al., 2004). Fumonisin contamination is a risk wherever maize is grown, although the severity of the risk varies with latitude (Munkvold, 2003b). The most influential factors in relation to fusarium ear rot and fumonisin risk are temperature, drought stress, insect damage, other fungal diseases and maize genotype (Miller, 2001). Insect wounds and exposed silks are the primary local infection sites upon which airborne F. verticillioides microconidia may land, germinate, colonize, and subsequently infect developing kernels through the stylar canal (Munkvold, 2003b; Duncan & Howard, 2010). Host genotypes with a predisposition for the 1130 ª 2012 The Authors Plant Pathology ª 2012 BSPP

2 Environmental effects on fusarium ear rot 1131 silk-cut or kernel-pop phenotypes are also at greater risk of infection by F. verticillioides (Odvody et al., 1997). Fusarium ear rot development is favoured by warm (>28 C), dry or droughty conditions during early reproductive stages (Shelby et al., 1994; Miller et al., 1995). Many insect pests can serve as vectors or create wounds that lead to infection by F. verticillioides (Dowd, 1998; Munkvold, 2003b). Insects implicated in fusarium ear rot and fumonisin contamination include western flower thrips (Frankliniella occidentalis; Farrar & Davis, 1991), European corn borer (Ostrinia nubilalis; Sobek & Munkvold, 1999) and other corn borers (Avantaggiato et al., 2002), corn earworm (Helicoverpa zea), and nitidulid beetles (Carpophilus spp.; Glischrochilus quadrisignatus; Dowd, 1998). Robust, field-based models to predict fusarium ear rot and fumonisin B 1 contamination in grain production environments have been elusive, most probably due to the complexity of interactions between numerous abiotic and biotic disease factors. A multiple regression model for predicting fumonisin B 1, from insect damage, temperature and precipitation data from Argentina and the Philippines, accounted for more than 82% of variation in fumonisin B 1 concentration (P 0Æ0001) (de la Campa et al., 2005). A multiple regression model to predict fusarium ear mould incidence based on Fusarium species, monthly air temperatures, monthly precipitation and the putative mode of fungal entry, accounted for 70% of variation of the disease across 56 locations in Ontario, Canada, from 1991 to 1993 (Vigier et al., 1997). Despite the relatively high precision of these models, the fact they are not broadly utilized in their current iterations to predict fusarium ear rot or fumonisin B 1 risks suggests they either (i) have not been broadly validated, or (ii) possess reduced predictive value when applied to environments outside those used to develop the models. There is a need for further investigation into the environmental and cultural factors that influence fusarium ear rot and fumonisin levels in maize grain. The goals of this multi-year study were to: (i) evaluate the variation in fusarium kernel rot symptoms and fumonisin B 1 contamination in relation to US locations, planting dates, hybrids and ear-infesting insect pests; and (ii) evaluate the association of two types of fusarium ear rot kernel symptoms (visible mould and starburst) with fumonisin B 1 contamination. Materials and methods Experimental plots and sampling A field study was conducted in California, Kansas, Iowa, Tennessee and Georgia, during the 2002 and 2003 growing seasons, as well as the winter season in Hawaii. Locations were near Woodland, CA (Yolo Co.), Garden City, KS (Finney Co.), Johnston, IA (Polk Co.), Union City, TN (Obion Co.), Tifton, GA (Tift Co.) and Waimea, HI (Kauai Co.). Three conventional, non-transgenic Pioneer Ò brand hybrids were used: one susceptible, one moderately susceptible, and one partially resistant to fusarium ear rot, according to sales literature from Pioneer Hi-Bred International, Inc. Cropping and tillage practices varied among locations. At the Woodland, CA location, the previous crop was sunflower and soil was turned thoroughly with a moldboard plough followed by smoothing with a field cultivator. The previous crop was maize at Garden City, KS, and soybean at Johnston, IA, Union City, TN, and Tifton, GA, with autumn disking and a field cultivator in the spring at these four locations. At Waimea, HI, the previous crop was maize and tillage consisted of deep subsoil ripping, followed by disking and field cultivation. Plots were sown to a density of approximately plants ha )1 on three dates at each site in each season, except Iowa, where only two planting dates were accomplished in 2002 due to excessive spring rains that precluded planting for several weeks. Fertilization and weed management were according to standard hybrid maize production practices for each region, with the goal of optimal plant nutrition and nearly weed-free conditions. Experiments were planted in a randomized complete block design at each location. Each open-pollinated plot measured two rows (1Æ52 m) by 6Æ10 m. Six and four blocks (replications) of the three hybrids were planted for each planting date in 2002 and 2003, respectively. Hybrids and planting dates were randomized within blocks. Plots were bordered by eight rows (6Æ10 m) of hybrid corn on the sides, and 6Æ10 m strips of border hybrid on the ends. Sites in Hawaii and California were drip and furrow irrigated, respectively, as per local practice throughout each season. Sites in Kansas and Georgia were irrigated by overhead sprinkler as needed during each season to minimize visible crop stress. Sites in Iowa and Tennessee were not irrigated. Insecticides were not applied at any of the locations. Ears were evaluated twice for presence of, and damage by, insects associated with fusarium ear rot. Approximately 21 days after pollination ( brownsilk ), three arbitrarily selected ears were manually removed from every plot, labelled, placed in sealed plastic Ziploc bags, and immediately chilled. Ears were then shipped overnight on ice to the Pioneer Hi-Bred International entomology lab in Johnston, Iowa. After receipt, and while still chilled, husk leaves and silks were carefully removed, and each ear was inspected under a 15 dissection microscope. The total number of thrips, regardless of development stage, was counted and recorded for each ear. Subsamples of thrips specimens were identified to species and confirmed as Frankliniella occidentalis or F. williamsi using a published dichotomous phenotypic key (Reed et al., 2006), but the two species were not differentiated during enumeration. Intra-ear thrips populations for three ears were averaged, to estimate the average number per ear for each plot. A second evaluation of insect damage occurred at harvest. The percentage of kernels damaged by lepidopteran larvae (primarily corn earworm and European corn borer), was visually estimated and recorded for five ears

3 1132 M. W. Parsons & G. P. Munkvold from each plot. After the mature ear evaluation, all ears from each plot were shelled with a single-ear mechanical research sheller (Agriculex Corporation), and a 454 g sample was retained. The 454 g sample was then fandried below 15% (if necessary), using heated air (40 C). Samples were then stored at 10 C and 50% relative humidity for subsequent kernel symptom assessments, mycotoxin analysis and fungal isolations. Symptom evaluation A 30 g subsample was taken by scooping from each wellmixed 454 g grain sample. Each kernel in the subsample was assessed in the laboratory for fusarium ear rot symptoms by inspection at 15 magnification. Kernels were classified as starburst if they exhibited white to pinkish streaks beneath the pericarp, or moulded if they exhibited visible fungal mycelium. At some locations, this symptom was predominantly associated with silk-cut. Kernels that exhibited both the starburst and moulded symptoms were counted as moulded. The fractional weights for each symptom type were then recorded and expressed as percentages of the total subsample weight (w w%). Fumonisin B 1 quantification From each plot grain sample, approximately 400 g were analysed for fumonisin B 1 by the Pioneer Hi-Bred Grain Analysis Laboratory (Pioneer Hi-Bred International, Inc.). Samples were finely ground (1 mm screen) and a 3 g sample was extracted for analysis by enzyme-linked immunosorbent assay (ELISA). The ELISA is a competition format constructed with proprietary antibodies to fumonisin B 1. Pre-coated, stabilized plates were prepared by Beacon Analytical Systems, Inc. Maize extract samples and horseradish peroxidase conjugated fumonisin B 1 were co-incubated at C for min with shaking in the dark. Substrate was added following washing of the plates. The substrate reaction was allowed to proceed for min at C with shaking in the dark. The reaction was stopped and the resulting colour intensity of the wells was read at 450 nm. The amount of fumonisin present in a sample was inversely proportional to the colour intensity in the assay well. Detection limits of the fumonisin ELISA ranged from 0Æ8 to 2000 mg kg )1. This method has been validated in comparison to HPLC (Kulisek & Hazebroek, 2000), using AOACapproved methods (Rice et al., 1995). Mean recovery of fumonisin B 1 from spiked samples was 92Æ1% (Kulisek & Hazebroek, 2000). In addition to fumonisin B 1 quantification, arbitrarily selected kernels from plot grain samples were cultured to confirm association of visible ear rot symptoms with F. verticillioides infection. Ten kernels per plot were surface-sterilized for 2 min in a dilute bleach solution, dried, and cultured on Nash-Snyder semi selective medium. After incubation for 5 days under ambient conditions in the laboratory, putative Fusarium colonies were transferred aseptically via hyphal tip to carnation leaf agar and incubated until sporulation. Each culture was then observed under magnification for purity, growth habit, spore, and conidiophore morphology characteristics of F. verticillioides (Leslie & Summerell, 2006). Data analysis Data were analysed using the mixed linear model function Fit Model-Standard Least Squares and Multivariate procedures in the JMP 7 software application (SAS Institute), and the Multiple Regression function in the STATISTICA software application (Statsoft Inc.). To protect against redundancy (i.e. lack of independence among predictor variables), the semi-partial correlation was reported. The semi-partial correlation is an accurate indicator of the practical relevance of a predictor, because it is scaled to (i.e. relative to) the total variability in the dependent (response) variable (Cohen, 2003). Analyses of residuals indicated the need for data transformation for some variables to reduce heterogeneity of variance. The 4th-root transformation gave the best improvement in homogeneity of variance for percentage starburst kernels, percentage moulded kernels, fumonisin B 1, and intra-ear thrips population variables. Data for percentage of kernels damaged by lepidopteran insects were not transformed. For each year (2002, 2003) and location (California, Georgia, Hawaii, Iowa, Kansas and Tennessee) a mixed linear model was used to estimate the influence of different effects on the variables of interest. Model effects were location, hybrid, planting date, the interactions of location with hybrid and hybrid with planting date, as well as the random block (rep) effect. Location interaction effects with planting date were not analysed because of imbalance across locations for levels of the planting date effect. Planting date means were compared within hybrids at each location, for each year, using Tukey s Honestly Significant Difference (HSD) method (P 0Æ05). Untransformed means for all variables are reported in the tables. Data from across locations, years, genotypes and planting dates were pooled, and linear correlation analysis was used to quantify the association of the fusarium ear rot symptoms (percentage moulded kernels and percentage starburst kernels), as well as insects (intra-ear thrips populations and percentage kernels damaged by lepidopteran species) with fumonisin B 1 concentration. Pooled data were similarly subjected to correlation analysis to evaluate the associations of insects with the two types of fusarium ear rot symptoms. Mean maximum daily temperature (T max ) and precipitation by base 10 C growing degree day (GDD) interval, location, intra-ear thrips population and percentage lepidopteran kernel damage were included as effects in a multiple linear regression model. Using local weather data, the mean maximum daily temperature and mean precipitation for each of four 700 GDD periods was calculated for each planting date at each location (Tables 1 & 2).

4 Environmental effects on fusarium ear rot 1133 Table 1 Temperature minima and maxima and mean daily precipitation for six locations in North America during four growing degree day (GDD) periods in 2002 following the planting of maize field experiments for assessment of fusarium ear rot and fumonisin accumulation Location Planting date GDD period a T max ( C) T min ( C) Garden City, KS 23 April Æ3 9Æ3 2Æ Æ4 18Æ3 4Æ Æ7 18Æ6 8Æ Æ3 17Æ4 6Æ9 Mean 31Æ7 15Æ9 5Æ6 2 May Æ9 11Æ3 2Æ Æ7 18Æ2 5Æ Æ4 18Æ6 7Æ Æ0 17Æ0 6Æ9 Mean 32Æ3 16Æ3 5Æ8 16 May Æ0 13Æ9 5Æ Æ1 18Æ8 3Æ Æ1 18Æ5 7Æ Æ5 16Æ3 6Æ7 Mean 32Æ4 16Æ9 5Æ Mean 32Æ1 16Æ4 5Æ7 Johnston, IA 20 April Æ6 9Æ6 14Æ Æ9 19Æ9 12Æ Æ8 19Æ1 3Æ Æ5 17Æ2 8Æ8 Mean 28Æ2 16Æ5 10Æ0 1 May Æ5 11Æ1 12Æ Æ1 20Æ1 12Æ Æ2 19Æ5 3Æ Æ1 16Æ5 8Æ6 Mean 28Æ7 16Æ8 9Æ Mean 28Æ5 16Æ6 9Æ7 Tifton, GA 15 March Æ1 13Æ2 9Æ Æ4 15Æ6 5Æ Æ0 19Æ8 7Æ Æ3 21Æ7 9Æ0 Mean 30Æ0 17Æ6 7Æ8 26 March Æ1 14Æ5 8Æ Æ0 16Æ1 6Æ Æ4 20Æ6 11Æ Æ8 22Æ0 19Æ6 Mean 30Æ6 18Æ3 11Æ5 13 April Æ2 17Æ5 6Æ Æ2 16Æ9 8Æ Æ8 20Æ8 6Æ Æ7 22Æ1 31Æ5 Mean 31Æ5 19Æ3 13Æ Mean 30Æ7 18Æ4 10Æ8 Union City, TN 3 April Æ2 11Æ3 20Æ Æ2 17Æ2 14Æ Æ6 20Æ8 2Æ Æ6 20Æ7 6Æ7 Mean 29Æ2 17Æ5 10Æ9 23 April Æ7 13Æ0 23Æ Æ8 19Æ4 10Æ Æ6 21Æ2 4Æ Æ2 20Æ4 9Æ9 Mean 30Æ1 18Æ5 12Æ0 10 May Æ6 15Æ4 30Æ Æ1 19Æ7 1Æ Æ4 21Æ5 3Æ Æ4 19Æ3 12Æ5 Mean 30Æ6 19Æ0 12Æ Mean 30Æ0 18Æ3 11Æ6 precipitation (mm)

5 1134 M. W. Parsons & G. P. Munkvold Table 1 (Continued) Location Planting date GDD period a T max ( C) T min ( C) precipitation (mm) Waimea, HI 5 November Æ0 19Æ6 1Æ Æ2 16Æ3 0Æ Æ4 16Æ1 6Æ Æ5 16Æ4 3Æ8 Mean 26Æ8 17Æ1 3Æ3 15 November Æ6 18Æ1 1Æ Æ3 15Æ6 2Æ Æ3 16Æ3 8Æ Æ0 17Æ1 0Æ0 Mean 26Æ8 16Æ8 3Æ1 1 December Æ2 16Æ3 0Æ Æ5 16Æ2 6Æ Æ4 16Æ4 4Æ Æ5 18Æ5 11Æ5 Mean 26Æ9 16Æ9 5Æ Mean 26Æ8 16Æ9 4Æ1 Woodland, CA 1 May Æ7 10Æ7 0Æ Æ4 15Æ5 0Æ Æ5 15Æ2 0Æ Æ4 15Æ8 0Æ0 Mean 32Æ0 14Æ3 0Æ2 10 May Æ3 12Æ4 0Æ Æ5 15Æ5 0Æ Æ9 16Æ6 0Æ Æ8 14Æ7 0Æ0 Mean 32Æ6 14Æ8 0Æ2 25 May Æ2 15Æ3 0Æ Æ4 15Æ1 0Æ Æ4 15Æ9 0Æ Æ6 15Æ1 0Æ0 Mean 33Æ9 15Æ4 0Æ Mean 32Æ8 14Æ8 0Æ1 a GDD were calculated on a basis of 10 C, and the four periods corresponded to the following maize growth periods: (early vegetative); (late vegetative); (early reproductive); (late reproductive). The GDD intervals were GDD ( early vegetative growth ), GDD ( late vegetative growth ), GDD ( early reproductive growth ) and GDD ( late reproductive growth ). The average GDD to flowering and physiological maturity for the test hybrids was 1400 and 2800, respectively. All factors were then considered in the multiple regression model as possible influencers of fusarium ear rot symptoms and fumonisin B 1 contamination. Results Location, hybrid and planting date were all frequently significant effects (P 0Æ05) influencing the percentage of moulded and starburst kernels, as well as fumonisin B 1 (Table 3). This was true especially in California and Hawaii, where the highest levels of disease severity and fumonisin B 1 contamination occurred (Table 4). The mean percentage of visibly moulded kernels was as high as 94Æ5% (2003) for hybrid A in California, and 78Æ5% (2002) in Hawaii (untransformed means). Maximum mean starburst symptom incidences in California and Hawaii were 75Æ0% (2002) and 32Æ5% (2002), respectively (hybrid B). Mean fumonisin B 1 concentrations in California and Hawaii were as high as 590Æ2 (2003) and 319Æ7 (2002) mg kg )1, respectively (hybrid A; untransformed means). In California and Hawaii, hybrid A tended to exhibit the highest proportion of visibly moulded kernels, hybrid B the highest proportion of starburst kernels, and hybrid C the lowest levels of both fusarium kernel rot symptoms. Hybrid A also accumulated the highest levels of fumonisin B 1 contamination in both California and Hawaii, although hybrid B accumulated similar concentrations in Hawaii. Hybrid C had the lowest concentrations of fumonisin B 1 in both locations. In both locations, fusarium kernel rot symptoms and fumonisin B 1 concentrations tended to be highest in the later plantings (Table 4). In Georgia, Iowa and Tennessee, fusarium kernel rot severity and fumonisin B 1 concentrations were lower (Table 4). Maximum values for visibly moulded kernel percentages were 7Æ8% (hybrid B, 2002), 5Æ7% (hybrid

6 Environmental effects on fusarium ear rot 1135 Table 2 Temperature minima and maxima and mean daily precipitation for six locations in North America during four growing degree day (GDD) periods in 2003 following the planting of maize field experiments for assessment of fusarium ear rot and fumonisin accumulation Location Planting date GDD period a T max ( C) T min ( C) Garden City, KS 26 April Æ8 9Æ9 10Æ Æ8 16Æ8 6Æ Æ9 18Æ8 2Æ Æ7 16Æ3 12Æ9 Mean 31Æ6 15Æ5 7Æ8 29 April Æ2 10Æ4 10Æ Æ9 16Æ8 5Æ Æ9 18Æ8 2Æ Æ7 16Æ3 12Æ9 Mean 31Æ7 15Æ6 7Æ8 14 May Æ7 12Æ9 13Æ Æ1 17Æ6 1Æ Æ0 18Æ3 6Æ Æ4 12Æ4 8Æ2 Mean 31Æ8 15Æ3 7Æ Mean 31Æ7 15Æ4 7Æ7 Johnston, IA 25 April Æ1 11Æ1 20Æ Æ5 17Æ2 16Æ Æ0 17Æ8 2Æ Æ0 16Æ2 3Æ7 Mean 27Æ4 15Æ6 10Æ6 3 May Æ8 11Æ7 15Æ Æ5 17Æ2 16Æ Æ5 18Æ3 2Æ Æ7 15Æ2 4Æ4 Mean 27Æ4 15Æ6 9Æ5 17 May Æ8 13Æ2 10Æ Æ6 17Æ8 13Æ Æ3 18Æ4 1Æ Æ3 12Æ9 7Æ6 Mean 27Æ5 15Æ6 8Æ Mean 27Æ4 15Æ6 9Æ4 Tifton, GA 12 March Æ7 12Æ0 17Æ Æ0 18Æ1 8Æ Æ0 19Æ9 24Æ Æ2 20Æ4 16Æ4 Mean 29Æ0 17Æ6 16Æ6 22 March Æ6 12Æ3 12Æ Æ1 18Æ3 3Æ Æ8 20Æ9 25Æ Æ4 21Æ3 19Æ3 Mean 29Æ0 18Æ2 15Æ1 2 April Æ0 14Æ2 12Æ Æ0 18Æ2 13Æ Æ0 21Æ1 28Æ Æ1 21Æ8 9Æ4 Mean 29Æ5 18Æ8 15Æ Mean 29Æ2 18Æ2 15Æ9 Union City, TN 18 April Æ2 13Æ3 31Æ Æ4 16Æ3 9Æ Æ1 19Æ9 14Æ Æ1 19Æ4 8Æ9 Mean 28Æ5 17Æ2 16Æ2 25 April Æ4 13Æ8 31Æ Æ7 17Æ6 11Æ Æ7 19Æ7 14Æ Æ4 19Æ5 5Æ6 Mean 28Æ8 17Æ7 15Æ7 precipitation (mm)

7 1136 M. W. Parsons & G. P. Munkvold Table 2 (Continued) Location Planting date GDD period a T max ( C) T min ( C) precipitation (mm) 7 May Æ8 14Æ8 23Æ Æ1 18Æ6 7Æ Æ3 19Æ1 13Æ Æ3 19Æ6 16Æ8 Mean 29Æ1 18Æ0 15Æ Mean 28Æ8 17Æ6 15Æ8 Woodland, CA 28 April Æ6 10Æ6 1Æ Æ0 14Æ2 0Æ Æ4 14Æ7 0Æ Æ6 17Æ3 0Æ0 Mean 32Æ4 14Æ2 0Æ3 15 May Æ6 14Æ1 0Æ Æ5 13Æ8 0Æ Æ8 15Æ1 0Æ Æ0 19Æ5 0Æ0 Mean 34Æ7 15Æ6 0Æ0 1 June Æ0 13Æ8 0Æ Æ6 15Æ1 0Æ Æ6 18Æ8 0Æ Æ1 16Æ2 0Æ1 Mean 35Æ1 16Æ0 0Æ Mean 34Æ1 15Æ3 0Æ1 a GDD were calculated on a basis of 10 C, and the four periods corresponded to the following maize growth periods: (early vegetative); (late vegetative); (early reproductive); (late reproductive). A, 2002) and 5Æ5% (hybrid A, 2002), in Iowa, Tennessee and Georgia, respectively (untransformed means). Maximum values for starburst kernel percentages were 8Æ5% (hybrid B, 2002), 11Æ5% (hybrid B, 2003) and 30Æ2% (hybrid B, 2002), in Iowa, Tennessee and Georgia, respectively. Hybrid was not a significant effect in Kansas, where fusarium kernel rot severity and fumonisin B 1 contamination levels were lowest. For Georgia, Iowa and Tennessee, differences among hybrids for fusarium ear rot symptom types were less profound, but hybrid A still tended to exhibit the highest percentages of visibly moulded kernels, hybrid B the highest percentages of starburst kernels, and hybrid C the lowest percentages of both symptoms, with exceptions (Table 4). Although planting date had a smaller effect for these locations, there was a similar association of later planting dates with higher levels of moulded kernels and fumonisin. In California and Hawaii, where intra-ear thrips populations were high, hybrid and planting date were also very significant factors influencing intra-ear thrips infestation (Tables 3 & 5). Maximum average intra-ear thrips populations were 161Æ8 (hybrid A, 2003), and 99Æ8 (hybrid B, 2002), in California and Hawaii, respectively (untransformed means). Similar to fusarium ear rot symptoms and fumonisin B 1 concentrations, later planting dates in both California and Hawaii experienced higher intra-ear thrips populations (Table 5). The only other location with moderate intra-ear thrips infestations was Georgia, where maximum mean intra-ear thrips populations reached 32Æ2 (hybrid B, 2002). Percentage kernel damage attributable to lepidopteran larvae was not significantly influenced by hybrid variety, except in Iowa. Although not always statistically significant, the trend across years and locations was for higher rates of kernel damage by lepidopteran pests with successively later planting (Table 5). None of the hybrids used in this study was engineered to express Cry proteins (i.e. Bt). Intra-ear thrips was more strongly associated with percentage visibly moulded kernels (r =0Æ78) than was percentage lepidopteran kernel damage (r = 0Æ37; Table 6). A similar trend was observed for the relationship between these two types of insects and the starburst symptom of fusarium ear rot (Table 6). The percentage of visibly moulded kernels was highly correlated (r = 0Æ89) with fumonisin B 1 concentration. Though significant, the correlation between starburst kernels and fumonisin B 1 (r = 0Æ18) was substantially weaker (Table 6). Intra-ear thrips populations were more strongly correlated with fumonisin B 1 concentration (r =0Æ83), compared to the correlation between percentage of lepidopteran kernel damage and fumonisin B 1 (r =0Æ34). The effects included in the multiple regression analysis accounted for a significant proportion of the variation in fumonisin B 1 concentrations for this data set (Adjusted R 2 =0Æ84, SE = 0Æ64, F =14Æ6, P <0Æ0001) (Table 7). Statistically significant model effects that influenced fumonisin B 1 concentrations were intra-ear thrips populations (b =0Æ86, P =0Æ0001), location (b =0Æ67, P = 0Æ0037), mean daily precipitation between 1401 and 2100 GDD (b = )0Æ254, P =0Æ0035), and mean daily precipitation between 2101 and 2800 GDD (b = )0Æ184, P =0Æ0251).

8 Environmental effects on fusarium ear rot 1137 Table 3 P-values from analysis of variance (ANOVA) for effects of hybrid and planting date on percentage of maize kernels with visible signs of fusarium ear rot, percentage of kernels with the starburst symptom of fusarium ear rot, fumonisin B 1 concentrations (mg kg )1 ), intra-ear thrips populations (#), and lepidopteran kernel damage (%), according to year and location a. Percentages of moulded and starburst kernels, fumonisin B 1 concentrations, and intra-ear thrips populations were subjected to 4th-root transformation to meet the assumptions of the ANOVA Year Variable Source CA GA HI h IA i KS TN 2002 Moulded kernels (%) b Hybrid f <0Æ001 0Æ546 <0Æ001 0Æ003 0Æ279 0Æ002 Planting date g 0Æ010 <0Æ001 <0Æ001 0Æ036 0Æ162 0Æ007 Hybrid Plant date 0Æ485 0Æ136 0Æ006 0Æ431 0Æ171 0Æ250 Starburst kernels (%) b Hybrid <0Æ001 <0Æ001 <0Æ001 0Æ172 0Æ487 0Æ665 Planting date 0Æ121 0Æ001 <0Æ001 0Æ050 0Æ702 0Æ127 Hybrid Plant date 0Æ005 0Æ849 0Æ547 0Æ615 0Æ415 0Æ920 Fumonisin B 1 (mg kg )1 ) c Hybrid <0Æ001 0Æ288 <0Æ001 0Æ012 0Æ280 0Æ181 Planting date <0Æ001 <0Æ001 <0Æ001 0Æ260 0Æ002 0Æ001 Hybrid Plant date 0Æ003 0Æ067 <0Æ001 0Æ372 0Æ521 0Æ483 Intra-ear thrips (#) d Hybrid 0Æ002 0Æ364 <0Æ001 0Æ616 0Æ124 0Æ043 Planting date 0Æ012 0Æ176 0Æ015 0Æ175 0Æ241 0Æ005 Hybrid Plant date 0Æ127 0Æ000 0Æ546 0Æ616 0Æ217 0Æ222 Lepidopteran kernel damage (%) e Hybrid 0Æ101 0Æ944 0Æ531 0Æ028 0Æ188 0Æ071 Planting date 0Æ244 <0Æ001 0Æ031 <0Æ001 0Æ120 <0Æ001 Hybrid Plant date 0Æ498 0Æ641 0Æ482 0Æ180 0Æ829 0Æ Moulded kernels (%) Hybrid <0Æ001 0Æ207 0Æ001 0Æ502 0Æ000 Planting date <0Æ001 0Æ065 <0Æ001 0Æ450 0Æ279 Hybrid Plant date <0Æ001 0Æ991 0Æ256 0Æ889 0Æ095 Starburst kernels (%) Hybrid <0Æ001 0Æ660 0Æ030 0Æ071 <0Æ001 Planting date 0Æ001 <0Æ001 0Æ266 0Æ056 0Æ030 Hybrid Plant date <0Æ001 0Æ216 0Æ592 0Æ992 0Æ709 Fumonisin B 1 (mg kg )1 ) Hybrid <0Æ001 0Æ182 0Æ006 0Æ566 0Æ006 Planting date <0Æ001 <0Æ001 <0Æ001 0Æ894 0Æ063 Hybrid Plant date 0Æ007 0Æ365 0Æ637 0Æ681 0Æ357 Intra-ear thrips (#) Hybrid <0Æ001 0Æ029 0Æ371 1Æ000 0Æ295 Planting date <0Æ001 0Æ029 0Æ371 1Æ000 0Æ305 Hybrid Plant date 0Æ339 0Æ065 0Æ410 1Æ000 0Æ304 Lepidopteran kernel damage (%) Hybrid 0Æ640 0Æ828 <0Æ001 0Æ668 0Æ781 Planting date <0Æ001 0Æ008 0Æ009 0Æ477 0Æ381 Hybrid Plant date 0Æ081 0Æ106 0Æ777 0Æ916 0Æ031 a Planted locations were Woodland (Yolo Co.), CA; Garden City (Finney Co.), KS; Johnston (Polk Co.), IA; Union City (Obion Co.), TN; Tifton (Tift Co.), GA; and Waimea (Kauai Co.), HI. b Based on a 30 g subsample of grain from each plot; expressed as a percentage by weight. c A 454 g grain sample was collected from each plot, ground, and subjected to analysis by ELISA to quantify fumonisin B 1 concentration. d Three arbitrarily selected ears were harvested from each plot 21 days after pollination; thrips of all developmental stages were counted for each ear, and counts averaged to estimate of the number of intra-ear thrips per plot. e Based on average of five ears per plot, percentage of kernels damaged by corn earworm feeding was estimated for each ear. f Commercially available grain hybrids from Pioneer Hi-Bred, International, Inc., rated as susceptible (A and B) or resistant (C) to fusarium ear rot in sales literature. g Plots were sown on planting dates considered early, normal, and late, compared to customary regional practice. h The study was conducted in Hawaii only during the 2002 season. i Due to adverse weather, there were only two planting dates in Iowa during the 2002 season. Discussion Fumonisin B 1 concentrations in late plantings of susceptible hybrids in California and Hawaii were substantially higher than reports from other maize production regions (Shelby et al., 1994; Munkvold & Desjardins, 1997), and in many cases, far exceeded the FDA recommendations for all feed and food applications (CFSAN, 2001). Fumonisin contamination of hybrid maize in Hawaii has been reported only recently (Parsons & Munkvold, 2010b). Thrips were found within ears at the Georgia location in 2002, although not at the high levels observed in California or Hawaii, and thrips counts were much lower in This is the first published report of thrips within maize ears outside California (Farrar & Davis, 1991) or Hawaii (Parsons & Munkvold, 2010b). Damage by lepidopteran pests occurred in all hybrids. Hybrid C was the most resistant hybrid in Tifton, GA, but differences from hybrids A and B for disease and fumonisin B 1 responses were not as dramatic, compared to the California and Hawaii environments. Disease levels during 2002 and 2003 in Iowa and Tennessee were lower than California, Hawaii or Georgia, though aligned with historical reports for their respective regions (Munkvold & Desjardins, 1997). Thrips were essentially absent from ears in both locations. Western

9 1138 M. W. Parsons & G. P. Munkvold Table 4 Hybrid and planting date means for ear rot symptoms and fumonisin B 1 levels. Values are back-transformed means (n = 6 for 2002 and n =4for 2003); statistical analyses were conducted on transformed data (4th-root). Means within years and locations followed by the same lowercase letter (within columns) or the same uppercase letter (within rows) are not significantly different (Tukey s HSD, P 0Æ05) Moulded kernels (%) d Starburst kernels (%) d Fumonisin B 1 (mg kg )1 ) Hybrid Hybrid Hybrid Year Location a Planting date b A c B C Mean A B C Mean A B C Mean 2002 CA 1 23Æ1 0Æ1 0Æ1 7Æ8b 10Æ6 3Æ6b 0Æ0 4Æ7 24Æ4b 0Æ6 0Æ0b 8Æ4 2 22Æ8 0Æ0 0Æ0 7Æ6b 10Æ7 5Æ3b 0Æ0 5Æ3 40Æ7b 5Æ1 0Æ0b 15Æ3 3 77Æ3 0Æ2 1Æ5 26Æ3a 0Æ2 74Æ7a 0Æ9 25Æ3 458Æ7a 11Æ8 20Æ7a 163Æ7 Mean 41Æ1A 0Æ1B 0Æ5B 7Æ2B 27Æ9A 0Æ3C 174Æ6A 5Æ8B 6Æ9B GA 1 0Æ0 0Æ2 0Æ0 0Æ1b 1Æ8 13Æ9 0Æ7 5Æ5b 0Æ0 0Æ2 0Æ0 0Æ1b 2 3Æ8 0Æ4 1Æ9 2Æ0a 12Æ4 27Æ0 4Æ6 14Æ6a 6Æ8 4Æ2 2Æ3 4Æ4a 3 0Æ8 0Æ4 0Æ9 0Æ7ab 1Æ6 12Æ1 1Æ0 4Æ9b 1Æ4 3Æ0 4Æ1 2Æ8a Mean 1Æ5 0Æ3 0Æ9 5Æ3B 17Æ7A 2Æ1B 2Æ7 2Æ5 2Æ1 HI e 1 6Æ4b 19Æ3b 0Æ2 8Æ7 0Æ1 2Æ2 0Æ1 0Æ8b 19Æ1b 33Æ5b 4Æ5 19Æ0 2 65Æ8a 56Æ7a 0Æ1 40Æ9 1Æ1 18Æ7 0Æ4 6Æ7ab 207Æ0a 168Æ4a 8Æ5 128Æ0 3 77Æ8a 57Æ5a 1Æ7 45Æ7 10Æ9 31Æ8 4Æ9 15Æ8a 317Æ4a 267Æ4a 18Æ0 200Æ9 Mean 50Æ0A 44Æ5A 0Æ7B 4Æ0B 17Æ6A 1Æ8B 181Æ2A 156Æ4A 10Æ4B IA f 1 3Æ2 4Æ8 0Æ1 2Æ7b 0Æ0 0Æ2 0Æ0 0Æ1b 5Æ9 8Æ1 1Æ6 5Æ2 2 5Æ8 6Æ1 2Æ1 4Æ7a 0Æ1 0Æ8 0Æ6 0Æ5a 1Æ5 11Æ4 0Æ1 4Æ3 Mean 4Æ5A 5Æ4A 1Æ1B 0Æ0 0Æ5 0Æ3 3Æ7B 9Æ7A 0Æ8B KS 1 0Æ1 0Æ1 0Æ0b 0Æ1 0Æ0 0Æ0 0Æ0 0Æ0 0Æ0 0Æ2 0Æ0 0Æ1b 2 0Æ5 0Æ0 0Æ0b 0Æ2 0Æ0 0Æ0 0Æ0 0Æ0 0Æ0 0Æ2 0Æ5 0Æ2b 3 0Æ1 0Æ1 0Æ3a 0Æ2 0Æ0 0Æ0 0Æ0 0Æ0 0Æ7 2Æ4 2Æ0 1Æ7a Mean 0Æ2 0Æ1 0Æ1 0Æ0 0Æ0 0Æ0 0Æ2 0Æ9 0Æ9 TN 1 2Æ3 0Æ1 0Æ0 0Æ8b 4Æ7 3Æ9 5Æ3 4Æ6 2Æ5 3Æ5 0Æ2 2Æ0b 2 4Æ9 0Æ2 2Æ5 2Æ5a 3Æ8 6Æ3 3Æ8 4Æ6 5Æ6 9Æ0 3Æ6 6Æ1a 3 3Æ0 1Æ5 1Æ0 1Æ8a 7Æ2 8Æ7 7Æ1 7Æ7 14Æ7 9Æ7 10Æ9 11Æ8a Mean 3Æ4A 0Æ6B 1Æ2B 5Æ2 6Æ3 5Æ4 7Æ6 7Æ4 4Æ CA 1 74Æ2b 4Æ0b 0Æ0b 26Æ1 7Æ7a 14Æ2b 6Æ1b 9Æ3 102Æ2b 22Æ6b 0Æ3b 41Æ7 2 86Æ3ab 16Æ8a 13Æ3a 38Æ8 3Æ7ab 50Æ5a 28Æ4a 27Æ5 301Æ8a 124Æ5a 86Æ9a 171Æ1 3 94Æ3a 8Æ1b 10Æ7a 37Æ7 2Æ0b 45Æ5a 18Æ5ab 22Æ0 578Æ2a 64Æ8ab 74Æ7a 239Æ3 Mean 84Æ9A 9Æ6B 8Æ0B 4Æ5C 36Æ7A 17Æ7B 327Æ4A 70Æ6B 54Æ0B GA 1 0Æ0 0Æ1 0Æ7 0Æ3 10Æ1 5Æ1 7Æ6 7Æ6a 1Æ1 6Æ5 7Æ5 5Æ0b 2 0Æ0 0Æ0 0Æ1 0Æ0 0Æ2 0Æ0 0Æ1 0Æ1b 0Æ4 0Æ1 1Æ8 0Æ8b 3 0Æ3 0Æ5 1Æ3 0Æ7 6Æ3 11Æ8 4Æ4 7Æ5a 11Æ4 17Æ8 13Æ7 14Æ3a Mean 0Æ1 0Æ2 0Æ7 5Æ5 5Æ6 4Æ0 4Æ3 8Æ1 7Æ7 IA 1 0Æ0 0Æ0 0Æ0 0Æ0b 0Æ0 0Æ1 0Æ1 0Æ1 0Æ0 0Æ0 0Æ0 0Æ0b 2 0Æ4 0Æ2 0Æ0 0Æ2ab 0Æ3 1Æ3 0Æ0 0Æ5 6Æ3 7Æ9 0Æ2 4Æ8ab 3 1Æ2 1Æ7 0Æ0 1Æ0a 0Æ0 0Æ8 0Æ0 0Æ3 8Æ6 9Æ7 2Æ0 6Æ8a Mean 0Æ5A 0Æ7A 0Æ0B 0Æ1B 0Æ7A 0Æ0B 5Æ0A 5Æ9A 0Æ7B KS 1 0Æ0 0Æ0 0Æ0 0Æ0 0Æ0 0Æ1 0Æ0 0Æ0 0Æ0 0Æ0 0Æ0 0Æ0 2 0Æ3 0Æ0 0Æ0 0Æ1 0Æ1 1Æ3 0Æ2 0Æ5 0Æ3 0Æ4 0Æ0 0Æ2 3 0Æ1 0Æ1 0Æ0 0Æ1 0Æ1 1Æ6 0Æ1 0Æ6 0Æ0 0Æ4 0Æ0 0Æ1 Mean 0Æ1 0Æ0 0Æ0 0Æ1 1Æ0 0Æ1 0Æ1 0Æ2 0Æ0 TN 1 0Æ1 0Æ4 0Æ0 0Æ1 0Æ0 1Æ0 0Æ0 0Æ4b 0Æ2 0Æ0 0Æ0 0Æ1b 2 1Æ3 0Æ2 0Æ0 0Æ5 0Æ1 8Æ0 0Æ1 2Æ7ab 0Æ4 0Æ0 0Æ0 0Æ1ab 3 2Æ3 0Æ1 0Æ0 0Æ8 0Æ2 10Æ7 0Æ1 3Æ7a 2Æ2 2Æ8 0Æ0 1Æ6a Mean 1Æ2A 0Æ2AB 0Æ0B 0Æ1B 6Æ6A 0Æ1B 0Æ9A 0Æ9A 0Æ0B a Planted locations were Woodland (Yolo Co.), CA; Garden City (Finney Co.), KS; Johnston (Polk Co.), IA; Union City (Obion Co.), TN; Tifton (Tift Co.), GA; and Waimea (Kauai Co.), HI. b Plots were sown on planting dates considered early (1), normal (2) and late (3), compared to customary regional practice. c Commercially available grain hybrids from Pioneer Hi-Bred, International, Inc., rated as susceptible (A and B) or resistant (C) to fusarium ear rot in sales literature. d Each kernel of a 30 g subsample of grain from each plot was categorized as moulded or starburst, and the weight of each category of kernels was expressed as a percentage of the total subsample weight for each plot. e The study was conducted in Hawaii only during the 2002 season. f Due to adverse weather, there were only two planting dates in Iowa during the 2002 season.

10 Environmental effects on fusarium ear rot 1139 Table 5 Hybrid and planting date means for insect activity. Values for thrips are back-transformed means (statistical analyses were conducted on 4th-root transformed data); values for lepidopteran insects were not transformed. n = 6 for 2002 and n = 4 for Means within years and locations followed by the same lowercase letter (within columns) or the same uppercase letter (within rows) are not significantly different (Tukey s HSD, P 0Æ05) Intra-ear thrips (#) Lepidopteran kernel damage (%) Hybrid Hybrid Year Location a Planting date b A c B C Mean A B C Mean 2002 CA 1 23Æ8 0Æ1 0Æ0 8Æ0b 16Æ7 16Æ7 6Æ7 13Æ3 2 0Æ7 0Æ0 2Æ6 1Æ1b 16Æ7 23Æ3 13Æ3 17Æ8 3 91Æ8 3Æ1 2Æ5 32Æ5a 23Æ3 16Æ7 16Æ7 18Æ9 Mean 38Æ8A 1Æ1B 1Æ7B 18Æ9 18Æ9 12Æ2 GA 1 22Æ1 21Æ7a 6Æ2b 16Æ7 3Æ3 1Æ7 3Æ3 2Æ8c 2 24Æ4 25Æ5a 9Æ0b 19Æ7 13Æ3 13Æ3 18Æ3 15Æ0b 3 8Æ9 5Æ4b 30Æ8a 15Æ0 35Æ5 38Æ3 33Æ3 35Æ7a Mean 18Æ5 17Æ5 15Æ3 17Æ4 17Æ8 18Æ3 HI d 1 14Æ0 58Æ8 2Æ7 25Æ2b 17Æ5 15Æ0 22Æ5 18Æ3b 2 31Æ8 56Æ7 2Æ7 30Æ4b 35Æ0 27Æ5 22Æ5 28Æ3a 3 70Æ4 97Æ5 6Æ2 58Æ1a 32Æ5 32Æ5 25Æ0 30Æ0a Mean 38Æ7B 71Æ0A 3Æ9C 28Æ3 25Æ0 23Æ3 IA e 1 0Æ0 0Æ0 0Æ0 0Æ0 12Æ5 5Æ0b 0Æ0b 2Æ5 2 0Æ0 0Æ0 0Æ0 0Æ0 22Æ5 20Æ0a 20Æ0a 20Æ0 Mean 0Æ0 0Æ0 0Æ0 0Æ0 17Æ5A 12Æ5AB 10Æ0B KS 1 0Æ0 0Æ0 0Æ0 0Æ0 5Æ0 0Æ2 1Æ7 2Æ2 2 0Æ0 0Æ0 0Æ0 0Æ0 6Æ0 3Æ7 3Æ3 4Æ3 3 0Æ0 0Æ0 0Æ0 0Æ0 15Æ0 8Æ5 4Æ0 9Æ2 Mean 0Æ0 0Æ0 0Æ0 0Æ0 8Æ7 4Æ0 3Æ0 TN 1 0Æ0 0Æ2 0Æ0 0Æ1a 0Æ0 1Æ7 0Æ0 0Æ6b 2 0Æ0 0Æ0 0Æ0 0Æ0b 0Æ0 0Æ0 0Æ0 0Æ0b 3 0Æ1 0Æ0 0Æ0 0Æ1a 6Æ7 11Æ7 3Æ3 7Æ2a Mean 0Æ0AB 0Æ1A 0Æ0B 2Æ2AB 4Æ4A 1Æ1B 2003 CA 1 69Æ2 11Æ1 3Æ7 28Æ0b 2Æ5 5Æ0 2Æ5 3Æ3b 2 119Æ4 64Æ6 45Æ7 76Æ6a 30Æ0 17Æ5 12Æ5 20Æ0a 3 160Æ2 78Æ0 40Æ4 92Æ9a 20Æ0 27Æ5 27Æ5 25Æ0a Mean 116Æ3A 51Æ2B 30Æ0B 17Æ5 16Æ7 14Æ2 GA 1 0Æ0 0Æ0 0Æ0 0Æ0b 12Æ5 2Æ5 15Æ0 10Æ0a 2 0Æ3 0Æ0 0Æ0 0Æ1a 2Æ5 0Æ0 0Æ0 0Æ8b 3 0Æ0 0Æ0 0Æ0 0Æ0b 7Æ5 15Æ0 7Æ5 10Æ0a Mean 0Æ1A 0Æ0B 0Æ0B 7Æ5 5Æ8 7Æ5 IA 1 0Æ0 0Æ0 0Æ0 0Æ0 5Æ0 10Æ0 0Æ0 5Æ0b 2 0Æ0 0Æ0 0Æ0 0Æ0 10Æ0 17Æ5 2Æ5 10Æ0ab 3 0Æ0 0Æ0 0Æ0 0Æ0 17Æ5 25Æ0 5Æ0 15Æ8a Mean 0Æ0 0Æ0 0Æ0 0Æ0 10Æ8 17Æ5 2Æ5 KS 1 0Æ0 0Æ0 0Æ0 0Æ0 2Æ5 2Æ5 0Æ0 1Æ7 2 0Æ0 0Æ0 0Æ0 0Æ0 5Æ0 5Æ0 2Æ5 4Æ2 3 0Æ0 0Æ0 0Æ0 0Æ0 7Æ5 2Æ5 5Æ0 5Æ0 Mean 0Æ0 0Æ0 0Æ0 0Æ0 5Æ0 3Æ3 2Æ5 TN 1 0Æ0 0Æ0 0Æ0 0Æ0 2Æ5b 2Æ5b 0Æ0b 1Æ7 2 0Æ0 0Æ0 0Æ0 0Æ0 7Æ5a 0Æ0b 7Æ5a 5Æ0 3 0Æ0 0Æ0 0Æ0 0Æ0 0Æ0b 10Æ0a 0Æ0b 3Æ3 Mean 0Æ0 0Æ0 0Æ0 0Æ0 3Æ3 4Æ2 2Æ5 a Planted locations were Woodland (Yolo Co.), CA; Garden City (Finney Co.), KS; Johnston (Polk Co.), IA; Union City (Obion Co.), TN; Tifton (Tift Co.), GA; and Waimea (Kauai Co.), HI. b Plots were sown on planting dates considered early (1), normal (2) and late (3), compared to customary regional practice. c Commercially available grain hybrids from Pioneer Hi-Bred, International, Inc., rated as susceptible (A and B) or resistant (C) to fusarium ear rot in sales literature. d The study was conducted in Hawaii only during the 2002 season. e Due to adverse weather, there were only two planting dates in Iowa during the 2002 season. Kansas experienced severe drought in 2002 and 2003, yet fusarium ear rot severity and fumonisin contamination were both very low. This result is at odds with many studies in the literature that associate increased fusarium ear rot severity and fumonisin B 1 contamination with excessively hot and dry conditions. Multiple studies report a negative correlation between precipitation near or following pollination, and fumonisin contamination and or

11 1140 M. W. Parsons & G. P. Munkvold Table 6 Linear correlation coefficients for insect data, fusarium ear rot symptoms, and fumonisin B 1 concentration (mg kg )1 ). Observations for all locations and years were pooled for the analysis (n = 423). Percentages of moulded and starburst kernels, as well as fumonisin B 1 concentration data, were transformed by the 4th-root method. Correlations shown were all significant (P 0Æ05) Variable Moulded kernels (%) a Starburst kernels (%) a Intra-ear thrips (#) b 0Æ78 0Æ33 0Æ83 Lepidopteran kernel 0Æ37 0Æ24 0Æ34 damage (%) c Moulded kernels (%) a 0Æ89 Starburst kernels (%) a 0Æ18 Fumonisin B 1 (mg kg )1 ) d a Each kernel of a 30 g subsample of grain from each plot was categorized as moulded or starburst, and the weight of each category of kernels was expressed as a percentage of the total subsample weight for each plot. b Three arbitrarily selected ears were harvested from each plot 21 days after pollination; thrips of all developmental stages were counted for each ear, and counts averaged to estimate the number of intra-ear thrips per plot. c Immediately prior to harvest, husks were removed from five arbitrarily selected ears in each plot, percentage of kernels damaged by corn earworm feeding was estimated for each ear, and individual ear estimates within each plot were averaged to represent corn earworm damage for the plot. d A 454 g grain sample was collected from each plot, ground, and subjected to analysis by ELISA to quantify fumonisin B 1 concentration. infection of maize grain by F. verticillioides (Shelby et al., 1994; Vigier et al., 1997). From studies in Ontario, Canada, fumonisin B 1 accumulation was associated with above average temperatures and below average rainfall following pollination (Miller et al., 1995). In Nebraska, Arino & Bullerman (1994) reported that kernel colonization by F. verticillioides was more severe in dryland versus irrigated plots. In Mississippi, seasons with above average temperatures and below average rainfall resulted in fumonisin B 1 concentrations significantly higher than in seasons with average rainfall and temperature (Abbas et al., 2002). Similar results were reported from a multiyear study in Poland (Pascale et al., 2002). The differing observations in the current study may be explained in part by the use of irrigation in the Kansas plots to reduce crop stress. No intra-ear thrips were observed, and lepidopteran insect damage was minimal. Given low disease levels, differences among hybrids were insignificant for the Kansas location. In locations with higher levels of fusarium ear rot, hybrids differed significantly in ear rot symptoms and fumonisin B 1 levels. In some cases, hybrids also differed significantly in starburst symptoms. The rank of susceptibility among these hybrids was as expected, based on seed company disease ratings (hybrid A > B > C), and these results reinforce the use of hybrid selection as an effective management practice for fusarium ear rot and fumonisins. In locations with significant thrips populations, hybrids also differed in intra-ear thrips infestation, which Table 7 Multiple linear regression analysis (adjusted R 2 =0Æ84, SE = 0Æ64, F = 14Æ6, P < 0Æ0001) of location, average daily high temperature and precipitation during defined periods of crop growth, intra-ear thrips, and lepidopteran kernel damage effects, as influencers of fumonisin B 1 concentration (mg kg )1 ). Intra-ear thrips and fumonisin B 1 data were transformed by the 4th-root method. Data were pooled across blocks and hybrids within each planting date for each location (n = 423) Effect Semi-partial correlation coefficient Beta SE P-level Location a 0Æ252 0Æ67 0Æ20 0Æ0037 b Mean T max1 0Æ060 0Æ17 0Æ22 0Æ4350 c Mean T max2 0Æ073 0Æ19 0Æ20 0Æ3473 d Mean T max3 )0Æ058 )0Æ21 0Æ28 0Æ4545 e Mean T max4 )0Æ001 0Æ00 0Æ18 0Æ9920 f precipitation 1 0Æ034 0Æ07 0Æ16 0Æ6539 g precipitation 2 0Æ013 0Æ03 0Æ15 0Æ8640 h precipitation 3 )0Æ254 )0Æ37 0Æ11 0Æ0035 i precipitation 4 )0Æ184 )0Æ26 0Æ10 0Æ0251 Intra-ear thrips j 0Æ386 0Æ86 0Æ17 0Æ0001 Lepidopteran kernel damage k 0Æ150 0Æ36 0Æ18 0Æ0626 a Planted locations were Woodland (Yolo Co.), CA; Garden City (Finney Co.), KS; Johnston (Polk Co.), IA; Union City (Obion Co.), TN; Tifton (Tift Co.), GA; and Waimea (Kauai Co.), HI. b Mean maximum daily temperature ( C) from 0 to 700 (early vegetative) GDD. c Mean maximum daily temperature ( C) from 701 to 1400 (late vegetative) GDD. d Mean maximum daily temperature ( C) from 1401 to 2100 (early reproductive) GDD. e Mean maximum daily temperature ( C) from 2101 to 2800 (late reproductive) GDD. f precipitation (mm) from 0 to 700 (early vegetative) GDD. g precipitation (mm) from 701 to 1400 (late vegetative) GDD. h precipitation (mm) from 1401 to 2100 (early reproductive) GDD. i precipitation (mm) from 2101 to 2800 (late reproductive) GDD. j Three arbitrarily selected ears were harvested from each plot 21 days after pollination; thrips of all developmental stages were counted for each ear, and counts averaged to estimate the number of intra-ear thrips per plot. k Immediately prior to harvest, husks were removed from five arbitrarily selected ears in each plot, percentage of kernels damaged by corn earworm feeding was estimated for each ear, and individual ear estimates within each plot were averaged to represent corn earworm damage for the plot. corresponded to their fusarium ear rot and fumonisin levels. The higher levels of ear rot and fumonisin B 1 for later planting dates are consistent with reports from California (Parsons & Munkvold, 2010a) and Italy (Battilani et al., 2008; Blandino et al., 2008). The planting date effect on fumonisin B 1 was significant in nine of 11 location-years in this study; the evidence presented here supports the practice of timely planting to reduce the risk of fumonisin contamination in most maize-growing areas. This is the first report showing the effects of planting date on

12 Environmental effects on fusarium ear rot 1141 fusarium ear rot and fumonisin B 1 in Georgia, Hawaii, Iowa, Kansas and Tennessee. Across locations, samples with visibly moulded kernels had significantly higher levels of fumonisin B 1 contamination, compared to grain samples with similar percentage of kernels showing the starburst symptom. Despite the inclusion of grain samples representing a wider range of fumonisin B 1 contamination, the correlation with visible fusarium kernel rot symptoms is consistent with reports from other studies (e.g. Munkvold et al., 1999; Presello et al., 2007b). However, these results suggest that the majority of fumonisin B 1 can be found in visibly moulded kernels, and indicate a weaker, though significant, association of the starburst symptom of fusarium ear rot with fumonisin B 1 contamination. Previous studies have not reported on the relationship of different fusarium ear rot symptoms with fumonisin concentrations; the results here suggest that kernels with starburst symptoms are far less contaminated with fumonisins than visibly moulded kernels. Multiple regression analysis suggested significant influences of location, intra-ear thrips infestations, and lower average daily precipitation after flowering, upon fumonisin B 1 concentrations. The effect of lepidopteran kernel damage upon fumonisin B 1 contamination approached a significant level (P = 0Æ06). The significant effect of location, presumed to be a unique combination and interaction of unmeasured factors, is consistent with recent reports (de la Campa et al., 2005; Battilani et al., 2008), although intra-ear thrips, if present, were not parsed from the location effect in those studies. In this study, it is doubtful that the location and intra-ear thrips effects are fully independent. Even with the possible mitigating impact of irrigation, the negative influence of precipitation during early and late plant reproductive stages upon fumonisin B 1 was significant in the multiple regression analysis. This is consistent with past reports, but the magnitude of this effect may have been diminished by the use of irrigation, and presumably the plants experienced little moisture stress. These results reinforce the importance of insect control in the management of fumonisins in maize (Munkvold et al., 1999; Dowd, 2003; Munkvold, 2003a). Given reports of the strong association of intra-ear thrips populations with fusarium kernel rot symptoms and fumonisin B 1 contamination, maize producers in regions within the international range of Frankliniella spp., especially F. occidentalis and F. williamsi, should be aware of the relative risk to maize posed by this insect (Farrar & Davis, 1991). Comprehensive regional and global surveys of the incidence and severity of maize intra-ear thrips infestation, and association with fusarium ear rot and fumonisins, are non-existent. Intra-ear thrips infestation should be considered in regions where fusarium kernel rot and fumonisin B 1 concentrations reduce yields or exceed advisable levels, respectively. Countries with potential for thrips to influence fusarium ear rot include Italy, Argentina, Mexico, Spain, South Africa and Australia (EPPO, 1989). Both thrips and lepidopteran insect injury also had significant positive correlations with starburst symptoms. Although development of the starburst symptom has been thoroughly described as a result of silk colonization and kernel entry through the stylar canal (Duncan & Howard, 2010), the present data suggest there may be a connection with insect activity, a possibility that warrants further investigation. This study also confirms the strong association of visible symptoms of fusarium ear rot with fumonisin B 1, and suggests that selection against reduced visible mould should reduce risk of fumonisin B 1 contamination to a greater magnitude than selection against starburst symptoms. Acknowledgements The authors are grateful to Pioneer Hi-Bred International, Inc., for supporting this research with financial and other resources. Mr Mark Mancl and other Pioneer co-workers provided instrumental help and support with experiment design, data collection and analysis. Thanks also to Drs Alicia Carriquiry and Petruza Caragea of Iowa State University for their guidance on statistical procedures. References Abbas HK, Williams WP, Windham GL, Pringle HD, Xie W, Shier WT, Aflatoxin and fumonisin contamination of commercial corn (Zea mays) hybrids in Mississippi. Journal of Agricultural and Food Chemistry 50, Arino AA, Bullerman LB, Fungal colonization of corn grown in Nebraska in relation to year, genotype and growing conditions. Journal of Food Protection 57, Avantaggiato G, Quaranta F, Desiderio E, Visconti A, Fumonisin contamination of maize hybrids visibly damaged by Sesamia. Journal of the Science of Food and Agriculture 83, Battilani P, Pietri A, Barbano C, Scandolara A, Bertuzzi T, Marocco A, Logistic regression modeling of cropping systems to predict fumonisin contamination in maize. Journal of Agricultural and Food Chemistry 56, Blandino M, Reyneri A, Vanara F, Pascale M, Haidukowski M, Saporiti M, Effect of sowing date and insecticide application against European corn borer (Lepidoptera: Crambidae) on fumonisin contamination in maize kernels. Crop Protection 27, de la Campa R, Hooker DC, Miller JD, Schaafsma AW, Hammond BG, Modeling effects of environment, insect damage, and Bt genotypes on fumonisin accumulation in maize in Argentina and the Phillipines. Mycopathologia 159, Center for Food Safety and Applied Nutrition, Guidance for the Industry: Fumonisin Levels in Corn and Corn Products Intended for Human Consumption. Center for Food Safety and Applied Nutrition. Online Publication. USFDA Center for Food Safety and Applied Nutrition. Docket No. 00D Washington, DC. Cohen J, Applied Multiple Regression Correlation Analysis for the Behavioral Sciences, 3rdedn.Mahwah,NJ,USA:L. Erlbaum Associates.