Aflatoxin and Fumonisin in Corn (Zea mays) Infected by Common Smut Ustilago maydis

Abbas, H.K., Zablotowicz, R.M., Shier,W. T., Johnson, B. J., Phillips, N.A.,Weaver,M. A., Abel, C. A., and Bruns, H.A. 2015. Aflatoxin and fumonisin in corn (Zea mays) infected by common smut Ustilago maydis. Plant Dis. 99:1236-1240. Corn infected with Ustilago maydis (common smut) produces galls that are valued as a delicacy in some cultures. During a 4-year period, aflatoxin levels in asymptomatic kernels of smutted ears were, on average, 45-fold higher than in kernels harvested from smut-free control ears and 99-fold higher than in smut galls. Aflatoxin levels in smut galls were lower than in kernels of smut-free control corn in all years combined. Fumonisin levels in asymptomatic kernels harvested from smutted ears were 5.2-fold higher than in kernels from smut-free control ears and 4.0-fold higher than in smut galls. Fumonisin levels in smut galls were not significantly different than in kernels of smut-free control corn. These studies indicate that, although corn smut was relatively free of the mycotoxins studied, the asymptomatic kernels of those ears contained mycotoxins at levels much higher than usually considered safe for direct human consumption. Common smut caused by Ustilago maydis (25) occurs frequently in corn-growing areas in the United States, including the Mississippi Delta. The fungus causes the formation of structures called galls that are filled with black teliospores. These galls are considered a delicacy in certain parts of the world, particularly in Mexico, and are marketed as “corn truffle”, “maizemushroom”, “huitlacoche”, and “cuitlachoche” (27). In those regions, smut galls are valued at about three times the normal corn crop; thus, farmers encourage smut formation by loosening corn husks in the field. Generally, U. maydis is not deleterious to corn productivity and profitability in the United States (7,25). However, other fungi, which may infect corn by similar routes, do impact productivity and profitability if they produce mycotoxins that contaminate corn kernels. Mycotoxin (particularly aflatoxin and fumonisin) contamination of corn is a serious food safety concern causing economic losses and is a major limitation for corn production (23). Aflatoxin ismore frequently a problem in the southern United States, while fumonisin can be a problem in both the Midwest and the South. The accumulation of aflatoxin in corn is influenced significantly by several environmental factors such as drought, heat stress, insects, and plant disease (2,4,8,9,13,21). A wider range of environmental parameters, including warm and wet conditions, insects, corn genotypes, and plant diseases, influence fumonisin accumulation in corn (2,4,8,9). Coexistence of both toxins in corn increases toxicological concerns (3,12,16,19). Little is known about the relationship between infection with U. maydis and mycotoxin contamination in corn. In one study evaluating fungal populations and mycotoxins in silage, silage made from smut-infected corn had increased mold and yeast (22). Aflatoxin was not detected in silage, while concentrations of other mycotoxins, including deoxynivalenol, ochratoxin, and zearalenone, were low and did not change during ensiling. Formation of smut galls on corn ears disrupts the husks and provides other fungi with an infection route to exposed, unsmutted kernels. In view of the limited understanding of the effect of common smut infection on plant stress in corn and on other factors that could contribute to infection by Aspergillus spp. and other fungi and to production of aflatoxin and fumonisin in corn, field studies were conducted in 2006, 2007, 2008, and 2009 in Mississippi to evaluate the incidence of common smut and its effect on (i) colonization of grain by Aspergillus flavus and (ii) aflatoxin and fumonisin levels in a Bt (34B24Bt) and a near-isogenic non-Bt (34B23) hybrid. Bt transgenic corn hybrids express Bacillus thuringiensis Cry insecticidal proteins that confer resistance to some ear-feeding insects and, thus, may reduce mycotoxins associated with insect-vectored fungal contamination (14). Materials and Methods Field trial experimental design. Experiments were conducted at the Southern Insect Management Research Unit Experimental farm at Elizabeth, MS, in 2006, 2007, 2008, and 2009. The study was conducted in corn planted for a study comparingmycotoxin and A. flavus levels in aging Bt and non-Bt corn residues under conventional till and Mississippi no-till conditions, which is described in detail by Abbas et al. (1). Briefly, the experimental design in the field was a randomized complete block design replicated in five blocks with corn hybrids 34B24 and 34B24Bt. The same treatment occurred on the same plots in each year of the study, except that in 2008 and 2009, the study was expanded to include a comparison of conventional till and Mississippi no-till conditions in a two-by-two randomized complete block design. The field had been planted in cotton for several years previously; therefore, it had no recent history of corn smut but it had a history of high incidences of Fusarium verticillioides and A. flavus (1). Because there is a high degree of variability in aflatoxin production in crops, the study used large plots (100 ft, 30.5 by 30.5 m, 32 rows in each plot, with soybean planted in a 30.5-m buffer area between plots) and large sample sizes (1). Natural infection byU. maydiswas relied on and husks were not opened. Corresponding author: H. K. Abbas, E-mail: [email protected] Disclaimer: Trade names are used in this publication solely for the purpose of providing specific information.Mention of a trade name, propriety product, or specific equipment does not constitute a guarantee or warranty by the USDAARS and does not imply approval of the named product to exclusion of other similar products. Accepted for publication 14 February 2015. http://dx.doi.org/10.1094/PDIS-03-14-0234-RE This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2015. 1236 Plant Disease /Vol. 99 No. 9 It was not determined whether the source ofU. maydiswas long-term survival in field soil or transmission from remote sources. Disease rating. Each block was divided into three sections of 10 rows each. The incidence of common smut-infected ears was counted at maturity on 100 plants from the middle two rows of each plot. Corn ears were considered infected with common smut only when one gall or more was visible to the naked eye. The percentage of common smut was recorded for each block based on an average of three sections per block. Aspergillus ear rot was not assessed. Experimental parameters. Corn was hand harvested at maturity (moisture content at 20% or less; 40 ears with smut galls per section and 40 ears with no visible smut galls per section) using several large paper bags (5 kg) for each sample. All samples were dried at 50°C in a forced air drier for 3 to 5 days to #12% moisture content. When high levels of aflatoxins in asymptomatic kernels of smutted ears were first observed in 2006, all field samples had been divided into two groups: (i) smutted ears from which common smut galls were removed by hand and (ii) ears with no visible smut galls. In 2007, 2008, and 2009, the scope of the study was expanded by dividing all field samples into three groups: (i) asymptomatic kernels from smutted ears from which common smut galls were removed by hand, (ii) apparently noninfected ears (ears with no visible smut galls), and (iii) common smut galls (teliospores). Variability in A. flavus infestation and aflatoxin production was addressed by taking large samples totaling at least 2 kg after shelling and using triplicate subsamples of ground kernels for chemical and biological determinations. The harvested, dried corn ears without galls were shelled, mixed, and ground in a Romer mill (20 mesh; Union, MO) before aflatoxin and fumonisin analysis and quantification of A. flavus. Galls of common smut were initially crushed with a hammer between two layers of paper, passed through a 2-mm sieve to remove any plant tissues, and ground to a fine powder in a coffee grinder individually to avoid cross contamination. Subsamples were taken for mycotoxin and fungal determination. Insect damage to corn was not assessed. A. flavus isolation. A. flavus was isolated from ground samples of asymptomatic kernels from smutted ears with the galls removed, kernels from apparently noninfected corn ears, and gall samples (described above) on modified dichloronitroaniline rose Bengal media (mDRBA) (5) supplemented with 3% NaCl. Field sample material (1 g) was added to 100 ml of 0.2% water agar and shaken on an Eberbach shaker for 30 min. The samples were serially diluted, and four replications per sample were plated on mDRBA plates and incubated for 5 days at 37°C. Aflatoxin analysis. Total aflatoxin concentrations were analyzed by high-performance liquid chromatography (HPLC) with confirmation in some years by enzyme-linked immunosorbent assay using commercially available kits (Neogen Inc., Lansing, MI) according to the manufacturer’s instructions. Shelled kernels or smut galls from each plot were pooled and ground, then oven dried at 50°C for 3 to 5 days. A representative subsample (10 g of sample for smut galls and 20 g of sample for kernels) was taken from each plot sample and extracted in 100 ml of 70% methanol, as previously described by Abbas et al. (1,2). Sample cleanup was carried out by diluting 500 ml of sample extract with 500 ml of acetonitrile in a microcentrifuge tube. The diluted aliquot was mixed on a vortex mixer, and 800 ml of the mixture was then applied to a 1.5-ml extract-clean reservoir minicolumn packed with aluminum oxide (1,2). After elution from the cleanup column, 20 ml of the eluate was injected onto a Waters HPLC system composed of aWaters 717Autosampler,Waters 600 Pump, Waters 2475 Fluorescence Detector, and Waters Temperature Control Module equipped with a 4-mm Nova-Pak C18 column (150 by 3.9 mm i.d.) at a column temperature of 30°C. Detection was achieved using a Photochemical Reactor for Enhanced Detection unit purchased from Aura Industries in combination with a fluorescent detector set at 365 nm (excitation) and 440 nm (emission). The mobile phase for this system was water/methanol/1-butanol (700:360:12.5 [vol/vol/vol]) with a flow rate of 0.9 ml/min. An aflatoxin standard mixture (number A9441; Sigma-Aldrich, St. Louis) and additional standard dilutions were studied using an methanol/water/acetic acid (310:190:0.5 [vol/vol/vol]) elutant. The same solution served as the blank during the HPLC run. The limit of detection was 0.1 ng g, and the standard curve was linear up to 200 ng g. Fumonisin cleanup. Methanol extracts were used for fumonisin with cleanup of samples using Bond-Elute SAX columns (Varian, Harbor City, CA), as described by Abbas et al. (2). Samples were evaporated under nitrogen and stored at 5°C until further analysis by liquid chromatography/mass spectrometry (LC/MS). The clean sample was reconstituted in 1 ml of acetonitrile/water (1:1). The limit of detection was 1 ng g. LC/MS analysis of fumonisins. The analytical method used for detecting various fumonisins was LC/MS (2). Fumonisin analysis was conducted using a MetaChem Intersil 5m ODS-3 column eluted with water/1% acetic acid/methanol (65:35:0) at 300 ml min for 10 min followed by a linear gradient to water/1% acetic acid in methanol/methanol (5:35:65), then held constant for 10 min. Between samples, the solvent was returned to water/1% acetic acid in methanol/methanol (65:35:0) within 1 min and held constant for 4 min for column equilibration. Analysis was carried out using an LTQ XL linear ion trap mass spectrometer with a Surveyor pump and autosampler (Thermo Electron Corporation, West Palm Beach, FL). Quantitation of FB1 and FB2 was carried out by the external standard method (linearity range of standards was 0.99). Statistical analysis. The percentage of ears with visible smut galls and A. flavus CFU were analyzed using Fisher’s least significant difference (LSD) in the PROC GLM procedure in the SAS statistical package (26). Mycotoxin concentrations in field samples were analyzed using analysis of variance (ANOVA) in the Microsoft Excel 2010 statistical package. Mycotoxin concentrations in field samples within the randomized complete block design were initially analyzed using LSD values estimated with PD MIXED 800 (24).