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Effect of Prothioconazole Application Timing on Fusarium Mycotoxin Content in Maize Grain Victor Limay-Rios, and Arthur Schaafsma J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00788 • Publication Date (Web): 22 Apr 2018 Downloaded from http://pubs.acs.org on April 22, 2018
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Effect of Prothioconazole Application Timing on Fusarium Mycotoxin Content in Maize Grain
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Victor Limay-Rios*† and Arthur W Schaafsma†
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University of Guelph, Ridgetown Campus, 120 Main St E, Ridgetown, Ontario N0P 2C0,
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Canada.
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*Corresponding author (Tel: +1 519-674-1500 x63567; Fax: +1 519-674-1515; E-mail:
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[email protected])
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Abstract
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In 2010 and 2011, studies to determine the optimal timing of prothioconazole application (200g
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a.i./ha) for reducing Fusarium mycotoxin accumulation in grain were conducted in controlled
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replicated experiments under small-plot mist-irrigated experiments and in field-scale
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experiments using two hybrids susceptible to F. gramineaerum infection. Significant decrease in
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total deoxynivalenol (DON) [DON+15-acetyl-DON+DON 3-glucoside+3-acetyl-DON] and
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zearalenone concentrations was observed when fungicide was sprayed at VT (tasseling) and
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R1 (silking; P80% of the ears were at the desired treatment stage using
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5 drop pipes, with 2 spray tips per drop pipe covering each side of the plant row, placed in each
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independent spray boom and separated by 0.76 m between row spacing. The drop pipe
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assembly consisted of a TeeJet QJ8600-2-1/4-NYB double swivel nozzle body attached to a
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76-cm, 610- mm diameter, drop pipe (Spraying Systems Co. Wheaton, IL). One end of the drop
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pipe was attached to the sprayer boom and the other end with the double swivel body held 2
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turbo wide-angle spray tips (TeeJet TT11002-VP). The drop pipe was stiffened by taping a 76-
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cm length of 6-mm fiberglass rod to the length of the pipe. Once the assembly was attached to
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the boom, the tip spray pattern was oriented to cover the entire ear zone. Temperature, relative
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humidity and wind speed were recorded during every treatment using a Kestrel 4000 pocket
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weather meter (Nielsen-Kellerman, Boothwyn, PA). The three center rows of each plot were
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harvested at maturity using a modified Gleaner K2 combine (AGCO Corp., Duluth, GA)
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equipped with a Harvest Master HM 1000 Grain Gage data collection system (Juniper Systems,
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Logan, UT) that recorded grain weight, test weight (kg/hL) and moisture (%) per plot. Composite
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samples (5 kg) were obtained from incremental grain samples collected over the course of
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harvesting the plot, 2-kg portions were taken using a calibrated sample splitter.
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Chemical reagents and equipment 8 ACS Paragon Plus Environment
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All reagents were LCMS grade or higher and, unless otherwise noted, obtained from Fisher
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Scientific (Oakville, ON, Canada). Methanol (MeOH) and acetonitrile (MeCN) were obtained
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from JT Baker (Phillipsburg, NJ). Water was obtained from OmniSolv (Billerica, MA). Glacial
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acetic acid (AcOH), ammonium acetate, formic acid, and ammonium formate were purchased
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from Fisher Scientific (Fairlawn, NJ). Deoxynivalenol, 1, deoxynivalenol 3-β-D-glucoside, 2, 3-
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acetyl-deoxynivalenol ,3, 15-acetyl-deoxynivalenol, 4, nivalenol, 5, fusarenon-X, 6, zearalenone,
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7, fumonisin B1, 8, fumonisin B2, 9, moniliformin, 10, HT-2 toxin, 11, T-2 toxin, 12,
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diacetoxyscirpenol, 13, beauvericin, 14 (dried and redissolved in MeCN), and isotope labeled
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internal standards [(13C34)fumonisin B1, (13C18)zearalenone and (13C15)deoxynivalenol) were
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obtained from Biopure standards (Romer Labs, Tulln, Austria). Enniatin A, 15, enniatin A1, 16,
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enniatin B, 17, and enniatin B1, 18, were dissolved in MeCN and obtained from Enzo Life
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Sciences (Farmingdale, NY).
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High performance liquid chromatography (HPLC) was performed using an 1100 Series HPLC
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unit with G1316A thermostat column oven, G1312A binary pump and G1322A degasser (Agilent
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Technologies, Santa Clara, CA) attached to a HTC Pal autosampler (CTC Analytics, Zwingen,
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Switzerland) equipped with a 100-µL injection syringe. Mass spectrometry (MS/MS) was
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performed using a PE Sciex API 365 triple quadrupole mass spectrometer (AB SCIEX,
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Concord, ON, Canada) modified with an EP10+ detector upgrade (IONICS, Concord, ON,
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Canada) equipped with a TurboIonSpray electrospray ion source (AB SCIEX). Nitrogen gas was
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used as the curtain, drying and collision gases during MS/MS analysis and was generated using
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a gas generator (purity > 99%). Eluents A and B consisted of H2O/MeOH/AcOH (89:10:1, v/v/v)
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with 5 mM ammonium acetate buffer and MeOH/H2O/AcOH (97:2:1, v/v/v) with 5 mM
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ammonium acetate buffer, respectively.
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Sample preparation, extraction and mycotoxin determination
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All samples were stored in burlap sacks, dried at 40°C for 24 h and stored at 2°C. Grain was
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coarsely ground using a No. 60 Power Grist Mill (C.S. Bell Co., Tiffin, OH), transferred to a
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clean 450 x 350-cm polypropylene tray and hand mixed for 30 s (layer < 1.5 cm in depth). A
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100-g subsample was obtained by taking 10 scoops from all four corners and the middle of the
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container. The subsample was further ground using a M2 Stein mill (Fred Stein Lab, Inc.,
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Atchison, KS) for 1 min to pass a 20-mesh sieve (0.989). LOD and
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LOQ were calculated as the mean peak height that could be detected using the mean height of
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the noise signal plus 3 and 10 times the standard deviation, respectively, around the analyte
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retention time. Relative standard variation for LOD and LOQ were below 7% and 18% in all
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analytes. Recovery tests were performed in triplicate by spiking a homogenized sample of 5 g of
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blank ground maize samples with the appropriate volume of analytical standards at two
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concentrations (50.0 and 100.0 ng/g). Spiked samples were allowed to equilibrate for 3 d at
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40°C in darkness followed by a standard extraction procedure and analysis. Recovery values
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ranged from 83±27% for fusarenon-X, 6, to 144±19% for beauvericin, 14, with an average of 11 ACS Paragon Plus Environment
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111±15%. All values below LOD, before being corrected for recovery rate, were considered
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negative.
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Statistical analysis
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Mycotoxin data were analyzed using the PROC MIXED procedure for randomized complete
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block design in SAS version 9.2 (SAS Institute, Cary, NC) after applying log (x+1)
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transformation to achieve approximate normality. Visual disease rating assessments, kernel
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weight and field yield data (adjusted to 15.5% moisture content) did not require transformation.
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Year, method, treatment, hybrid, and their interactions were included in the model as fixed
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factors to implement restricted maximum likelihood to estimate variance components. To study
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the relative contribution of DON-related compounds to the overall DON concentration, the
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contributions of DON, 1, DON 3-Gluc, 2, 3-acetyl-DON, 3, and 15-acetyl-DON, 4, and were
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expressed as ratios related to their sum or total DON, 1+2+3+4,. zearalenone, 7, was also
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compared separately from total DON. Fungicide efficacy was calculated as one minus the ratio
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of mycotoxin content in each fungicide-treated plot to their respective untreated control within
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each block. To avoid negative numbers on toxin reduction, a log (x+12) transformation was
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added prior to analysis. Results were expressed as the percentage of mycotoxin reduction and
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percentage of DON-related compounds relative to total DON. Means separation and standard
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error calculation were performed using the SAS macro “pdmix800” with a Fisher's Least
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Significant Difference test type option after Tukey–Kramer adjustment for multiple comparison
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with p75%.29 The mean Gibberella ear rot rating in the misted experiments was 3.3
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± 0.5. Only mycotoxins produced by F. graminearum were significantly correlated with disease
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severity symptoms [total DON, 1+2+3+4, (R=0.55, pF F 46.0
