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The fungicidal activity of tebuconazole enantiomers against Fusarium graminearum and its selective effect on DON production under different conditions Xue Diao, Yiye Hang, and chenglan liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05483 • Publication Date (Web): 21 Mar 2018 Downloaded from http://pubs.acs.org on March 25, 2018
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The fungicidal activity of tebuconazole enantiomers against
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Fusarium graminearum and its selective effect on DON production
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under different conditions
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Xue Diao†, Yiye Hang†, Chenglan Liu†, *
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†Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of
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Agriculture& Key Laboratory of Bio-Pesticide Innovation and Application of
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Guangdong Province, South China Agricultural University, Wushan Road 483, Tianhe
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District, Guangzhou, 510642, China
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ABSTRACT:
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Tebuconazole, which consists of a pair of enantiomers with different fungicidal
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activities, is one of the most common fungicides used in the control of Fusarium
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graminearum. In this study, the fungicidal activity of rac-tebuconazole and its
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enantiomers against F. graminearum was determined at 0.997, 0.975, 0.950 aw and at
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20, 25, 30 °C on wheat-based media. Then, F. graminearum treated with
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rac-tebuconazole and its enantiomers at the EC10, EC50 and EC90 levels under
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different culture conditions, and DON production was measured. Finally, expression
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of the DON biosynthetic genes (TRI5 and TRI6) was quantified by real-time RT-PCR
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after incubation with EC50 doses of rac-tebuconazole and its enantiomers for 4, 8 and
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14 days at 30 °C and aw 0.997. The results showed that the fungicidal activity of
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tebuconazole was strongly influenced by temperature, aw and the combined factors.
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(-)-tebuconazole
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rac-tebuconazole with 24–99-fold and 1.8–6.7-fold, respectively.
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(-)-tebuconazole was generally more favorable for DON production than
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(+)-tebuconazole under the same conditions. Additionally, (-)-tebuconazole and
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rac-tebuconazole induced significantly increased expression of the DON biosynthetic
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genes (TRI5 and TRI6) compared to the control by the 14th day of treatment. In this
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research, the combination condition of 30 °C and 0.997 aw are the most suitable for
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DON production by F. graminearum. The test strains of F. graminearum treated with
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the EC10 dose of (-)-tebuconazole produced the most amounts of DON.
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KEYWORDS: stereoisomers, biological activity, deoxynivalenol, TRI5, TRI6
is
higher
fungicidal
activity
than
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(+)-tebuconazole
and
However,
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INTRODUCTION
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Fusarium graminearum (teleomorph Gibberella zeae) is one of the most
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common pathogens causing Fusarium head blight (FHB) in wheat and other small
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grains.
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Fusarium spp., seriously threatens crop yield and food safety.4-6 These hazardous
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toxins frequently cause acute symptoms and chronic toxicity of in humans and
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livestock.7, 8 Therefore, mycotoxins such as deoxynivalenol (DON) present a growing
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concern. DON is the main trichothecene mycotoxin produced by F. graminearum; at
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the same time, DON has been confirmed to be a virulence factor in the process of F.
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graminearum infection of wheat.9-11 DON impairs the immune response and has been
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listed as a Group 3 agent (Group 3 is not classifiable as to its carcinogenicity to
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humans).7, 12 In recent years, surveys conducted in China found that the incidence of
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DON in cereal products is much higher than that of other mycotoxins, and the
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detection rate can even up to 93.3% in some investigation reports, thereby seriously
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affecting food safety.13-15
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The spread of FHB, along with a range of mycotoxins produced by
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Fungicide application is considered an important control measure for decreasing
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the incidence of FHB.16-18 However, the fungicide-related decline in FHB incidence
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does not always correspond to mycotoxins levels.19,20 Apart from fungicides, other
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environmental factors such as temperature and water activity also greatly influence on
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the production of mycotoxins, both pre-harvest and postharvest.21-23 Therefore,
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evaluation of mycotoxin production after application of fungicide under different
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environment is necessary. Some studies have observed that mycotoxin levels increase
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after treatment with tebuconazole, epoxiconazole and azoxystrobin under different
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water activities, temperatures or fungicide concentrations 24-26
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Tebuconazole, a fungicide useful for controlling FHB, is a demethylation
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inhibitors (DMIs), which acts on the cytochrome P450-dependent enzyme and thereby
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inhibits ergosterol biosynthesis.27 Like most of chiral fungicides, tebuconazole is
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produced and applied as a racemate, although the fungicidal activity and
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environmental behaviors of the two enantiomers have been shown to differ.28-30 So far,
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many reports have revealed the enantioselective behaviors of chiral fungicides in
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environmental degradation and toxicity for non-target organisms. For example,
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(-)-tebuconazole has higher fungicidal activity than (+)-tebuconazole against Botrytis
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cinerea and F. graminearum, while (+)-tebuconazole is more readily enriched in
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earthworms and exhibits slower to degradation in soil and grapes than
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(-)-tebuconazole. 30-32 However, to our knowledge, studies reporting enantioselectivity
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related to mycotoxins production are scarce. DON contamination of cereal is one of
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the major problems caused by FHB. Tebuconazole has been widely used to control
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FHB. Hence, it is necessary to study the effect of tebuconazole enantiomers on DON
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production.
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Therefore, the aims of the current study were to first assess the fungicidal
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activity of rac-tebuconazole and its enantiomers against F. graminearum in a
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wheat-based medium under different aw and temperature conditions. Then, the EC10,
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EC50 and EC90 values for the tested compounds in relation to DON production under
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the experimental conditions were determined. Lastly, the relative expression of the
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Tri5 and Tri6 genes was measured to evaluate the enantioselective effects of
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rac-tebuconazole and its enantiomers on the DON biosynthetic genes.
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MATERIALS AND METHODS
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Fungal strains and media. The DON-producing strain of Fusarium graminearum
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(KK082465.1) used in this study was provided by Prof. Mingguo Zhou, Nanjing
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Agricultural University, China. The fungi were maintained on potato dextrose agar
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(PDA) slants at 4 °C. Prior to experiments, the strain was inoculated on 3% wheat
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agar medium (3 g whole-wheat meal, 2 g agar, 100 mL deionized water) for 5 days at
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25 °C.
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Determination of EC10, EC50 and EC90 values under different conditions.
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Glycerol was used to adjust the water activity of the 3% wheat agar medium to obtain
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the target levels. The water activity of the medium is 0.997 without adding any
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glycerol. When the water activity is 0.975 and 0.950, 100 mL of medium need to add
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14 and 25.5 g of glycerol, respectively. The compounds of (+)-tebuconazole,
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(-)-tebuconazole and rac-tebuconazole were purchased from Shanghai Chiralway
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Biotech Co., Ltd. (Shanghai, China), and were dissolved in acetone and diluted with
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sterile deionized water. Next, 1 mL of the solutions of the tested compounds was
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mixed with molten agar medium, and the mixtures were poured into 9 cm sterile Petri
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dishes (10 mL per plate). 1 mL of sterile deionized water was added to 9 mL molten
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medium for use as a blank control. The aw level of the media was determined with an
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AquaLab 4 Water Activity Meter. A mycelial agar disk 5 mm in diameter from the
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margins of the aforementioned growing colonies was excised, and all plates were
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inoculated in the center of the media. Each treatment was repeated three times, and
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the plates were wrapped with cling film. According the Ramirez et al. report,23 the
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plates were incubated at the combination conditions of each temperature (20, 25 and
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30 °C) and water activity (0.997, 0.975 and 0.950), respectively. Colony diameters
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were measured until the blank control cover two-thirds of the plate. The EC10, EC50
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and EC90 concentrations for rac-tebuconazole and its enantiomers at each combination
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of temperature and water activity were calculated by comparison with the controls.33
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The EC10 and EC90 values are listed in Table S1.
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F. graminearum cultivation under different culture conditions. The tested
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compounds were added to the media at the EC10, EC50 or EC90 concentration. All
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treatments were carried out in triplicate. After inoculation, the sealed plates were
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incubated for 14 days at a series of temperatures (20, 25 and 30 °C) and water activity
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(0.997, 0.975 and 0.950) combinations. All plates were preserved at -20 °C until DON
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was extracted from the media.
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DON quantification. For DON extraction, 8 g of a representative sample was
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homogenized in 20 mL acetonitrile. After shaking for 30 min at 250 rpm, the sample
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was filtered through filter paper (Whatman No. 1), and 5 mL of filtrate was then
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transferred to a centrifuge tube containing 1 g of NaCl. Then, the mixture was
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vortexed briefly and centrifuged at 3500 rpm for 5 min. The supernatant was
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transferred to a rotary flask and was evaporated with a rotary evaporator at 45 °C. The
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dry residue was redissolved in 1 mL of a methanol/water mixture (1:1, v/v) and
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filtered through a nylon filter (0.22 µm). The HPLC system included an Waters 2695
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modular system, a reverse-phase symmetry C18 column (5 µm, 150×4.6 mm, Agilent)
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and a Waters 2489 UV detector. The column temperature was set thermostated at
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30 °C, and the detection wavelength was 218 nm. A mixture of water and methanol
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(82:18, v/v) was used as the mobile phase at a flow rate of 0.7 mL min−1 with an
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injection volume of 20 µL. When necessary, the samples were diluted using a
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methanol/water mixture (1:1, v/v). The recovery rate was 92%, with a range of 79
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–107%. The quantification limit of the system was 0.05 mg/kg.
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RNA extraction, reverse transcription and qRT-PCR. Cultures with EC50
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concentrations of the tested compounds and blank controls cultures were prepared as
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previously described. Mycelia grown at 30 °C and 0.997 aw, were collected at 4, 8 and
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14 d after inoculation and stored at -20 °C with an appropriate amount of RNA Later.
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Total RNA was extracted using a Trizol Total RNA Purification Kit (Simgen, China)
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following the manufacturer’s instructions. The concentration and purity of the total
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RNA were determined with a NanoDrop (Bio-Rad, USA). The M-MLV reverse
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transcriptase was used to synthesize first-strand cDNA. Next, the real-time
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quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was
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performed using the SYBR® Green PCR Master Mix (Bio-Rad). Primer sequences for
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the endogenous reference gene and the target gene are listed in Table 1. The relative
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expression of the Tri6 and Tri5 genes was calculated according to the 2-∆∆Ct method.
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Statistical analysis. All data were executed to one-way ANOVA and Duncan's test.
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All analyses were performed with SPSS16.0 and GraphPad Prism 5.0 software. The
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EC50 values are shown in Table 2. Multifactor (three-way) ANOVA was performed
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to analyze the effects of temperature (20, 25 and 30 oC), aw (0.997, 0.975 and 0.950),
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and enantiomeric composition on the EC50 values. The results are shown in Table 3.
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DON production of F. graminearum, which treated with rac-tebuconazole and its
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stereoisomers at three water activities (0.997, 0.975 and 0.950) and temperatures (20,
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25 and 30 oC), was analyzed using the non-parametric Kruskal-Wallis test with a
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sequential Bonferroni correction for multiple comparisons. The analysis results are
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shown in Table 4.
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RESULTS
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Fungicidal activity test of tebuconazole under different conditions. The
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fungicidal activity of rac-tebuconazole and its stereoisomers against F. graminearum
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(KK082465.1) was evaluated under different conditions by the EC50 values (Table 2).
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Obviously, (+)-tebuconazole was far less active than (-)-tebuconazole and
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rac-tebuconazole. The activity of (-)-tebuconazole against F. graminearum was
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approximately 24–99-fold and 1.8–6.7-fold more than that of (+)-tebuconazole and
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rac-tebuconazole for the F. graminearum, respectively. Duncan's test showed that
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each compound formed a separate homogeneous group. Additionally, the EC50 values
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generally increased with the increasing aw and temperature (Table 2). Duncan's test
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revealed that temperature and water activity rooted in three homogeneous,
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respectively, and non-overlapping groups. The result of multifactorial ANOVA ACS Paragon Plus Environment
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(Table 3) indicated that there are significant (p0.05) (Table 4). Moreover, the multiple comparisons showed that the
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temperature factor separated into two homogeneous non-overlapping groups (30 and
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25 °C, 20 °C) and the aw factor classified into three different non-overlapping clusters
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(Table 4). The production of DON generally increased with the increasing aw and
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temperature (Fig 1), although DON production was not significantly different at 30
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and 25 °C (p>0.05) (Table 4).
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Effect of rac-tebuconazole and its stereoisomers on TRI5 and TRI6 gene
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expression in F. graminearum. qRT-PCR was performed to observe the effects of
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rac-tebuconazole and its stereoisomers on the expression of genes related to DON
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synthesis in F. graminearum. As Fig. 2 showed, the relative expression of the TRI5
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and TRI6 genes changed with respect to incubation duration. On the fourth and eighth
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day of incubation, the expression of TRI5 and TRI6 was significantly higher in the
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sample
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(−)-tebuconazole and rac-tebuconazole (p (+)-tebuconazole, our research demonstrated a
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similar tebuconazole activity for F. graminearum. Against F. graminearum, the
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activity of (-)-tebuconazole was approximately 24-99-fold and 1.8-6.7-fold higher
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than that of (+)-tebuconazole and rac-tebuconazole, respectively. In addition,
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environmental conditions are the important factors impacting the fungicidal activity of
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fungicides. Mateo et al.36 reported that the fungicidal activity of tebuconazole against
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Fusarium langsethiae varies with aw and temperature. The maximum EC50 values
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were obtained under the optimal growth conditions for this study, at 25 °C and 0.96
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aw. In the present study, the fungicidal activity of tebuconazole against F.
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graminearum generally increased with the aw and temperature. This result is
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consistent with the growth patterns of F. graminearum and may explain the result that
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a higher aw (0.997) and temperature (30 °C) were more favorable for the growth of
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this strain, hence a higher fungicides dose is required to control growth.37 However, it
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is interesting that the EC50 value of (−)-tebuconazole at 0.950 aw and 30 °C was
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statistically lower than that at 25 and 20 °C. According to the multifactorial ANOVA,
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the EC50 was significantly affected by water activity, temperature and enantiomeric
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composition, and by all interactions among these factors. This result may provide a
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reference for the control of F. graminearum and other fungi using tebuconazole under
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different environmental conditions. We have not found any previous studies that
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compared the fungicidal activity of (−)-tebuconazole and (+)-tebuconazole against
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fungi under different aw and temperature conditions. Indeed, the disparity in the
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fungicidal activity of these of enantiomers varies under diverse conditions.
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The ability of rac-tebuconazole and its enantiomers to affect the production of
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DON by F. graminearum under different environmental conditions and at different
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doses was tested. The data showed that DON production was significantly influenced
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by temperature, water activity, compound and dose (p
