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. 2015 Mar 5;7(3):728–742. doi: 10.3390/toxins7030728

Relationship of Deoxynivalenol Content in Grain, Chaff, and Straw with Fusarium Head Blight Severity in Wheat Varieties with Various Levels of Resistance

Fang Ji 1,2,3,4, Jirong Wu 1,2,3,4, Hongyan Zhao 1,2,3,4, Jianhong Xu 1,2,3,4, Jianrong Shi 1,2,3,4,*
Editor: Richard A Manderville
PMCID: PMC4379521  PMID: 25751146

Abstract

A total of 122 wheat varieties obtained from the Nordic Genetic Resource Center were infected artificially with an aggressive Fusariumasiaticum strain in a field experiment. We calculated the severity of Fusarium head blight (FHB) and determined the deoxynivalenol (DON) content of wheat grain, straw and glumes. We found DON contamination levels to be highest in the glumes, intermediate in the straw, and lowest in the grain in most samples. The DON contamination levels did not increase consistently with increased FHB incidence. The DON levels in the wheat varieties with high FHB resistance were not necessarily low, and those in the wheat varieties with high FHB sensitivity were not necessarily high. We selected 50 wheat genotypes with reduced DON content for future research. This study will be helpful in breeding new wheat varieties with low levels of DON accumulation.

Keywords: deoxynivalenol, FHB severity, wheat varieties

1. Introduction

Fusarium head blight (FHB) is a fungal disease that affects numerous small grain species [1,2,3,4]. This disease is caused by members of the Fusarium graminearum species complex, which consists of at least 16 phylogenetically distinct species [5]. FHB reduces kernel set and kernel weight, which contribute to significant grain yield losses. The disease also decreases the nutritive and baking quality of grain through degradation of proteins. Mycotoxins may accumulate to unacceptable levels, making harvested grain and their byproducts unsuitable for human and animal consumption.

Deoxynivalenol (DON) is one of the most economically important trichothecenes produced by F. graminearum. It is stable at high temperatures and is soluble in water and some polar solvents [6]. DON is also referred to as vomitoxin, due to its ability to induce vomiting, especially in pigs [7]. In plants, DON delays seed germination and subsequent plant growth and development [8]. At the cellular level, the main toxic effect of DON is inhibition of protein synthesis via binding to ribosomes [9]. Additionally, DON acts as a virulence factor and plays an essential role in the spread of F. graminearum after the initial injection of a wheat plant [10].

Since the 1990s, interest in the health benefits and safety of cereal products has increased. Alimentary mycotoxin accumulation is a risk associated with the consumption of infected grain [11], and this has resulted in the establishment of maximum permissible DON levels for grain destined for human consumption. In China, the maximum permissible level of DON in cereals and their byproducts is 1 mg/kg [12].

Wheat straw and chaff are often used for feedstuff, but may be unsuitable for animal consumption if contaminated by Fusarium toxins. Kang and Buchenauer (2000) and Matthäus et al. (2004) reported that natural contamination of feed by Fusarium toxins resulted in altered nutrient composition and marked modification of cell wall compounds in the infected grain due to higher activities of protease, amylase, and several NSP-degrading enzymes [13,14]. K. Seeling et al. demonstrated that feeding animals with wheat and wheat chaff contaminated with the Fusarium toxins DON and zearalenone (ZON) caused slight changes in ruminal nutrient degradability and in rumen physiological parameters, probably due to fungus-related modifications of the grain [15].

The development of FHB-resistant cultivars has been effective in reducing damage caused by the disease, However, resistance to FHB is affected by the relationship between symptom intensity and toxin production [16,17,18]. Understanding the relationship between FHB and DON accumulation could be important in predicting the risks of mycotoxins in food, and may aid in the development of a more reliable and rational strategy for managing the FHB disease complex and reducing DON production. Numerous attempts have been made to relate disease incidence or severity to mycotoxin concentrations and the results have varied [19]. Despite the fact that most studies reported positive correlations between disease incidence and mycotoxin accumulation, the quantity of mycotoxins per unit of disease index differed considerably and some studies failed to find a significant relationship [20]. The warm and humid climate in Jiangsu Province, China, during wheat flowering is favorable for the development of FHB epidemics. In this study, 250 winter wheat varieties obtained from the Nordic Genetic Resource Center in Alnarp, Sweden, for FHB resistance in China (data not shown), we selected 122 varieties that exhibited stable type II resistance to examine the relationship between DON content and FHB severity and to screen for wheat genotypes with reduced DON content. In addition, we measured the concentrations of DON in the grain, chaff and straw of these varieties. Our results mayfacilitate the selection of varieties used for animal feed.

2. Results

2.1. FHB Severity in Wheat Cultivars

During an FHB epidemic in Jiangsu Province, China, in 2012 [21], the rate of infected spikelets was as low as 24% in the Sumai-3 cultivar, which exhibited the highest resistance, but was as high as 89% in susceptible cultivars. In this study, we found a large range of genetic variation among the varieties in the severity of FHB infection. The occurrence of infected spikelets was less than 25% in eleven varieties and greater than 89% in 26 varieties, and the remaining varieties exhibited intermediate levels of resistance to FHB (Table 1).

Table 1.

Fusarium head blight (FHB) severity for selected wheat varieties from Sweden based on field-grown plants in 2012.

NO. Accession name Origin PIS 1
1 Loyal Sweden 31.69 ± 10.56
2 Hereford Sweden 24.34 ± 7.64
3 Skagen Sweden 23.35 ± 6.04
4 PAJ706-575A Sweden 45.82 ± 22.35
5 Mariboss Sweden 29.96 ± 22.27
6 Harnesk-7 Sweden 65.15 ± 11.67
7 IDUNA Sweden 39.17 ± 16.00
8 ANKAR Sweden 38.53 ± 20.22
9 ÅRING III Sweden 100.00
10 EROICA Sweden 22.61 ± 19.92
11 AROS Sweden 33.08 ± 22.40
12 BANCO Sweden 16.86 ± 8.73
13 NORRE Sweden 12.19 ± 8.80
14 STURE Sweden 56.28 ± 31.01
15 HELGE Sweden 14.40 ± 0.73
16 LINNA Sweden 29.39 ± 12.89
17 JYVÄ Sweden 95.14 ± 6.82
18 SIGYN II Sweden 100 ± 0.00
19 FOLKE Sweden 32.61 ± 13.48
20 HOLGER Sweden 24.12 ± 9.90
21 ALEMAR UST. HV. SG. Sweden 61.48 ± 13.55
22 GUSALEK K.17 Sweden 96.07 ± 6.79
23 LANTVETE FRÅN UPPSALA Sweden 100 ± 0.00
24 KOTTE Sweden 53.70 ± 11.88
25 EXTRA SQUAREHEAD Sweden 47.02 ± 11.75
26 RENODLAT SAMMETSVETE Sweden 91.61 ± 6.20
27 THULE II Sweden 92.81 ± 11.84
28 PANSAR II Sweden 29.68 ± 6.63
29 SVEA I Sweden 97.27 ± 6.10
30 PANSAR III Sweden 79.84 ± 10.06
31 GYLLEN II Sweden 85.29 ± 10.23
32 GLUTEN Sweden 93.75 ± 12.50
33 BORG Sweden 89.91 ± 14.39
34 PÄRL II Sweden 37.50 ± 17.68
35 ROBUR Sweden 26.34 ± 10.49
36 SEBA Sweden 68.30 ± 11.44
37 VIRGO Sweden 88.38 ± 18.02
38 SOLID Sweden 40.82 ± 15.72
39 HILDUR Sweden 17.09 ± 6.66
40 HANKKIJAN ILVES Sweden 39.52 ± 10.72
41 Harnesk-5 Sweden 24.68 ± 9.89
42 GUSALEK K.10 A Sweden 38.41 ± 23.08
43 KOSACK Sweden 46.04 ± 11.00
44 MK 2-114 Sweden 74.57 ± 11.86
45 MK 2-302 Sweden 32.31 ± 6.84
46 MK 2-304 Sweden 85.23 ± 17.94
47 MK 2-306 Sweden 95.56 ± 9.94
48 MK 2-313 Sweden 27.27 ± 6.43
49 MK 2-502 Sweden 41.36 ± 10.15
50 MK 2-503 Sweden 100.00 ± 0.00
51 MK 2-506 Sweden 46.11 ± 16.35
52 MK 2-508 Sweden 31.11 ± 12.57
53 MK 2-542 Sweden 59.82 ± 15.41
54 MK 2-543 Sweden 83.43 ± 9.19
55 MK 2-547 Sweden 97.37 ± 5.26
56 MK 2-548 Sweden 21.67 ± 21.62
57 MK 2-556 Sweden 90.73 ± 11.73
58 MK 2-557 Sweden 77.06 ± 11.22
59 MK 2-558 Sweden 84.31 ± 13.25
60 MK 2-564 Sweden 63.92 ± 25.45
61 SALUT Sweden 53.91 ± 12.70
62 ØSTBY Sweden 30.81 ± 9.26
63 MK 2-501 Sweden 59.04 ± 11.22
64 MK 2-510 Sweden 94.18 ± 8.85
65 PORTAL Sweden 30.01 ± 10.00
66 TJELVAR Sweden 45.51 ± 9.95
67 TRYGGVE Sweden 88.81 ± 10.62
68 MK 2-549 Sweden 79.50 ± 11.95
69 MK 2-566 Sweden 91.67 ± 14.43
70 MK 2-651 Sweden 42.84 ± 11.66
71 MK 2-656 Sweden 66.51 ± 28.36
72 MK 2-659 Sweden 39.18 ± 13.15
73 MK 2-660 Sweden 61.33 ± 21.33
74 MK 2-775 Sweden 44.96 ± 7.58
75 MK 2-780 Sweden 88.18 ± 21.70
76 MK 2-786 Sweden 86.93 ± 15.12
77 MK 2-787 Sweden 44.74 ± 12.32
78 PANU Sweden 65.90 ± 34.88
79 KONGE III Sweden 37.50 ± 15.52
80 TYSTOFTE SMAAHVEDE Sweden 34.03 ± 10.88
81 RENTAL Sweden 57.33 ± 13.20
82 STAVA Sweden 53.88 ± 15.44
83 RUDOLF RUBIN Sweden 90.58 ± 13.79
84 KIRSTEN Sweden 42.80 ± 11.91
85 LONE Sweden 43.68 ± 8.71
86 BRANDT Sweden 40.56 ± 11.73
87 KARAT Sweden 39.61 ± 17.13
88 MK 2-101 Sweden 41.43 ± 17.05
89 MK 2-113 Sweden 49.57 ± 24.98
90 MK 2-116 Sweden 71.81 ± 24.64
91 MK 2-122 Sweden 94.44 ± 13.61
92 MK 2-130 Sweden 29.91 ± 10.39
93 MK 2-316 Sweden 47.09 ± 15.38
94 MK 2-317 Sweden 69.01 ± 13.76
95 MK 2-337 Sweden 64.83 ± 15.38
96 MK 2-567 Sweden 57.99 ± 39.13
97 MK 2-655 Sweden 68.97 ± 26.56
98 MK 2-138 Sweden 32.40 ± 16.47
99 MK 2-679 Sweden 81.00 ± 7.12
100 MK 2-788 Sweden 70.70 ± 10.31
101 MK 2-847 Sweden 25.99 ± 13.17
102 Saxild Sweden 37.17 ± 18.92
103 Abba Sweden 33.86 ± 4.74
104 Konsul Sweden 100.00 ± 0.00
105 Rektor Sweden 35.89 ± 8.00
106 Stakado Sweden 100.00 ± 0.00
107 Kerimäkeläinen Sweden 95.45 ± 9.09
108 Sampo Sweden 95.83 ± 9.32
109 Väinö Sweden 87.20 ± 11.37
110 Speltti Vaalea Sweden 67.79 ± 19.61
111 Speltti Ruskea Baulander Sweden 56.30 ± 18.53
112 Kökar Sweden 31.96 ± 8.09
113 Olympia Sweden 77.45 ± 13.20
114 H 8703 Sweden 100.00 ± 0.00
115 H 8196 Sweden 100.00 ± 0.00
116 H 8202 Sweden 100.00 ± 0.00
117 H 8222 Sweden 76.78 ± 35.98
118 H 8296 Sweden 34.72 ± 28.69
119 H 8300 Sweden 88.75 ± 15.56
120 H 8305 Sweden 19.57 ± 7.03
121 H 8310 Sweden 94.44 ± 9.62
122 H 8311 Sweden 33.55 ± 7.08
- Control cultivar - -
- Annong 8455 China 89.65 ± 2.33
- Sumai 3 China 24.21 ± 9.04
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1 PIS is the percentage of infected spikelets.

2.2. Deoxynivalenol (DON) Levels in Wheat Grain, Straw, and Glume Tissues after Artificial Inoculation of Wheat Ears

A total of 122 wheat varieties were inoculated, and DON levels were determined in a total of 366 grain, straw, and glume samples. DON content was higher in glumes than in straw in 90.16% of the samples, and higher in straw than in grain samples in 73.77%. The DON content was higher in glumes than in grain in 95.90% of the samples (Figure 1). One-way analysis of variance (ANOVA) showed significant differences in DON content among grain, straw, and glumes (F = 42.20, p < 0.0001). The different plant sample types showed positive correlations (rgrain vs. straw = 0.261 **, rgrain vs. glume = 0.251 **, and rstraw vs. glume = 0.879 **). Although differences in mycotoxin content were large, the distribution of DON in plant tissues exhibited a distinct pattern (Table 2).

Figure 1.

Figure 1

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Distribution of deoxynivalenol in grain, glume and straw of wheat with different varieties.

Table 2.

Deoxynivalenol (DON) levels in samples of various wheat tissues determined using High Performance Liquid Chromatography-tandem Mass Spectrometry.

DON (mg/kg)
Tissue n Mean * Median Max Min
grain 122 3.88 A 2.38 23.46 0.16
straw 122 15.30 B 7.85 123.74 0.04
glume 122 20.95 C 19.13 55.42 1.15
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*: Values in the same column with different capital letters were significantly different (α = 0.01); Different letters within a column represent significant difference at p = 0.01.

2.3. DON Levels in Tissues from Wheat Varieties Exhibiting Differing FHB Disease Severity

To better understand how the level of DON contamination varied with FHB severity, we divided the entire dataset into three levels of FHB severity relative to the severity of FHB according to the control cultivars (Figure 2): <25% (n = 11, mean = 20.08%), 25%–89% (n = 85, mean = 52.94%), and >89% (n = 26, mean = 96.30%).

Figure 2.

Figure 2

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Box plots of the distribution of deoxynivalenol (DON) levels in wheat tissues ((A): grain; (B): straw; (C): glumes) inoculated with Fusarium graminearum at various levels of Fusarium head blight disease severity. Solid and dashed lines indicate medians and means, respectively. The box boundaries indicate the 75% and 25% quartiles. The whisker caps indicate 90th and 10th percentiles and the circles indicate the 95th and 5th percentiles.

The DON contamination levels did not increase consistently with increasing FHB severity (Figure 2). The average and median DON levels were the highest in all three wheat tissues when the FHB severity was greater than 89%. In the grain and the straw, the average and median DON levels were lowest when the FHB severity was between 25% and 89%, whereas the lowest average and median levels in the glume occurred when the FHB severity was below 25%. In general, the DON levels in the wheat varieties that were highly resistant to FHB were not necessarily low, and the DON levels in the varieties that were highly sensitive to FHB were not necessarily high.

2.4. Relationship between FHB Severity and DON Levels among Wheat Varieties and Selection of Wheat Genotypes with Reduced DON Content

To further compare the variation of DON contamination levels with FHB severity among wheat varieties, the data were allocated into four groups according to average FHB severity and DON contamination level: (1) varieties with low FHB severity and low DON contamination; (2) varieties with low FHB severity and high DON contamination; (3) varieties with high FHB severity and low DON contamination; and (4) varieties with high FHB severity and high DON contamination (Figure 2). The horizontal and vertical lines represent the y- and x-axis average values, respectively.

The relationship between DON content in grain and FHB severity is shown in Figure 3A. Groups 1, 2, 3, and 4 included 45, 25, 36, and 16 varieties, respectively. For the straw samples of the 122 wheat varieties, 41% were in group 1, 16% in group 2, 30% in group 3, and 13% in group 4 (Figure 3B). For the glume samples, 33% were in group 1, 25% in group 2, 26% in group 3, and 16% in group 4 (Figure 3C). Many of the samples exhibited low DON contamination levels (groups 1 and 3) and may correspond to varieties that would be useful for breeding new wheat varieties with low levels of DON accumulation.

Figure 3.

Figure 3

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Relationship between Fusarium head blight (FHB) severity and deoxynivalenol levels in wheat tissues ((A): grain; (B): straw; (C): glumes). Dashed lines indicate the average FHB severity (59%) and average concentration of DON ((A): 3.78 mg/kg, (B): 14.77 mg/kg, (C): 20.65 mg/kg) in 366 samples (grain samples = 122, straw samples = 122, glume samples = 122).

Among the 122 varieties, 27 were consistently in group 1, six were consistently in group 2, 23 were consistently in group 3, and seven were consistently in group 4. For both straw and glumes, 13 varieties were consistently in group 1, 14 were consistently in group 2, nine were consistently in group 3, and nine were consistently in group 4. For both straw and grain, four varieties were consistently in group 1 and four varieties were consistently in group 3. For both grain and glumes, only six varieties were consistently in group 2.The varieties in group 1 had low DON contamination levels not only in the grain but also in the straw and glume. These varieties were Skagen, IDUNA, ANKA, EROICA, BANCO, NORRE, ROBUR, HILDUR, Harnesk-5, MK 2-302, MK2-313, MK 2-506, MK 2-508, MK 2-547, SALUT, MK 2-549, MK 2-775, MK 2-787, KONGE III, TYSTOFTE SMAAHVEDE, MK 2-316, MK 2-317, MK 2-567, Abba, Speltti Vaalea, Kökar, and Olympia.

The varieties in group 3 had low DON contamination levels in all three wheat tissues while exhibiting high FHB severity. These varieties were Harnesk-7, ÅRING III, JYVÄ, SIGYN II, LANTVETE FRÅN UPPSALA, RENODLAT SAMMETSVETE, SVEA I, GUSALEK K.10 A, MK 2-304, MK 2-306, MK 2-557, MK 2-558, MK 2-564, MK 2-501, TJELVAR, MK 2-651, MK 2-780, PANU, MK 2-113, MK 2-788, Konsul, Sampo and Speltti Ruskea Baulander. We selected these 50 varieties as wheat genotypes with reduced DON content for future studies.

3. Discussion

Resistance to FHB can be characterized as consisting of two components: resistance to initial penetration and resistance to pathogen spread in the host tissue [22]. In addition to the resistance mechanisms that determine the severity of FHB, other mechanisms may influence DON content in kernels, including degradation or conjugation of DON and tolerance to DON [8,23,24]. DON tolerance results in an increased level of resistance and inherent prevention of trichothecene accumulation [25]. DON production is an important factor in the overall pathogenesis of FHB. Several studies have reported a significant positive correlation between the incidence of FHB and DON concentration [20,26]. Snijders and Perkowski reported that reduced disease incidence in the field ensured reduced mycotoxin content of the grain [8]. Correlations between FHB ratings and DON content were high in segregating material and in a collection of varieties with different levels of FHB resistance [27,28]. Others have demonstrated a significant correlation between DON and the fungal biomass of the grain [18,20,29]. These results suggest that new cultivars could be selected based on disease symptoms to ensure low levels of DON.

Bai et al. identified some wheat cultivars with severe FHB symptoms and low DON levels, particularly in cultivars with moderate resistance to F. graminearum [20]. Chen et al. inoculated five wheat varieties with a strain of F. graminearum and found no correlation between DON concentration and FHB severity [30]. Liu also found no correlation between DON concentration and FHB severity in wheat, barley (Hordeum vulgare L.) and oats (Avena sativa L.) after inoculation with a complex of Fusarium species [23]. In natural contamination, there was also no correlation between FHB severity in wheat and DON concentration or between the presence of Fusarium and DON concentration [31,32,33]. These differences in results may be due to the differences in the genotypes planted, pathogen populations, weather conditions, or management practices among the various studies.

Certain genotypes can limit the development of mycelium in the grain, thereby protecting it from degradation and limiting the visual signs of attack, but those genotypes often have low tolerance to mycotoxins [20]. Conversely, other cultivars may exhibit severe FHB symptoms and low mycotoxin levels. Beyer et al. studied the moderately susceptible wheat cultivar Dekan, which can partially avoid infection and then limit DON accumulation in the kernels when infections cannot be avoided [34].

We found DON contamination to be highest in glumes, intermediate in the straw, and lowest in the grain. DON produced by FHB in wheat can also have physiological effects in other parts of the plant [25]. Between the chaff and the kernel, several physical barriers could potentially limit the movement of the fungus and therefore prevent DON to move from the point of initial infection on the glumes or chaff to the kernel [35]. Snijiders et al. and Doohan et al. reported the ability of the toxin to translocate from chaff to the grain [36,37]. Data from previous studies suggest that the highest accumulation of mycotoxins in the barley hull [38]. Legzdina et al. reported that hulless barley tends to be less prone to mycotoxin contamination than covered barley, assuming a similar level of field FHB severity [39]. Wang and Miller reported higher levels of DON in the chaff than in the kernel of wheat [40].

In mature plants, DON appears to circulate in the phloem, with concentration following a descending gradient from the rachis, through the lemmas and grain, to the peduncle [41]. In our results, we found that the DON content was lowest in grain, then in straw and highest in glume. Schroeder and Christensen injected spores into the rachis and showed that the pathogen was able to migrate within the plant and propagate more rapidly longitudinally than transversely. Contaminated straw and glume residues remaining on the soil surface can infect seedlings of the following crop during the vegetative growth stage [42].

In conclusion, understanding the relationship between FHB severity and mycotoxin contamination is important. The conditions under which this relationship is purely qualitative rather than quantitative are unclear. In China, the increased use of high-yielding wheat varieties with greater susceptibility to FHB has increased the incidence of mycotoxins, indicating that the goal of a breeding program should be to select for both mycotoxin resistance and grain yield.

4. Materials and Methods

4.1. Reagents and Chemicals

DON was obtained from Sigma-Aldrich (Shanghai, China) at a concentration of 100 µg/mL in acetonitrile. HPLC-grade acetonitrile and methanol were obtained from Merck (Darmstadt, Germany). Deionized water (<8 MΩ cm−1 resistivity) was produced using a Milli-Q water purification system (Millipore, Bedford, MA, USA). All solvents were passed through 0.22-µm cellulose filters (Jinteng, Tianjin, China) before use.

4.2. Plant Material

Winter wheat seeds were obtained from the Nordic Genetic Resource Center in Alnarp, Sweden. Sumai-3 (high resistance) and Annong-8455 (high susceptibility) cultivars were used as control varieties to define levels of FHB resistance [43,44].

Winter wheat was grown according to current recommendations of integrated pest management, although applications of fungicide against Fusarium species were omitted. The field experiment was conducted at the Luhe experimental station, Jiangsu Academy of Agricultural Science (Luhe), Nanjing, China (32°28.583′N, 118°38′E) in 2011. Wheat was sown in late October in rows 150 cm in length and 33 cm apart with three replicates using a randomized block design.

An aggressive strain, F0613, was used to produce macroconidia in mung bean extraction liquid medium, as previously described [45]. The F0613 strain was isolated originally from diseased wheat grain in 2006 in Jiangsu Province. It belongs to Fusarium asiaticum, a member of the Fusarium graminearum species complex. At the heading stage, the middle spikelets of wheat flowers were inoculated with 10 μL of the conidial suspension containing 1 × 105 spores mL−1. Twenty days after inoculation, the numbers of infected spikelets and spikelets per head were recorded. The percentage of infected spikelets was calculated for each head. The mean FHB severity of 50 heads was calculated and all the 50 heads were pool into one sample for further analysis [46].

4.3. Mycotoxin Analysis

4.3.1. Preparation of Analytical Standard Solutions

Individual stock standard solutions were prepared as prescribed by the manufacturer. The working dilution for the calibration curve was prepared using a sample extract from a blank wheat sample (matrix-matched standard curve).

4.3.2. Sample Preparation

Harvested grain samples were cleaned manually. The glume was separated from the kernel. Straw was chopped into 3.0-cm segments that were subsequently pulverized. The grain was ground to a powder of 20-meshe fineness in a laboratory mill (Ika Werke, Staufen, Germany), and the chaff and straw were treated with liquid nitrogen and pulverized using a Moulinette 320-grinder (Moulinex, Barcelona, Spain). All samples were stored at 4 °C for a maximum of 7 day.

The finely ground samples (10 g) were weighed and extracted with 40 mL of acetonitrile:water:acetic acid (79:20:1 v/v/v) at 180 rpm for 30 min [47].

After centrifugation at 3000 rpm for 10 min, 0.5 mL of each final extract was diluted with acetonitrile:water:acetic acid (20:79:1, v/v/v) and filtered through a nylon filter (13 mm in diameter, 0.22-µm pore size) into an autosampler vial, capped and analyzed by LC-MS/MS [48].

4.3.3. LC-MS/MS Analysis

The LC-MS/MS system consisted of an Agilent 1200 HPLC, an Agilent 6410B triple-quadrupole mass spectrometer, and an Agilent MassHunter Workstation running Qualitative Analysis Version B.01.03 software (Agilent, Shanghai, China, 2001) for data acquisition and analysis. The analytical column was an XDB-C18 (2.1 × 150 mm, 3.5-µm bead diameter; Agilent, Shanghai, China) column and the column temperature was held constant at 30 °C. Nitrogen was used as a drying gas at 10 L/min. The capillary voltage was 4 kV, the nebulizer pressure was 25 psi, and the drying gas temperature was 350 °C. Mycotoxins were analyzed via multiple reaction monitoring (MRM). Mass spectrometric parameters of the mycotoxins and the composition of the mobile phase have been described by Soleimany et al. [49].

Quantification was performed against a matrix-matched calibration curve. During method development, limits of detection (LOD) and quantification (LOQ) scores for each analyte were determined on the basis of the signal-to-noise rations of 3:1 and 10:1. The LOD and LOQ scores were 10 and 20 µg/kg, respectively.

4.4. Statistical Analysis

All data are expressed as percentages or means ± relative standard deviation (RSD) using MS Excel.

Acknowledgments

The authors wish to acknowledge the Nordic Genetic Resource Center in Alnarp, Sweden, for providing winter wheat seeds. The work was supported financially by the National Institute of Food and Agriculture, Special Fund for Agro-scientific Research in the Public Interest (201303088), the National Natural Science Foundation of China (31271988), the Jiangsu Agriculture Science and Technology Innovation Fund of China (CX(13)3092), the Science and Technology Planning Project of Jiangsu Province, China (BE2014738), and the National Agricultural Products Quality Safety Risk Assessment Program of China (GJFP2014006)

Author Contributions

Jianrong Shi and Fang Ji conceived and designed the experiments; Fang Ji performed the experiments, analyzed the data and wrote the paper; Jirong Wu conducted the field experiment and calculated the Fusarium head blight severity; Hongyan Zhao and Jianhong Xu contributed reagents/materials/analysis tools.

Conflicts of Interest

The authors declare no conflict of interest.

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