Interactions among Fungal Community, Fusarium Mycotoxins, and

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Interactions among Fungal Community, Fusarium Mycotoxins, and Components of Harvested Wheat under Simulated Storage Conditions Yingyue Zhang,†,‡,⊗ Fei Pei,†,⊗ Yong Fang,*,† Peng Li,† Ji Xia,† Lei Sun,† Yanyu Zou,† Fei Shen,† and Qiuhui Hu†

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College of Food Science and Engineering, Nanjing University of Finance and Economics, Collaborative Innovation Center for Modern Grain Circulation and Safety, Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing 210023, China ‡ College of Food and Pharmaceutical Engineering, Nanjing Normal University, Nanjing 210023, China ABSTRACT: Economic loss of postharvest wheat under poor storage conditions due to fungal spoilage and mycotoxin contamination is severe. In order to study the influencing factors of the aggravation of mildew in natural wheat during storage, we assessed changes in Fusarium mycotoxins by high performance liquid chromatography, changes in fungal communities by high-throughput sequencing, and changes in biochemical components in wheat stored under artificial simulation conditions. Deoxynivalenol was the dominant Fusarium mycotoxin, reaching 1103 μg/kg at 25 °C with 75% relative humidity after 30 weeks. Under these conditions, Fusarium dominated the fungal communities, and Fusarium graminearum was significantly negatively correlated with glutenin (p < 0.05). Low storage temperatures and low humidity result in lower levels of Fusarium mycotoxins. Different fungi tended to consume different wheat components, and the interaction between environmental and biological factors eventually leads to the deterioration of wheat quality. These findings might provide valuable information for control strategies of mildew occurrence during grain storage. KEYWORDS: Fusarium, mycotoxins, fungal community, interaction, wheat storage

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subsequent mycotoxin production.5 It was reported that significant semipartial correlations were observed between relative humidity and DON production.14 Another study showed that regardless of spore type, DON production increased with relative humidity.15 However, DON and ZEN are usually produced along with their derivative mycotoxins, which are also severely toxic, such as 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), zearalanone (ZAN), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), αzearalanol (α-ZAL), and β-zearalanol (β-ZAL). Cumulative exposure to these derivatives was shown to cause to health risks in our previous study.16 Consequently, to monitor content changes of derivative mycotoxins is also necessary during wheat storage. Furthermore, individual environmental factors are not sufficient to predict disease and mycotoxin contamination, and previous studies have mostly been done in sterilized grain or culture media with pure fungal species. Therefore, it is hard to represent how postharvest wheat is further infected with Fusarium and its mycotoxins under longterm storage conditions where microbial or fungal communities may have competitive relationships. Actually, fungal physiological activities not only produce mycotoxins but also cause deterioration of wheat quality. Wang et al. reported that F. culmorum could destroy starch granules in winter wheat kernels and further cause weak dough

INTRODUCTION Wheat is one of the most important food crops worldwide, with annual consumption reaching approximately 697 million tons in recent years.1 Fusarium head blight, as known as scab, can easily occur in wheat and lead to extensive losses in crop production and yield.2,3 It is mainly caused by members of the Fusarium graminearum (F. graminearum) species complex and is often accompanied by mycotoxins that are harmful to human and animal health. In the United States alone, the annual economic loss of postharvest wheat due to fungal spoilage and mycotoxin contamination is more than $300 million.4 When wheat is in the field, Fusarium head blight is mainly affected by the weather, which cannot be altered artificially. After entering the storage stage, Fusarium attaches to the surface of wheat grain, and the disease may still occur, but it is influenced greatly by storage conditions, including temperature, relative humidity, pH, and nutrient availability.5,6 In particular, temperature and humidity are the key environmental factors determining the safety of stored grain.7−9 At low temperatures, the growth and reproduction of most fungi are inhibited, and spores and mycotoxins are produced at warmer temperatures. Previous studies found that the optimum temperature range for F. graminearum growth is 20−25 °C, and deoxynivalenol (DON) is optimally produced at 15−30 °C.10−12 In addition, both warm temperatures (20− 28 °C) and cooler temperatures (15 and 17 °C) were reported to promote zearalenone (ZEN) synthesis by F. graminearum and Fusarium culmorum (F. culmorum).13 On the other hand, relative humidity affects the moisture content of the grain, which in turn influences the availability of water and © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

April 1, 2019 June 5, 2019 June 27, 2019 June 27, 2019 DOI: 10.1021/acs.jafc.9b02021 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Changes in mycotoxin contents in wheat under different storage conditions: (a−d) 15 °C and 50% RH, 20 °C and 65% RH, 25 °C and 75% RH, and 30 °C and 80% RH storage conditions, respectively. Different letters (a−e) indicate significant differences (p < 0.05) among contents of a certain mycotoxin at different storage times.

properties and unsatisfactory bread quality.17 Furthermore, studies have shown that carbohydrates, lipids, and proteins significantly change in wheat kernels after infection with F. graminearum.18 Correspondingly, with the deterioration of the nutritional quality of wheat, the sensory quality also changes. Volatile compounds have been proven to be good early indicators of grain spoilage.19 Lippolis et al. detected 70 related microbial volatile organic compounds in wheat infected with Fusarium head blight and found that trichodiene, longifolene, 3-methylbutanal, tridecane, γ-caprolactone, and 6,10,14trimethyl-2-pentadecanone are positively correlated with DON.20 In summary, both mycotoxin contamination and quality deterioration cause declines in the commodity value of wheat. However, the interaction of factors including environmental temperature, environmental relative humidity, the fungal community, and wheat biochemical components during storage is unclear, especially in high-risk storage environments. Therefore, the objectives of this study were to monitor changes in Fusarium mycotoxins by multiple immunoaffinity column−high performance liquid chromatography, changes in fungal communities by high-throughput sequencing, and changes in biochemical components in wheat in different storage temperatures and humidity levels over 30 weeks and study the correlation among fungal communities, mycotoxins, and wheat quality parameters in high-risk storage conditions.

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Company, Ltd. (Shanghai, China). Liquid chromatography grade acetonitrile and methanol were obtained from Merck (Darmstadt, Germany). Immunoaffinity columns were purchased from Huan Magnech Bio-Tech Company, Ltd. (Beijing, China). Ultrapure water was obtained by a Milli-Q Element System (18 MΩ cm, Millipore-Q, Milford, MA). Sampling and Artificial Simulation of Storage of Wheat. Freshly harvested wheat grains were obtained from Jiangsu Province, China, in July 2017. In the laboratory, the total wheat samples (24.0 kg) were divided into four equal parts (for three replicates each). We simulated the summer average temperature and humidity conditions for four major grain storage ecological regions in China and successively stored wheat at 15 °C and 50% relative humidity (RH), at 20 °C and 65% RH, at 25 °C and 75% RH and at 30 °C and 80% RH. Samples were stored in constant temperature and humidity test chambers that were purchased from Wuhu Huace Instrument and Equipment Company, Ltd. (Anhui, China). Wheat grains were stored from July 2017 to February 2018, and samples were taken every 6 weeks and analyzed immediately. Mycotoxin Analysis. Our previous method was applied to mycotoxin analysis.16 Briefly, 5.0 g of milled wheat sample was extracted by 25 mL of 80% acetonitrile solvent, and then 20 mL of diluted supernatant passed through a multiple immunoaffinity column (Huan Magnech Bio-Tech Company, Ltd., Beijing, China). Afterward, the column was eluted with 3.0 mL of eluent (acetic acid/ methanol, 2:98, v/v), and then it was dried with nitrogen gas. The residues were redissolved in 1.0 mL of 10% acetonitrile solvent, after which they were ready for high-performance liquid chromatography analysis with a ZORBAX StableBond-C18 column (250 × 4.6 mm, 5 μm). Ultrapure water was used as eluent A, and acetonitrile was eluent B. The elution procedure started with 10% eluent B for 8 min, increased to 45% eluent B over 9 min, stayed at 45% eluent B for 5 min, gradually increased to 65% eluent B over 10 min, and finally decreased to 10% eluent B over 1 min.

MATERIALS AND METHODS

Chemicals and Reagents. Standard solutions of DON, 3AcDON, 15-AcDON, ZEN, α-ZOL, β-ZOL, ZAN, α-ZAL, and βZAL were purchased from Sigma-Aldrich Chemicals (St. Louis, MO). Glucose standard, 3,5-dinitrosalicylic acid (DNS), and other analytical grade chemicals were all bought from Sinopharm Chemical Reagent B

DOI: 10.1021/acs.jafc.9b02021 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article


Figure 2. (a) Microbial compositions of wheat with different storage conditions at the class level. (b,c) Microbial community structure based on (b) principal component analysis and (c) cluster analysis. The original sample is marked as 0 Day; 18WA, 18WB, 18WC, and 18WD represent the samples stored at 15 °C and 50% RH, 20 °C and 65% RH, 25 °C and 75% RH, and 30 °C and 80% RH for 18 weeks; 30WA, 30WB, 30WC, and 30WD represent the samples stored at 15 °C and 50% RH, 20 °C and 65% RH, 25 °C and 75% RH, and 30 °C and 80% RH for 30 weeks. Chemical Analysis. Reducing Sugar. The amount of reducing sugar was determined by the 3,5-dinitrosalicylic acid (DNS) method.21 Approximatively 1.0 g of milled wheat sample was soaked in 10 mL of hexane for 3 h at room temperature, and then the mixture was centrifuged at 1000g for 10 min at 4 °C. Successively, 10 mL of ultrapure water was added to the precipitate, which was mixed and centrifuged. Next, 150 μL of the supernatant was pipetted into a 10 mL polypropylene tube and diluted to 800 μL with ultrapure water. Then, 600 μL of 4.4 mmol/L DNS solution was added, and the mixture was placed in a water bath (Prima YB12, Prima Instrument Company, Ltd., Shanghai, China) at 100 °C for 5 min. The sample was cooled down rapidly to room temperature and diluted to 10 mL with ultrapure water; then, the absorbance was measured at 540 nm. The water-soluble reducing sugar content was calculated using a glucose standard curve with 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, and 0.90 mg/mL. Fatty Acid Value. The fatty acid value was detected according to previous studies.22 Briefly, 2.0 g of milled wheat sample was extracted with 10 mL of benzol for 30 min; after centrifugation, 5.0 mL of supernatant was mixed with 5.0 mL of ethanol (containing 0.04% phenolphthalein, v/v). The mixture was well shaken and immediately titrated with 0.01 mol/L KOH−ethanol solution until it turned

reddish and did not fade for 30 s. The fatty acid value was expressed in milligrams of KOH that was required to neutralize the fatty acids in 100 g of dry weight sample. Soluble Proteins. According to the method of Fang et al.,23 1.0 g of milled wheat was soaked in 10 mL of hexane. After being air-dried, the defatted wheat flour was extracted with 10 mL of distilled water in a water bath at 50 °C for 0.5 h and then centrifuged at 4000g for 20 min to get the albumin. Following water extraction, the flour was extracted with 10 mL of 10% NaCl at 20 °C for 3 h to get the globulin. Glutelin was then extracted from the sediment with 10 mL of 0.02 M NaOH at 20 °C for 1 h; this was followed by gliadin extraction with 10 mL of 75% ethanol at 20 °C for 3 h. The contents of four soluble proteins were determined by the Bradford method.24 Moisture Content. The moisture content of the sample was detected by an HX204 rapid moisture meter (Mettler Toledo Instruments Company, Ltd., Shanghai, China). Microbial Community Analysis. Wheat grain surface microbes were extracted with sterilized water under aseptic operating conditions. The DNA of the total fungi in the sample was extracted with a Fungal Isolation DNA Kit (Sangon Biotech Company, Ltd., Shanghai, China), and then the extracted DNA was evaluated using a 1% agarose gel. For ITS2 sequencing, the DNA samples were sent to C


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Figure 3. Microbial community distribution in wheat under different storage conditions by heatmap (a) and changes in the relative abundance of each community (b). The top 5 communities at genus level with an average relative abundance >1% are shown. The original sample is marked as 0 Day; 18WA, 18WB, 18WC and 18WD represent the sample stored at 15 °C and 50% RH, 20 °C and 65% RH, 25 °C and 75% RH and 30 °C and 80% RH for 18 weeks; 30WA, 30WB, 30WC and 30WD represent the sample stored at 15 °C and 50% RH, 20 °C and 65% RH, 25 °C and 75% RH and 30 °C and 80% RH for 30 weeks. Different letters (A-E) differ significantly (p < 0.05) among a fungi at different storage conditions in 18 weeks; Different letters (a-e) differ significantly (p < 0.05) among a fungi at different storage conditions in 30 weeks; Significant differences (p < 0.05) in samples stored in same condition but different storage time are indicated by asterisk (*).

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Genesky Bio-Tech Company, Ltd. (Shanghai, China) at −20 °C and under dry ice conditions. High-throughput sequencing was performed on an Illumina Miseq platform with the 2 × 250 bp paired-end method. All the results were based on sequenced reads and operational taxonomic units (OTUs). Statistical Analysis. Statistical comparisons of data were estimated by one way single factor analysis of variance (ANOVA) and Duncan’s multiple-range test by using SPSS 20, and p < 0.05 was considered statistically significant. All the analysis was done in triplicate, and the data are expressed as means ± standard deviations (SD).

RESULTS Mycotoxin Content Changes under Different Simulated Storage Conditions. Mycotoxin changes in stored wheat under four artificial simulation storage conditions, including 15 °C and 50% RH, 20 °C and 65% RH, 25 °C and 75% RH, and 30 °C and 80% RH, are shown in Figure 1. Only one mycotoxin, DON, was detected in the original wheat sample, and its concentration was 383.1 μg/kg. As storage time prolonged, three additional mycotoxins were detected in the D


Article

Journal of Agricultural and Food Chemistry Table 1. Changes in the Chemical Substance of Wheat at 25 °C and 75% Relative Humidity for 30 Weeksa protein (mg/g) storage time (weeks) 0 6 12 18 24 30

moisture content (%) 10.74 13.30 12.71 15.05 14.18 17.13

± ± ± ± ± ±

0.51f 0.41d 0.71e 0.51b 0.67c 0.67a

reducing sugar (mg/g) 25.10 26.11 27.70 30.37 28.15 27.47

± ± ± ± ± ±

0.02d 0.04d 0.16a 0.71c 0.07b 1.18a

fatty acid value (mg of KOH per 100 g) 16.08 19.40 20.62 24.60 33.30 45.57

± ± ± ± ± ±

0.07f 0.59e 1.74d 1.38c 6.77b 8.55a

albumin 31.60 31.88 33.13 32.32 33.86 33.07

± ± ± ± ± ±

0.33c 0.96c 0.90a 1.44b 1.00a 0.54a

globulin 14.15 14.26 15.81 16.04 16.28 15.13

± ± ± ± ± ±

0.25c 0.59c 1.16b 0.15a 0.59a 0.91b

gliadin 42.00 41.24 39.46 38.46 36.53 35.63

± ± ± ± ± ±

1.67a 1.70a 0.48b 1.12c 0.54d 1.43e

glutenin 54.76 53.78 53.25 50.83 49.94 47.99

± ± ± ± ± ±

1.07a 0.72a 1.11a 0.87b 1.29b 0.64c

Data are expressed as means ± SD (n = 3). Different letters in the same row indicate significant differences at p < 0.05.

a

sixth week under all storage conditions: 3-AcDON, 15AcDON, and ZEN. At the 15 °C and 50% RH storage condition (Figure 1a), DON increased slowly, reaching a maximum content of 519.1 μg/kg in the 18th week, after which it remained unchanged (p > 0.05). Similar trends could be observed at the 20 °C and 65% RH (Figure 1b) and 30 °C and 80% RH storage conditions (Figure 1d), in which DON contents were relatively high in the 18th week, at 608.1 and 793.0 μg/kg, respectively. Additionally, at the 25 °C and 75% RH storage condition (Figure 1c), DON increased rapidly, and it reached the maximum level of 1103 μg/kg in the 30th week, exceeding the maximum limit of 1000 μg/kg. On the other hand, 3-AcDON, 15-AcDON, and ZEN concentrations were lower than that of DON, but there were different contents under different storage conditions. In the 30th week, the lowest values of 3-AcDON, 15-AcDON, and ZEN were observed at the 15 °C and 50% RH storage condition (Figure 1a), with contents of 50.97, 25.40, and 30.25 μg/kg, respectively; the highest values appeared at the 25 °C and 75% RH storage condition (Figure 1c), with contents up to 84.13, 47.21, and 42.14 μg/kg, respectively. DON was the mycotoxin with the highest frequency and highest concentration, and the 25 °C and 75% RH condition was the most suitable one for mycotoxin production. Overall Structural Changes and Diversity of Fungal Communities in Wheat. The original wheat sample is marked as “0 Day”, and samples that were stored at 15 °C and 50% RH, 20 °C and 65% RH, 25 °C and 75% RH, and 30 °C and 80% RH for 18 weeks are marked as 18WA, 18WB, 18WC, and 18WD, respectively. Similarly, 30WA, 30WB, 30WC, and 30WD represent the samples stored at 15 °C and 50% RH, 20 °C and 65% RH, 25 °C and 75% RH, and 30 °C and 80% RH for 30 weeks, respectively. The relative abundance (%) of each fungus at the class level is described in Figure 2a. Tremellomycetes (35.77%), Dothideomycetes (34.30%), and Sordariomycetes (16.17%) were predominant in the initial wheat sample. As the storage time increased, the fungal composition changed differently in the four storage conditions. In particular, at 25 °C and 75% RH and at 30 °C and 80% RH in the 30th week, the relative abundance of Eurotiomycetes was significantly increased compared with that of the original sample (from 0.57 to 7.1% at 25 °C and 75% RH and from 0.57 to 23.07% at 30 °C and 80% RH), whereas the other fungi, such as Wallemiomycetes, were significantly reduced (from 6.01 to 0.67% at 25 °C and 75% RH and from 6.01 to 0.72% at 30 °C and 80% RH). The overall differences in the fungal communities were compared at OTU level, and the results are shown in Figure 2. After 30 weeks of storage, the composition of fungus significantly changed at 25 °C and 75% RH and at 30 °C

and 80% RH compared with that of the 0 day sample. In addition, the compositions of the samples stored at 15 °C and 50% RH and at 20 °C and 65% RH were close to that of the 0 day sample on the basis of principal component analysis (PCA, Figure 2b). As shown in Figure 2c, the samples stored at 20 °C and 65% RH were most similar to the 0 day group in terms of wheat grain surface fungal composition, and the 15 °C and 50% RH group was the second most similar group to the 0 day group, whereas the 30 °C and 80% RH group was the most different from the 0 day group. The results were consistent with the results obtained by PCA. Moreover, it was obvious that Fusarium dominated the fungal communities at 25 °C and 75% RH from the heatmap of cluster stacking at the genus level (Figure 3a). Additionally, high temperature and humidity increased the relative abundances of Aspergillus and Penicillium; conversely, the relative abundance of Papiliotrema was significantly decreased at 25 °C and 75% RH and at 30 °C and 80% RH (Figure 3b). Changes of Biochemical Components in Wheat Stored under Simulated Conditions. Under 25 °C and 75% RH storage conditions, the composition of the fungi, especially Fusarium spp., in the wheat was significantly different from that of the initial wheat sample, and DON, 3-AcDON, 15-AcDON, and ZEN had the highest contents under these conditions. Therefore, we screened these conditions as the optimal ones for Fusarium growth and Fusarium mycotoxin production and further studied the changes in the chemical composition of wheat at 25 °C and 75% RH. Changes in moisture content, reducing sugar level, fatty acid value, and protein components over 30 weeks are shown in Table 1. The moisture content of the wheat fluctuated at 25 °C and 75% RH, with a maximum of 17.13% in the 30th week. The content of reducing sugar increased first, reached a maximum of 30.37 mg/g in the 18th week, and then decreased to 27.47 mg/g in the 30th week. Four soluble proteins changed differently, with increases for albumin and globulin but decreases for gliadin and glutenin. In addition, fatty acid values increased during storage, reaching the highest level of 45.57 mg of KOH per 100 g in the 30th week.

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DISCUSSION Dynamic changes of mycotoxin in wheat were greatly affected by storage conditions. Although four mycotoxins (DON, 3AcDON, 15-AcDON, and ZEN) were detected simultaneously, DON was the dominant mycotoxin, which was consistent with other studies.25,26 The production of DON in naturally harvested wheat stored under high temperature and humidity conditions was more accelerated compared with that in wheat stored under low temperature and humidity conditions. This may be related to the optimal conditions for E


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Figure 4. Heatmap of deoxynivalenol (DON) contents, chemical substances, and microbiota correlation coefficients in wheat. The top 10 microbial communities at the species level with average relative abundances >1% are shown. Colors represent the correlation coefficients, with orange indicating a positive correlation and green indicating a negative correlation. Statistically significant correlations (p < 0.05) are indicated with asterisks (*).

graminearum could be detected.38 Hasan et al. conducted a field control trial of Fusarium head blight by using a screened strain. The results showed that Trichoderma harzianum could effectively inhibit infection with F. graminearum.39 Elucidation of the antifungal mechanisms of antagonistic strains is still in its infancy. Most researchers believe that some cell wall degrading enzymes and secondary metabolites such as antibiotics are produced by antagonistic strains against pathogenic fungi and thus control Fusarium head blight.40 In our study, the abundance of Papiliotrema decreased rapidly, whereas that of Fusarium sharply increased at 25 °C and 75% RH; this was accompanied by an increase in DON content. Therefore, F. graminearum may inhibit the growth of P. f lavescens under these conditions, and the effects of environmental conditions should also be considered when applying biological antagonists. Wheat infected with F. graminearum not only brings mycotoxins but also declines in quality. No significant correlation was found between moisture content and the presence of these fungi, and this may be due to the fluctuations caused by the interactions between high humidity and wheat physiological activities. However, there is no doubt that an increase in moisture content in wheat is conducive to the growth of fungi, resulting in quality deterioration. The carbohydrates in wheat are mainly polysaccharides, such as starch, cellulose, and sucrose, and carbohydrate changes in wheat are mainly changes in the contents of soluble sugars. In the present study, the content of reducing sugar in wheat first increased and then decreased (Table 1). This may be due to the fact that most of the molds have strong physiological activity in the early stage of storage, and starch can be decomposed into reducing sugars by hydrolase. As the storage time was extended, the reducing sugar was used in the respiration of the wheat itself or by the microorganisms, resulting in a decrease in the reducing sugar content. The fatty acid value is used as an indicator of cereal deterioration, and it increased in this study in the high-risk storage conditions (25

microbial growth and toxin production. According to Wu et al., who studied the optimal conditions for toxin production of Fusarium graminearum (F. graminearum) originating in China, the maximum DON level was obtained when the fungi was cultivated at 24 °C.27 Similarly, the highest DON contents and the highest relative abundance of Fusarium spp. were at 25 °C and 75% RH in our study. Obviously, it was beneficial to slow down the production of DON at both of 15 °C and 50% RH and at 20 °C and 65% RH in our study. At the same time, Aspergillus and Penicillium become the dominant fungi, and the relative abundance of Fusarium decreased at 30 °C and 80% RH (Figure 3). Aspergillus and Penicillium species can grow at higher temperatures than Fusarium species,28 and these storage fungi could negatively influence the survival of field fungi (Fusarium), according to the study of Scariot et al.29 However, these fungi can also produce mycotoxins, such as aflatoxin and ochratoxin A.30 Therefore, the temperature and humidity should be kept as low as possible during wheat storage. In addition to the temperature and humidity conditions affecting mycotoxin production, the complicated microbial environment of the sample may also have an impact on it. As shown in Figure 4, F. graminearum and DON were significantly positively correlated at 25 °C and 75% RH, but interestingly, a significant negative correlation was observed between DON and Papiliotrema f lavescens (P. f lavescens). Studies have shown that the use of P. f lavescens alone31−33 or in combination with other biocontrol agents34,35 can reduce the incidence of Fusarium head blight and DON in greenhouse and field environments. In other studies, doses ranging from 5 × 107 to 5 × 108 CFU/mL of this yeast had reducing effects but were not stable.36,37 On the other hand, many fungi have also been developed as biological control agents for Fusarium head blight. Palazzini et al. tested the control effects of Clonostachys rosea on Fusarium head blight in two different regions of Argentina for 2 consecutive years. The results showed that after 90 days, this fungus could reduce the content of F. graminearum by 73%, and after 180 days, almost no F. F


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Journal of Agricultural and Food Chemistry °C and 75% RH). Additionally, there was a significant correlation between the fatty acid value and Aspergillus flavus (A. f lavus). The reason for this result may be that the lipase in wheat is activated by the actions of microorganisms, and thus the fat in wheat is hydrolyzed into glycerol and fatty acid.4 With the extension of storage time, the fatty acid production rate is greater than the loss rate, and fatty acids are gradually accumulated. Therefore, A. flavus was probably the main factor causing rancidity of fats in wheat. Wheat protein is mainly composed of albumin, globulin, gliadin, and glutenin. Gliadin and glutenin are the main components of gluten, the main factor in dough viscoelasticity, and they play an important role in the evaluation of wheat nutritional quality.41 In the present study, albumin and globulin increased, but gliadin and glutenin decreased (Table 1). Albumin and globulin are mainly enzyme proteins in wheat grains, including α-amylase, protease inhibitor, regulatory enzymes, and synthetases of special pathways, among others, and their roles are to regulate various metabolic processes in wheat. The increases in albumin and globulin may be due to infection with Fusarium, which affects some synthetic catabolism in wheat grains. The change in gliadin was consistent with the results of Dexter’s study.42 However, another study found that the contents of gliadin in wheat infected by Fusarium increased.43 Furthermore, glutenin was significantly negatively correlated with F. graminearum (Figure 4), indicating a preference of F. graminearum for glutenin. Previous studies have reported that Fusarium proteases are mainly trypsin-like serine proteases that can cut lysines or arginines on proteins.44,45 Eggert et al. investigated the effects of Fusarium on wheat proteins in an in vitro model system and found that F. graminearum proteases showed a preference for glutenin compared with gliadin.43 Therefore, cereal mildew is actually the result of interactions between microorganisms and the storage environment. Undesirable storage temperature and humidity cause changes in fungal communities, subsequently resulting in mycotoxin production and quality decline, but the contributions of various fungi are different. In conclusion, this study highlights the interactions of various factors that influence Fusarium infection and mycotoxin production during storage. It is more beneficial to reduce infection at lower storage temperatures and humidity. On the other hand, field fungi (especially Fusarium) and stored fungi (especially Penicillium and Aspergillus) have different suitable storage conditions. Notably, during storage, F. graminearum exhibits a tendentious consumption of glutenin, whereas A. f lavus is related to the rancidity of fats. In the future, it is necessary to develop multiple crucial control points for prevention of mildew in grain during the storage process to reduce economic loss and health risks.

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Jiangsu Agriculture Science and Technology Innovation Fund (CX(17)1003, CX(182023)), the National Research of Quality and Safety Risk Assessment for Grain and Oil Products of China (GJFP2018001), the National Youth Talent in Grain Area Support Program of China, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Notes

The authors declare no competing financial interest.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Y.F.). ORCID

Yong Fang: 0000-0001-8310-5673 Fei Shen: 0000-0002-8749-7472 Author Contributions ⊗

Yingyue Zhang and Fei Pei contributed equally to this work.

Funding

This study was supported by the National Key Research and Development Program of China (2016YFD0401203), the G


Article

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