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Distribution and variation of fungi and major mycotoxins in pre- and post-nature drying maize in North China Plain
Accepted Manuscript Distribution and variation of fungi and major mycotoxins in pre- and post-nature drying maize in North China Plain
Fuguo Xing, Xiao Liu, Limin Wang, Jonathan Nimal Selvaraj, Nuo Jin, Yan Wang, Yueju Zhao, Yang Liu PII:
S0956-7135(17)30136-6
DOI:
10.1016/j.foodcont.2017.03.055
Reference:
JFCO 5595
To appear in:
Food Control
Received Date:
06 January 2017
Revised Date:
12 March 2017
Accepted Date:
14 March 2017
Please cite this article as: Fuguo Xing, Xiao Liu, Limin Wang, Jonathan Nimal Selvaraj, Nuo Jin, Yan Wang, Yueju Zhao, Yang Liu, Distribution and variation of fungi and major mycotoxins in preand post-nature drying maize in North China Plain, Food Control (2017), doi: 10.1016/j.foodcont. 2017.03.055
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ACCEPTED MANUSCRIPT Highlights
Fungi infection was significantly higher in pre-drying samples than post-drying.
F. verticillioides and F. graminearum were predominant species in maize kernels.
FB1 and DON were the major mycotoxins presented in the samples, followed by ZEN.
DON contamination was significantly higher in post-drying samples than pre-drying.
Occurrence of mycotoxins is highly in accordance with incidence of relevant fungi.
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Title:
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Distribution and variation of fungi and major mycotoxins in pre- and
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post-nature drying maize in North China Plain
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Author names and affiliations:
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Fuguo Xing1*, Xiao Liu1, Limin Wang, Jonathan Nimal Selvaraj, Nuo Jin, Yan Wang,
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Yueju Zhao, Yang Liu*
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Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences
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/Key Laboratory of Agro-Products Processing, Ministry of Agriculture, Beijing 100193,
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P. R. China
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1These
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*Corresponding Author
authors contributed equally to this work.
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Abstract
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Forty-four pre- and post-nature drying maize kernels were collected from North China
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Plain and assessed for fungi infection and mycotoxins contamination. The percentage of
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fungi infection was significantly higher in pre-nature drying samples than post-nature
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drying samples except for Fusariuim graminearum, which increases from 6.06% to
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24.09%. Fusarium, Aspergillus, Alternaria and Trichoderma were main genera.
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Fusarium verticillioides (24.77%) and F. graminearum (15.08%) were predominant
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species, followed by Aspergillus niger (7.51%) and Aspergillus flavus (4.93%). FB1 and
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DON were the major mycotoxins presented in the samples, followed by ZEN. All
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samples showed FB1 ranging from 16.5 to 315.9 μg/kg. All post-nature drying maize
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kernels showed DON ranging from 5.8 to 9843.3 μg/kg, while 7 of 22 pre-nature drying
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samples contaminated with DON ranging from 50.7 to 776.6 μg/kg. The samples
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contaminated with ZEN in pre- and post-nature drying maize were 3, with the content
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ranging from 60.5 to 147.6 μg/kg and from 40.7 to 1056.8 μg/kg, respectively. Only 1
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sample contaminated with AFB1 of 148.4 μg/kg. The occurrence of mycotoxins is highly
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in accordance with the incidence of the corresponding mycotoxin-producing fungi. This
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is the first comprehensive comparison of fungi infection and mycotoxins contamination
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between pre- and post-nature drying maize kernels.
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Key words: Maize kernels; Pre- and post-nature drying; Fungi; Mycotoxins
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1. Introduction
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Maize (Zea mays L.) is one of the most widely grown cereal crops, which is a staple
37
food in many regions around the world, and large quantity of maize is produced each year
38
than any other grain (International grains council market report, 2013). The United States
39
produces 40% of the world’s maize harvest and other top producing countries include
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China, Brazil, Mexico, Indonesia, India, France and Argentina. Global production was
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963 million tons in 2014, which is more than wheat (719 million tons) and rice (495
42
million tons). In China, the annual maize production was 216 million tons (data from
43
National Bureau of Statistics of China) which was more than rice (206 million tons) and
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wheat (126 million tons). In China, approximately 70% of produced maize is used as
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livestock feed. In addition, maize is increasingly used as a raw material for ethanol
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production and produces a greater quantity of biomass than other cereal plants, which is
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used for fodder.
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The Food and Agriculture Organization (1995) reported that approximately 25% of
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the cereal-based foods produced in the world are contaminated with mycotoxins
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produced by filamentous fungi as secondary metabolites as they contaminate the foods.
51
After discovering the hazardous nature of aflatoxin in early 1960s, many countries began
52
conducting surveys on the occurrence of mycotoxins in their agricultural products in last
53
few decades (Cui et al., 2013; Selvaraj et al., 2015). Some key mycotoxins that cause
54
huge health issues and agricultural loss are aflatoxins, ochratoxins, trichothecenes,
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zearalenone, fumonisins, tremorgenic toxins and ergot alkaloids. These mycotoxins are
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mainly produced by the Fusarium, Aspergillus, Penicillium and Alternaria genera. The
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simultaneous presence of different mycotoxins in a single commodity produced by
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different fungal genera is not uncommon (Aresta, Cioffi, Palmisano, & Zambonin, 2003).
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Among the grains, maize is highly contaminated with many fungi and the subsequent
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mycotoxins.
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Numerous fungi infect maize, approximately 19 genera of fungal species, among
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them Fusarium, Aspergillus, Alternaria, Penicillium and Stenocarpella were the main
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genera (Payne, 1999). These fungi produce mycotoxins that can cause toxic and/or
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carcinogenic effects in humans and animals. Fusarium species produce many mycotoxins
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such
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Fusarium contamination on maize in temperate and semi-tropical areas are very common
67
as they cause ear rot, which affects the crop yield to a greater extent. Aspergillus species
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can cause the food to spoil leading to enormous economic loss. Furthermore, some
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Aspergillus species can produce toxic secondary metabolites: like aflatoxins and
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ochratoxins which affect the food safety (Marín, Ramos, Cano-Sancho, & Sanchis, 2012).
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Recent studies show that change in climatic trends may pose longer-term impacts on
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the distribution of fungi, the occurrence of mycotoxins and host crop plants (Wu et al.,
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2011). However, apart from weather parameters, agronomic factors such as drying
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methods and time may affect the nature and degree of mycobiota contamination and the
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occurrence of mycotoxins in the grains (Storm, Sørensen, Rasmussen, Nielsen, & Thrane,
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2008). Drying makes it easy for farmers to bring down the moisture content to a safe
77
level as a result, the produce, with sufficiently low water content restrains fungal invasion,
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spore germination and fungal growth.
as
zearalenone,
fumonisins
and
trichothecenes.
Occurrences
of
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The aim of this study was to evaluate the mycoflora and the occurrence of main
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mycotoxins in maize before and after drying. As fungal variation mainly occurs during
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drying process, it was assumed that the effective control of fungi must be taken, which
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influences the growth of toxigenic fungi and the production of mycotoxins during post-
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harvest period.
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2. Materials and methods
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2.1. Chemicals
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High performance liquid chromatography (HPLC) grade methanol and acetonitrile
87
were purchased from Fisher Scientific (Fisher Chemicals HPLC, USA). Aflatoxin B1
88
(AFB1), Zearalenone (ZEN), Deoxynivanol (DON) and Fumonisin B1 (FB1) standards
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were procured from Sigma-Aldrich Chemicals (USA). ToxinFast immunoaffinity
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columns for AFB1 and ZEN were purchased from HuaanMagnech Bio-tech (Beijing,
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China); MycosepTM 227 columns were from Romer Labs, Inc., (USA).
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2.2. Sample collection and preparation
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A total of 44 maize samples were collected from the local farmers of three major
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maize production provinces (Shandong, Hebei and Henan) in North China Plain in the
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autumn of 2014. The samples were taken from eight areas such as Southwestern
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(Nanyang), Southeastern (Linyi), Central (Liaocheng, Heze, Zibo, Xingtai and Handan),
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and Northern (Langfang) regions of North China Plain. The amount of samples from each
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place is 2 or 3 according to the maize yield. The detailed samples information was
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presented in Table 1. Of 44 maize samples, 22 samples of fresh maize without drying
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treatment in the October were collected, which were named as pre-drying samples. After
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2 months, other 22 samples were collected again from the same group of farmers. For
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each sample, at least ten incremental samples of 100 g each were taken and combined,
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according to EC 401/2006 norms (European Commission, 2006). All samples were taken 5
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up immediately for analyzing the mycobiota. Each 200 g of a sample was ground to a
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fine powder and stored at -20 ºC until further detection of four major mycotoxins.
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2.3. Determination of water activity (aw) and moisture content
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The aw and moisture content of kernels of maize were determined by automatic
108
analysis, using Aqualab 4TE (Decagon Devices, Pullman, WA, USA).
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2.4. Recovery, identification and enumeration of the mycoflora from kernels of maize
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From each sample, approximately 30 g subsamples of maize kernels was disinfected
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by immersion in 1% sodium hypochlorite solution for 3 min, followed by three rinses
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with sterile distilled water. After disinfection, 60 kernels were randomly separated and
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directly seeded onto 5 Petri dishes (bottom diameter: 90 mm, 6 kernels per dish)
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containing Potato Dextrose Agar（PDA）and 5 Petri dishes (6 kernels per dish)
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containing Dichloran 18% Glycerol Agar (DG18) for the isolation of internal mycoflora
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(Pitt & Hocking, 2009). The plates were incubated at 25 ºC for 5 days, but the
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observations were made daily. The fungi grew from maize kernels internal. The average
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percentage of kernels infected with fungi was then calculated.
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Speciation of isolated fungal strains was performed according to Tournas, Rivera Calo,
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& Sapp (2013) with minor modifications. Recovered isolates was purified by re-culturing
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on PDA and identified to genus or species level using the conventional methods and keys
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described in “Fungi and Food Spoilage” (Pitt & Hocking, 2009), “Introduction to Food-
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and Airborne Fungi” (Samson, Hoekstra, & Frisvad, 2004), “Identification of Common
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Aspergillus species” (Klich, 2002), “Fusarium Species: An Illustrated Manual for
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Identification” (Nelson, Touson, & Marasas, 1983) and “A Laboratory Guide to Common
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Penicillium Species” (Pitt, 1985). Isolates which are not identified at the species level by
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different conventional plating methods were identified using molecular method as
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described below.
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2.5. Identification of isolated fungal strains using molecular method
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To extract genomic DNA, fungal isolates were grown in YES liquid medium
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inoculated with 1 × 105 spores. After cultivating at 200 rpm (28 ºC) for 5 days, mycelia
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were collected by filtration through Whatman filter paper, and washed with distilled
133
water. Genomic DNA was isolated by using the Fungal DNA Kit (Omega Bio-Tek, Inc.
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Norcross, GA, USA) according to the manufacturer’s instructions. The extracted DNA
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was quantified by electrophoresis in 0.8% (w/v) agarose gels with 1 × TAE buffer
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containing 0.1‰ GelRed Nucleic Acid Stain (10,000 ×, Biotium, Bay Area, Cal, USA).
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DNA was diluted to a concentration of 50 ng/µl for further use in PCR reactions.
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Fungal universal primers ITS1 (5’ –TCCGTAGGTGAACCTGCGG-3’) and ITS4
139
(5’-TCCTCCGCTTATTGATATGC-3’) were used to amplify the ITS region of fungal
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rDNA. PCR reactions were carried out in a total volume of 30 µl consisting of 15 µl of
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Gotaq PCR Master Mix (Promega, USA), 1 µl of fungal genomic DNA, 1 µl of forward
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and reverse primer (10 µmol). PCR conditions were as follows: denaturation at 95 ºC for
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3 min, 35 cycles of denaturation at 95 ºC for 30 s, annealing at 55 ºC for 30 s, extension
144
at 72 ºC for 1min, and final extension at 72 ºC for 7 min. Amplification products were
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purified and sequenced. Basic Local Alignment Search Tool (BLAST) was used to
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identify the closest affiliated sequence in the GenBank/NCBI dataset.
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2.6. Determination of mycotoxins
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A 50 g sub-sample of maize kernels was finely ground with a grinder until they could
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pass through a 29 mm mesh screen, or they were rendered into a paste and stored at 4 ºC 7
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in suitable glass container before determination of mycotoxins.
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2.6.1 Determination of AFB1
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AFB1 in the maize powder was detected by the HPLC (high performance liquid
153
chromatography). The specific methodology and technology were refer to the method
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reported by Ding et al. (2015).
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2.6.2 Determination of DON
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Determination of DON in different maize samples was performed by HPLC according
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to the method reported by Cui et al. (2013) with minor modifications. Finely ground
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samples (5.0 g) were extracted with 25 ml of acetonitrile : water (84 : 16, v/v), and the
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mixtures were blended for 3 min at 10,000 rpm using a blender (IKA, ULTRA-TURRAX
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T25 digital, Germany). The extract was filtered through Whatman No.4 paper and 10 ml
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of the defatted extract was purified through BondElute column (Agilent, USA), according
162
to the manufacturer’s instructions. 2 ml of purified extract was transferred to another tube
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and evaporated to dryness under a steam of nitrogen at 40 ºC. The dry residue was
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dissolved and mixed well in 1 ml of HPLC diluting solution (acetonitrile : methanol :
165
water, 5 : 5 : 90, v/v/v), after centrifuged at 12,000 rpm for 6 min, and was taken up
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further for HPLC/UV analysis.
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HPLC analysis was performed by using Waters 2695 with a Waters 2478 UV detector
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(220 nm) according to the method reported by Cui et al. (2013).
169
2.6.3 Determination of ZEN
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Determination of ZEN in different maize samples was performed by HPLC according
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to the method reported by Langseth, Ellingsen, Nymoen & Okland (1989) and Bakan,
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Melcion, Richard-Molard & Cahagnier (2002) with minor modifications. Finely ground
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samples (5.0 g) were extracted with 25 ml of methanol : water (80 : 20, v/v), and the
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mixtures were shaken for 30 min at 200 rpm. The extract was filtered through Whatman
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No. 4 paper and 5 ml of filtered extract was diluted into 25 ml with deionized water.
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Then the dilution was passed through immunoaffinity columns (ToxinFast Columns, Cat.
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No. HCM0525, Huaan Magnech Biotech, Beijing, China) with a flow rate of one droplet
178
per second, and was eluted with 2 ml of methanol into glass tubes. The purified extract
179
was quantified by HPLC-FD.
180
HPLC analysis was performed by using Waters 2695 coupled to a Waters 2475
181
fluorescence detector (λexc 360 nm; λem 440 nm) and post-column derivation system,
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and an Agilent TC-C18 column. The mobile phase (acetonitrile : water, 60 : 40) was
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pumped at a flow rate of 0.5 ml/min. ZEN (Sigma-Aldrich, St. Louis, MO, USA) was
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used as standards. Quantification of ZEN levels was performed by the measurement of
185
peak area compared with the standards solutions used for the calibration curve. LOD
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(limit of detection) was 1 µg/kg samples.
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2.6.3 Determination of FB1
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Determination of FB1 in different maize samples was performed by HPLC according to
189
the method reported by González Pereyra et al. (2008) with minor modifications. Finely
190
ground samples (5.0 g) were extracted with 20 ml of methanol : water (3 : 1, v/v), and the
191
mixtures were shaken for 30 min at 200 rpm. The extract was filtered through Whatman
192
No. 4 paper. 10 ml of the filtered extract was passed through immunoaffinity columns
193
(ToxinFast Columns, Cat. No. HCM0825, Huaan Magnech Biotech, Beijing, China) with
194
a flow rate of one droplet per second, and was eluted with 4 ml of 0.5% acetic acid in
195
methanol. The eluate was evaporated to dryness, redissolved in acetonitrile : water (1 : 1,
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v/v) and analysed for FB1 by HPLC using the methodology proposed by Shephard,
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Sydenham, Thiel & Gelderblom (1990) and modified by Doko, Rapior, Visconti &
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Schjoth (1995). An aliquot (50 μl) of this solution was derivated with 200 μl of o-
199
phthaldialdehyde (OPA). The OPA solution was obtained by adding 5 ml of 0.1 mol/l
200
sodium tetraborate and 50 μl of 2-mercaptoethanol to 1 ml of methanol containing 40 mg
201
of OPA. The FB1 OPA derivatives (20 μl solution) were analyzed using Waters 2695
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coupled to a Waters 2475 fluorescence detector (λexc 335 nm; λem 440 nm), and an
203
Agilent TC-C18 column. The mobile phase (methanol : 0.1mol/l sodium dihydrogen
204
phosphate) was pumped at flow rate of 0.7 ml/min. FB1 (Sigma-Aldrich, St. Louis, MO,
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USA) was used as standard. Quantification of FB1 levels was performed by the
206
measurement of peak area compared with the standards solutions used for the calibration
207
curve. LOD (limit of detection) was 1µg/kg samples.
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2.7. Statistical analysis
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The data were analyzed with the SAS (statistical analysis software) program using
210
analysis of variance (ANOVA) for multiple comparisons followed by the Turkey test.
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The mycotoxins concentrations were expressed as percentage and mean ± RSD.
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3. Results and discussion
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3.1. Water activity (aw) and moisture content in pre- and post-nature drying maize
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Table 2 showed that the aw of pre-drying maize was higher than 0.90 except some
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samples (No. 6, 8, 18, 20) and the average value of aw was 0.917. Except some samples
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(No. 3, 9, 10, 11, 13, 14, 15), aw of post-drying maize was lower than 0.70 which was
217
below the minimum range (0.70-0.80) required for the growth A. flavus (Hocking, 2007)
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and the minimum range (0.80-0.90) required for aflatoxins production (Passone, Rosso,
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& Etcheverry, 2012). Furthermore, the moisture contents of most maize kernels were
220
under the standard of storage maize in China (≤14%) and were confirmed safe for long-
221
term storage (Magan & Aldred, 2007a, b).
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3.2. Distribution and variation of mycobiota in pre- and post-nature drying maize
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As shown in Fig. 1, the percentage of kernels infected by fungi was significantly
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higher in pre-nature drying maize than in post-nature drying maize. For the former, the
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average percentages of kernels infected by fungi on PDA medium and DG18 medium
226
were 90.76% and 86.52%, respectively. For post-nature drying maize, the average
227
percentages of kernels infected by fungi on PDA and DG18 were 59.24% and 63.33%,
228
respectively. In maize with high aw, the percentage of kernels infected by fungi was
229
higher on PDA than on DG18. While for maize with low aw, the percentage of kernels
230
infected by fungi was lower on PDA than on DG18. The results suggested DG18 is more
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adequate for the numeration of fungi in food with low aw than PDA and confirmed that
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DG18 is medium for xerophilic fungi (Samson, Hoekstra, & Frisvad, 2004; Pitt &
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Hocking, 2009).
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As shown in Table 3, in the maize kernels the following mycobiota were isolated on
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DG18: F. verticillioides (24.77%), F. graminearum (15.08%), Fusarium spp. (5.54%), A.
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flavus (4.93%), A. niger (7.51%), Penicillium spp. (3.86%), Eupenicilliium sp. (1.82%),
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Alternaria sp. (3.57%), Trichoderma sp. (2.20%). Of the Fusarium, F. verticillioides and
238
F. graminearum were predominant and the most important because of their known
239
toxigenic potential. F. verticillioides and F. graminearum were the predominant fungal
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species detected in maize, and were well-known fumonisin-producing species (Thiel et
241
al., 1991) and deoxynivalenol-producing species (Snijders, 1990), respectively. This
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result is in accordance with the finding of Bakan, Melcion, Richard-Molard & Cahagnier
243
(2002).
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The results showed that fungal genera occurring in pre-nature drying maize kernels
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were Fusarium, Aspergillus, Alternaria and Penicillium, similar with many other studies
246
(Streit et al., 2012). Fewer fungi were recovered from the post-nature dried maize kernels
247
than pre-nature drying ones. Except for F. graminearum, the percentages of kernels
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infected by fungal species in pre-nature drying maize were higher than that in post-nature
249
dried maize. The results suggested that the value of aw significantly influenced the
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percentages of kernels infected by fungi, especially Aspergillus spp. Of the Aspergillus, A.
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flavus was the dominant fungi isolated from maize kernels. Many studies have reported
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that high temperature and drought stress directly favor the growth, conidiation, dispersal
253
of A. flavus (Cotty & Jaime-Garcia, 2007; Payne & Widstrom, 1992). Abdel-Hadi,
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Schmidt-Heydt, Parra, Geisen & Magan (2012) summarized the optimum aw for the
255
growth of A. flavus is 0.90-0.99, while the sporulation, germination, and growth of F.
256
verticillioides are optimized at 25-30 ºC and aw 0.70-0.80 (Vittorio, Andrea, & Paola,
257
2009). Furthermore, F. verticillioides has been reported as a species that infects all the
258
stages of plant development, infecting the roots, stalks and kernels (Munkvold, McGee,
259
& Carlton, 1997). So the contamination of F. verticillioides was higher than that of A.
260
flavus in the maize samples.
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In pre-nature drying maize kernels, the predominant fungi species was F.
262
verticillioides, and the percentage of kernels infected by F. verticillioides was higher than
263
27%. The result was in accordance with the earlier published works (Kedera, Plattner, &
264
Desjardins, 1999). The Fusarium species produces large quantities of microconidia,
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which most probably allow them to disseminate and easily contaminate maize ears in the
266
field. In post-nature drying maize kernels, the predominant fungi species was F.
267
graminearum, the percentage of kernels infected by it was higher than 24%. The result
268
suggested that the growth of F. graminearum is rapid and easily contaminated maize
269
kernels during nature drying process. This means that mycotoxins (DON and ZEN)
270
produced by the Fusarium species should be emphasized after harvest. The result is
271
inconsistent with other researchers who considered that DON was mainly produced
272
before harvest (Bensassi, Zaied, Abid, Hajlaoui, & Bacha, 2010; Bottalico & Perrone,
273
2002).
274
3.3. Occurrence of major mycotoxins in pre- and post-nature drying maize kernels
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The mycotoxins levels in pre- and post-nature drying maize kernels are presented in
276
Table 5. FB1 and DON were the major mycotoxins present in the samples. All pre- and
277
post-nature drying maize kernels tested were found to contain FB1, with the mean level of
278
132.8 μg/kg and 100.2 μg/kg, respectively. FB1 concentrations ranged from 16.5 to 315.9
279
μg/kg for pre-nature drying maize and from 17.2 to 220.6 μg/kg for post-nature drying
280
maize, which are lower than the recommended standard of US Food and Drug
281
Administration (FDA) (2000 μg/kg). All post-nature maize kernels tested were found to
282
contain DON with the mean level of 1581.2 μg/kg, which is significantly higher than the
283
limit standard (1000 μg/kg) of China and US FDA. While only 7 of 22 pre-nature maize
284
kernels contaminated with DON ranged from 50.7 to 776.6 μg/kg. This result suggested
285
that DON could be produced during the nature drying process. The results of FB1 and
286
DON contamination are also in accordance with the results of the mycological analysis,
287
because F. verticillioides and F. graminearum, which were the predominant fungal
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species detected, are well-known fumonisin-producing species (Thiel et al., 1991) and
289
DON-producing species, respectively.
290
The frequencies of AFB1 and ZEN were low in all the samples. For pre-nature
291
drying maize kernels, only one sample (No. 18) was found to contain AFB1 with the
292
concentration of 148.4 μg/kg, which is more than 7-folds of the Chinese standard limit of
293
20μg/kg. For post-nature drying maize kernels, AFB1 was undetectable and the
294
contamination rates of A. flavus on DG18 medium in all the samples except No. 4, 5 and
295
18 were zero or lower than 5%. The result mainly be attributed to low aw (< 0.70, Table 2)
296
of most samples which was below the minimum range of 0.70-0.80 established for the
297
growth of A. flavus (Hocking, 2007) and the minimum range of 0.80-0.90 for aflatoxin
298
production (Passone et al., 2010, 2012). Furthermore, the mean temperature ranged from
299
9-27 ºC and the RH ranged from 49 to 73% in the collection location in the autumn. Thus,
300
the temperature was below 32-33 ºC considered to be the optimum temperature for the
301
growth of A. flavus by Hocking (2007), the RH was lower than 83-85% considered to
302
favor the growth of A. flavus (Oyeka, 2004). Liu, Gao & Yu (2006) obtained the similar
303
results. They found that the average content in maize kernels of Liaoning province in
304
China was found to be very low (0.99 μg/kg) although almost all samples collected
305
contained aflatoxins.
306
For pre-nature drying maize kernels, three samples were found to contain ZEN
307
ranging from 60.5 to 147.6 μg/kg, which all exceed the Chinese regulatory limit of 60
308
μg/kg. For post-nature drying maize kernels, also three samples were found to contain
309
ZEN ranging from 40.7 to 1056.8 μg/kg, ZEN levels in two samples heavily exceed the
310
Chinese regulatory limit. The occurrence of ZEN in this study was lower than that of a 14
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previous publication based on investigation in North Asia (Binder, Tan, Chin, Handl, &
312
Richard, 2007). In that report, ZEN occurrence was detected in 47.0% for different
313
commodities, feeds and feed ingredients with a much higher average concentration of
314
494.0 μg/kg. Recent studies by Li et al. (2014) for the presence of AFB1, OTA, DON and
315
ZEN in cereal and oil products from the Yangtze Delta region showed that ZEN was the
316
major contaminants in the region and maize products had the highest incidences of ZEN
317
contaminations with the incidence at 35.7%, which is significantly higher than the
318
occurrence of ZEN in this study. A similar study by Ji, Xu, Liu, Yin, & Shi (2014) on
319
wheat samples from Jiangsu province of Yangtze Delta region harvested during 2010-12
320
showed 74% and 12.8% contamination with DON and ZEN, respectively. These results
321
show that ZEN has become a serious safety problem in cereal although the incidence of
322
ZEN contaminations in North China Plain is significantly lower than that in Yangtze
323
Delta region.
324
4. Conclusions
325
This study demonstrated the distribution and variation of fungi and four major
326
mycotoxins in pre- and post-nature drying maize samples collected from North China
327
Plain. The percentage of maize kernels infected by fungi was significantly higher in pre-
328
nature drying samples than that in post-nature drying samples except for F. graminearum,
329
which increase from 6.06% to 24.09%. In maize, Fusarium, Aspergillus, Penicillium,
330
Eupenicillium, Alternaria and Trichoderma were main genera. F. verticillioides and F.
331
graminearum were the predominant fungal species, followed by A. niger and A. flavus.
332
We found that aw played a significant role on the occurrence of fungi. FB1 and DON were
333
the major mycotoxins presented in the samples, followed by ZEN and AFB1. The DON
15
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contaminations in post-nature drying maize kernels were significantly higher than that in
335
pre-nature drying maize. The results of mycotoxins contamination are in accordance with
336
the results of the mycological analysis.
337
Acknowledgments
338
We gratefully acknowledge the financial support of National Key Research and
339
Development Program of China (2016YFD0400105), the National Program of China
340
Basic Science and Technology Research (2013FY113400), the National Natural Science
341
Foundation of China (31571938) and Fundamental Research Funds for Central Non-
342
profit Scientific Institution. The funders had no role in the study design, data collection
343
and analysis, decision to publish, or preparation of the manuscript.
344 345
Conflicts of Interest
346
The authors declare no conflict of interest.
347
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Table 1 The detailed samples information of maize Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
City
Province
Heze
Shandong
Liaocheng
Shandong
Zibo
Shandong
Xingtai
Hebei
Handan
Hebei
Langfang
Hebei
Nanyang
Henan
Linyi
Shandong
475
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Table 2 Water (aw) and moisture content of pre- and post-nature drying maize Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Average
aw Post-drying
Pre-drying
0.973±0.003 0.983±0.004 0.944±0.004 0.975±0.004 0.917±0.004 0.537±0.034 0.923±0.001 0.765±0.002 0.988±0.002 0.986±0.001 0.987±0.003 0.964±0.002 0.975±0.003 0.984±0.004 0.904±0.002 0.914±0.003 0.973±0.001 0.822±0.001 0.928±0.001 0.783±0.002 0.978±0.001 0.962±0.003
0.695±0.004 0.731±0.035 0.602±0.001 0.676±0.003 0.680±0.004 0.460±0.002 0.513±0.018 0.560±0.023 0.912±0.003 0.749±0.039 0.765±0.023 0.651±0.003 0.734±0.001 0.864±0.002 0.848±0.002 0.699±0.004 0.622±0.003 0.699±0.035 0.683±0.003 0.633±0.002 0.423±0.004 0.554±0.045
22.39±0.56 25.48±0.23 19.06±0.45 26.10±0.78 18.98±1.01 9.85±0.34 18.79±1.32 13.22±0.86 31.63±0.23 30.25±1.24 31.32±0.63 24.3±0.57 24.78±0.43 29.34±0.79 18.77±0.88 18.29±0.54 24.3±1.00 13.99±0.35 17.46±0.28 13.37±0.48 27.63±0.53 27.77±0.43
12.73±0.78 12.05±0.98 9.98±1.2 12.33±1.01 10.78±0.68 8.85±0.24 9.81±0.02 10.47±0.40 17.79±0.71 12.14±0.35 13.18±0.64 12.22±0.47 14.86±0.81 15.32±0.32 15.59±0.53 12.00±0.37 10.73±0.24 12.44±0.64 12.18±0.42 11.38±0.28 9.85±0.19 10.19±0.46
0.917
0.671
22.14
12.13
Pre-drying
478
24
MC(%) Post-drying
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479 480
Table 3 List of fungal species from pre- and post-nature drying maize and there isolation frequencies on DG18
Samples Pre Post 2 Pre Post Pre 3 Post 4 Pre Post Pre 5 Post 6 Pre Post Pre 7 Post Pre 8 Post 9 Pre Post 10 Pre Post 11 Pre Post 12 Pre Post 13 Pre Post 14 Pre Post 15 Pre Post 16 Pre Post 17 Pre Post 18 Pre Post 19 Pre Post 20 Pre Post 21 Pre Post 22 Pre Post Pre Av Post Total 1
Fva 33.33 33.33 40.00 60.00 6.67 13.33 70.00 16.67 56.67 ND ND ND 30.00 73.33 10.00 43.33 ND 30.00 6.67 26.67 6.67 3.33 66.67 ND 40.00 6.67 23.33 3.33 30.00 10.00 36.67 6.67 36.67 33.33 26.67 43.33 6.67 56.67 ND 23.33 26.67 3.33 46.67 3.33 27.27 22.27 24.77
Fusarium Fgb 6.67 33.33 3.33 16.67 ND 16.67 ND 33.33 ND 70.00 ND 3.33 ND 3.33 ND ND 20 53.33 46.67 20 ND 36.67 ND 6.67 ND 30.00 30.00 60.00 ND 60 6.67 36.67 6.67 ND 6.67 6.67 ND 20 ND 6.67 3.33 10 3.33 6.67 6.06 24.09 15.08
Fsc ND 3.33 43.33 10 ND 20 ND 3.33 ND ND 3.33 ND ND ND 10 ND ND ND 3.33 ND ND ND 3.33 ND ND ND 23.33 10 10 10 23.33 3.33 23.33 3.57 ND 6.67 ND ND ND ND 10 ND 20 ND 7.88 3.19 5.54
Aspergillus Afd Ane 3.33 36.67 3.33 ND ND 6.67 ND 3.33 ND 13.33 ND 3.33 ND 3.33 23.33 ND 3.33 6.67 6.67 3.33 6.67 10 ND 6.67 ND 33.33 ND ND 13.33 3.33 ND ND 10.00 ND ND 3.33 3.33 6.67 3.33 ND 3.33 6.67 ND ND ND ND ND ND ND ND ND ND 3.33 6.67 3.33 ND 6.67 26.67 ND ND 6.67 ND ND 6.67 6.67 16.67 3.57 3.57 10.00 16.67 6.67 ND 43.33 40 3.33 10 40.00 50 3.33 6.67 ND ND ND ND ND ND ND ND 7.27 12.88 2.59 2.13 4.93 7.51
% kernels infected by fungi Penicillium Eupenicillium Psf Esg 3.33 3.33 ND ND ND ND ND ND 3.33 ND 3.33 ND 3.33 ND ND 3.33 6.67 ND ND ND ND ND ND ND 3.33 3.33 10 3.33 6.67 3.33 3.33 ND ND ND ND ND 3.33 ND 3.33 ND 3.33 ND 3.33 ND 6.67 13.33 ND ND 3.33 23.33 50 ND 6.67 6.67 ND ND 13.33 6.67 ND ND ND ND 3.33 ND ND ND ND 3.57 6.67 ND ND ND 3.33 6.67 6.67 ND 3.33 3.33 6.67 ND ND ND ND ND 3.33 ND ND ND 3.64 3.18 4.09 0.47 3.86 1.82
25
Alternaria Ash ND 3.33 ND ND 26.67 ND 6.67 ND 6.67 ND 3.33 3.33 10 ND ND ND 3.33 3.33 10 3.33 43.33 ND 6.67 ND 6.67 ND ND 3.33 ND ND ND ND ND 3.57 ND 3.33 ND ND ND 6.67 3.33 ND ND ND 5.76 1.37 3.57
Trichoderma Tsi ND ND ND ND ND ND ND ND ND ND ND ND ND ND 16.67 3.33 ND ND ND ND ND ND 3.33 3.33 26.67 ND ND ND ND ND 10 ND ND ND 33.33 ND ND ND ND ND ND ND ND ND 4.09 0.30 2.20
Other 10 ND 3.45 3.57 25.00 5.56 ND 4.17 ND ND 36.36 33.33 14.29 3.57 36.67 6.25 37.50 ND ND 5.56 5.00 ND ND ND ND ND ND ND 6.67 ND 16.66 ND 3.33 ND ND ND ND ND 3.33 ND 10 ND 26.67 3.33 10.68 2.97 6.82
ACCEPTED MANUSCRIPT
481
a=Fusarium verticillioides, b=Fusarium graminearum, c=Fusarium sp., d=Aspergillus
482
flavus, e=Aspergillus niger, f=Penicillium sp., g=Eupenicillium sp., h=Alternaria sp.,
483
i=Trichoderma sp., Av=Average
484
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486 487 Samples 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Av
Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Total
Table 4 List of fungal species from pre- and post-nature drying maize and there isolation frequencies on PDA Fusarium Fva Fgb Fsc 36.67 3.33 30.00 30.00 53.33 ND 56.67 6.67 33.33 36.67 40.00 ND 63.33 10.00 13.33 10.00 23.33 ND 63.33 ND 13.33 33.33 40.00 ND 93.33 ND 6.67 30.00 6.67 ND 10.00 ND ND 3.33 ND ND 10.00 ND 6.67 80.00 ND ND 30.00 ND 13.33 40.00 ND 6.67 3.33 ND ND 6.67 36.67 ND ND 40.00 6.67 26.67 20.00 ND ND 3.33 ND 3.33 33.33 ND 46.67 ND 30.00 10.00 3.33 ND 53.33 3.33 10.00 46.67 33.33 ND 16.67 46.67 6.67 20.00 53.33 ND 63.33 ND ND 50.00 43.33 3.33 33.33 ND 26.67 33.33 13.33 3.33 60.00 ND 10.00 10.00 3.33 ND 6.67 ND ND 26.67 23.33 ND 10.00 26.67 3.33 26.67 13.33 ND 3.33 ND 6.67 16.67 10.00 6.67 53.33 ND 43.33 ND 10.00 3.33 ND 60.00 30.00 6.67 16.67 ND 32.42 9.09 13.18 24.85 21.67 1.06 28.64 15.38 7.12
Aspergillus Afd Ane ND 3.33 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 10.00 13.33 3.33 3.33 ND ND ND ND ND 10.00 ND ND 20.00 3.33 ND ND ND 6.67 3.33 0.00 13.33 13.33 ND ND 3.33 10.00 ND ND ND 3.33 ND 3.33 ND 3.33 ND ND ND ND ND ND ND ND ND ND ND ND ND ND 10.00 ND ND ND 43.33 13.33 6.67 ND 36.67 43.33 10.00 13.33 ND ND ND ND ND ND ND ND 6.21 5.61 1.06 0.91 3.64 3.26
% kernels infected by fungi Penicillium Eupenicillium Psf Esg 16.67 ND ND ND ND ND ND ND ND ND 3.33 ND ND ND 3.33 ND ND ND ND ND 16.67 ND 6.67 ND ND ND 3.33 ND ND 3.33 ND ND 6.67 ND ND ND ND ND ND ND ND 6.67 3.33 ND 3.33 ND 3.33 ND 13.33 13.33 3.33 ND ND 3.33 3.33 ND 6.67 ND ND ND ND ND 3.33 ND 3.33 ND ND ND ND ND ND ND ND ND ND ND 3.33 6.67 6.67 ND ND ND ND ND ND ND ND ND 3.18 1.52 1.82 0.00 2.50 0.76
27
Alternaria Ash ND ND ND ND ND 3.33 ND ND ND 3.33 ND ND ND ND ND ND 20.00 0.00 13.33 3.33 30.00 ND ND 6.67 ND ND ND ND ND ND 3.33 ND ND ND ND 3.33 ND ND ND 3.33 ND ND ND ND 3.03 1.06 2.05
Trichoderma Tsi ND ND ND ND ND ND 20.00 ND ND 46.67 33.33 ND ND ND 26.67 ND ND 23.33 ND ND ND ND ND ND ND ND 6.67 ND ND ND 36.67 ND ND ND 76.67 36.67 3.33 30.00 ND ND ND ND ND ND 9.24 6.21 7.73
Other 10.00 6.67 3.33 3.33 10.00 ND 3.33 6.67 ND ND 10.00 3.33 3.33 13.33 6.67 16.67 ND ND 3.33 ND 13.33 ND 3.33 3.33 3.33 10.00 16.67 ND 30.00 ND ND ND 26.67 3.33 ND 3.33 ND ND ND ND 3.33 ND 10.00 13.33 7.12 3.79 5.45
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a=Fusarium verticillioides, b=Fusarium graminearum, c=Fusarium sp., d=Aspergillus
489
flavus, e=Aspergillus niger, f=Penicillium sp., g=Eupenicillium sp., h=Alternaria sp.,
490
i=Trichoderma sp. Av=Average
28
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492 493 Samples 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Mean
Table 5 Mycotoxins levels (µg/kg) in samples from pre- and post-nature drying maize. AFB1 prepost148.4±22.0 -
Zen pre141.1±37.0 60.5±12.4 147.6±17.6 15.9
DON
post788.3±46.3 1056.8±267.9 40.7±8.3 85.7
pre279.8±69.8 226.5±45.4 102.1±27.6 107.6±38.8 776.6±98.7 232.7±73.2 50.7±12.3 80.7
494
29
post1503.7±504.1 210.7±64.7 4244.3±356.8 476.6±102.4 9843.3±780.4 8.7±4.7 11.0±8.9 5.8±6.5 792.2±356.4 818.4±367.8 771.9±459.2 47.4±7.0 938.3±278.5 6287.5±503.7 1873.0±620.6 4968.1±236.4 59.7±17.7 87.0±25.9 72.9±30.2 32.4±18.9 1627.6±423.5 105.7±32.3 1581.2
FB1 pre152.5±30.1 35.6 ±2.9 16.5 ±1.7 260.9 ±29.2 70.2±2.0 32.6 ±3.2 29.7 ±1.6 68.3 ±4.5 41.2 ±1.8 56.2 ±3.4 36.0 ±2.2 52.7 ±2.0 238.0 ±5.0 57.1 ±2.7 43.9±2.3 196.9 ±2.5 315.9 ±52.2 268.3 ±83.3 264.1 ±150.1 156.8 ±35.2 303.5 ±82.2 224.7 ±69.0 132.8
post162.9 ±47.6 195.6 ±85.5 33.0 ±3.9 86.2 ±4.7 32.6 ±1.6 17.2 ±1.9 162.7 ±24.2 46.5 ±15.5 52.7 ±9.3 135.1 ±33.3 23.6 ±7.9 40.2 ±6.4 101.5 ±3.3 73.3 ±4.7 154.7±23.2 35.3 ±116.4 220.6 ±18.3 167.7 ±27.3 164.4 ±61.3 128.1 ±26.3 133.3 ±65.1 36.6 ±12.2 100.2
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Figure Legends Figure 1. Percentages of kernels infected by fungi in pre- and post-drying maize kernels.
30
Fuguo Xing, Xiao Liu, Limin Wang, Jonathan Nimal Selvaraj, Nuo Jin, Yan Wang, Yueju Zhao, Yang Liu PII:
S0956-7135(17)30136-6
DOI:
10.1016/j.foodcont.2017.03.055
Reference:
JFCO 5595
To appear in:
Food Control
Received Date:
06 January 2017
Revised Date:
12 March 2017
Accepted Date:
14 March 2017
Please cite this article as: Fuguo Xing, Xiao Liu, Limin Wang, Jonathan Nimal Selvaraj, Nuo Jin, Yan Wang, Yueju Zhao, Yang Liu, Distribution and variation of fungi and major mycotoxins in preand post-nature drying maize in North China Plain, Food Control (2017), doi: 10.1016/j.foodcont. 2017.03.055
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ACCEPTED MANUSCRIPT Highlights
Fungi infection was significantly higher in pre-drying samples than post-drying.
F. verticillioides and F. graminearum were predominant species in maize kernels.
FB1 and DON were the major mycotoxins presented in the samples, followed by ZEN.
DON contamination was significantly higher in post-drying samples than pre-drying.
Occurrence of mycotoxins is highly in accordance with incidence of relevant fungi.
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Title:
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Distribution and variation of fungi and major mycotoxins in pre- and
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post-nature drying maize in North China Plain
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Author names and affiliations:
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Fuguo Xing1*, Xiao Liu1, Limin Wang, Jonathan Nimal Selvaraj, Nuo Jin, Yan Wang,
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Yueju Zhao, Yang Liu*
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Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences
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/Key Laboratory of Agro-Products Processing, Ministry of Agriculture, Beijing 100193,
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P. R. China
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1These
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*Corresponding Author
authors contributed equally to this work.
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Abstract
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Forty-four pre- and post-nature drying maize kernels were collected from North China
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Plain and assessed for fungi infection and mycotoxins contamination. The percentage of
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fungi infection was significantly higher in pre-nature drying samples than post-nature
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drying samples except for Fusariuim graminearum, which increases from 6.06% to
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24.09%. Fusarium, Aspergillus, Alternaria and Trichoderma were main genera.
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Fusarium verticillioides (24.77%) and F. graminearum (15.08%) were predominant
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species, followed by Aspergillus niger (7.51%) and Aspergillus flavus (4.93%). FB1 and
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DON were the major mycotoxins presented in the samples, followed by ZEN. All
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samples showed FB1 ranging from 16.5 to 315.9 μg/kg. All post-nature drying maize
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kernels showed DON ranging from 5.8 to 9843.3 μg/kg, while 7 of 22 pre-nature drying
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samples contaminated with DON ranging from 50.7 to 776.6 μg/kg. The samples
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contaminated with ZEN in pre- and post-nature drying maize were 3, with the content
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ranging from 60.5 to 147.6 μg/kg and from 40.7 to 1056.8 μg/kg, respectively. Only 1
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sample contaminated with AFB1 of 148.4 μg/kg. The occurrence of mycotoxins is highly
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in accordance with the incidence of the corresponding mycotoxin-producing fungi. This
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is the first comprehensive comparison of fungi infection and mycotoxins contamination
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between pre- and post-nature drying maize kernels.
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Key words: Maize kernels; Pre- and post-nature drying; Fungi; Mycotoxins
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1. Introduction
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Maize (Zea mays L.) is one of the most widely grown cereal crops, which is a staple
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food in many regions around the world, and large quantity of maize is produced each year
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than any other grain (International grains council market report, 2013). The United States
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produces 40% of the world’s maize harvest and other top producing countries include
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China, Brazil, Mexico, Indonesia, India, France and Argentina. Global production was
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963 million tons in 2014, which is more than wheat (719 million tons) and rice (495
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million tons). In China, the annual maize production was 216 million tons (data from
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National Bureau of Statistics of China) which was more than rice (206 million tons) and
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wheat (126 million tons). In China, approximately 70% of produced maize is used as
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livestock feed. In addition, maize is increasingly used as a raw material for ethanol
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production and produces a greater quantity of biomass than other cereal plants, which is
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used for fodder.
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The Food and Agriculture Organization (1995) reported that approximately 25% of
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the cereal-based foods produced in the world are contaminated with mycotoxins
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produced by filamentous fungi as secondary metabolites as they contaminate the foods.
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After discovering the hazardous nature of aflatoxin in early 1960s, many countries began
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conducting surveys on the occurrence of mycotoxins in their agricultural products in last
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few decades (Cui et al., 2013; Selvaraj et al., 2015). Some key mycotoxins that cause
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huge health issues and agricultural loss are aflatoxins, ochratoxins, trichothecenes,
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zearalenone, fumonisins, tremorgenic toxins and ergot alkaloids. These mycotoxins are
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mainly produced by the Fusarium, Aspergillus, Penicillium and Alternaria genera. The
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simultaneous presence of different mycotoxins in a single commodity produced by
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different fungal genera is not uncommon (Aresta, Cioffi, Palmisano, & Zambonin, 2003).
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Among the grains, maize is highly contaminated with many fungi and the subsequent
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mycotoxins.
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Numerous fungi infect maize, approximately 19 genera of fungal species, among
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them Fusarium, Aspergillus, Alternaria, Penicillium and Stenocarpella were the main
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genera (Payne, 1999). These fungi produce mycotoxins that can cause toxic and/or
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carcinogenic effects in humans and animals. Fusarium species produce many mycotoxins
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such
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Fusarium contamination on maize in temperate and semi-tropical areas are very common
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as they cause ear rot, which affects the crop yield to a greater extent. Aspergillus species
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can cause the food to spoil leading to enormous economic loss. Furthermore, some
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Aspergillus species can produce toxic secondary metabolites: like aflatoxins and
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ochratoxins which affect the food safety (Marín, Ramos, Cano-Sancho, & Sanchis, 2012).
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Recent studies show that change in climatic trends may pose longer-term impacts on
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the distribution of fungi, the occurrence of mycotoxins and host crop plants (Wu et al.,
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2011). However, apart from weather parameters, agronomic factors such as drying
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methods and time may affect the nature and degree of mycobiota contamination and the
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occurrence of mycotoxins in the grains (Storm, Sørensen, Rasmussen, Nielsen, & Thrane,
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2008). Drying makes it easy for farmers to bring down the moisture content to a safe
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level as a result, the produce, with sufficiently low water content restrains fungal invasion,
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spore germination and fungal growth.
as
zearalenone,
fumonisins
and
trichothecenes.
Occurrences
of
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The aim of this study was to evaluate the mycoflora and the occurrence of main
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mycotoxins in maize before and after drying. As fungal variation mainly occurs during
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drying process, it was assumed that the effective control of fungi must be taken, which
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influences the growth of toxigenic fungi and the production of mycotoxins during post-
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harvest period.
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2. Materials and methods
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2.1. Chemicals
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High performance liquid chromatography (HPLC) grade methanol and acetonitrile
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were purchased from Fisher Scientific (Fisher Chemicals HPLC, USA). Aflatoxin B1
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(AFB1), Zearalenone (ZEN), Deoxynivanol (DON) and Fumonisin B1 (FB1) standards
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were procured from Sigma-Aldrich Chemicals (USA). ToxinFast immunoaffinity
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columns for AFB1 and ZEN were purchased from HuaanMagnech Bio-tech (Beijing,
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China); MycosepTM 227 columns were from Romer Labs, Inc., (USA).
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2.2. Sample collection and preparation
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A total of 44 maize samples were collected from the local farmers of three major
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maize production provinces (Shandong, Hebei and Henan) in North China Plain in the
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autumn of 2014. The samples were taken from eight areas such as Southwestern
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(Nanyang), Southeastern (Linyi), Central (Liaocheng, Heze, Zibo, Xingtai and Handan),
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and Northern (Langfang) regions of North China Plain. The amount of samples from each
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place is 2 or 3 according to the maize yield. The detailed samples information was
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presented in Table 1. Of 44 maize samples, 22 samples of fresh maize without drying
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treatment in the October were collected, which were named as pre-drying samples. After
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2 months, other 22 samples were collected again from the same group of farmers. For
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each sample, at least ten incremental samples of 100 g each were taken and combined,
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according to EC 401/2006 norms (European Commission, 2006). All samples were taken 5
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up immediately for analyzing the mycobiota. Each 200 g of a sample was ground to a
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fine powder and stored at -20 ºC until further detection of four major mycotoxins.
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2.3. Determination of water activity (aw) and moisture content
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The aw and moisture content of kernels of maize were determined by automatic
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analysis, using Aqualab 4TE (Decagon Devices, Pullman, WA, USA).
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2.4. Recovery, identification and enumeration of the mycoflora from kernels of maize
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From each sample, approximately 30 g subsamples of maize kernels was disinfected
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by immersion in 1% sodium hypochlorite solution for 3 min, followed by three rinses
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with sterile distilled water. After disinfection, 60 kernels were randomly separated and
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directly seeded onto 5 Petri dishes (bottom diameter: 90 mm, 6 kernels per dish)
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containing Potato Dextrose Agar（PDA）and 5 Petri dishes (6 kernels per dish)
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containing Dichloran 18% Glycerol Agar (DG18) for the isolation of internal mycoflora
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(Pitt & Hocking, 2009). The plates were incubated at 25 ºC for 5 days, but the
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observations were made daily. The fungi grew from maize kernels internal. The average
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percentage of kernels infected with fungi was then calculated.
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Speciation of isolated fungal strains was performed according to Tournas, Rivera Calo,
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& Sapp (2013) with minor modifications. Recovered isolates was purified by re-culturing
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on PDA and identified to genus or species level using the conventional methods and keys
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described in “Fungi and Food Spoilage” (Pitt & Hocking, 2009), “Introduction to Food-
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and Airborne Fungi” (Samson, Hoekstra, & Frisvad, 2004), “Identification of Common
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Aspergillus species” (Klich, 2002), “Fusarium Species: An Illustrated Manual for
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Identification” (Nelson, Touson, & Marasas, 1983) and “A Laboratory Guide to Common
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Penicillium Species” (Pitt, 1985). Isolates which are not identified at the species level by
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different conventional plating methods were identified using molecular method as
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described below.
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2.5. Identification of isolated fungal strains using molecular method
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To extract genomic DNA, fungal isolates were grown in YES liquid medium
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inoculated with 1 × 105 spores. After cultivating at 200 rpm (28 ºC) for 5 days, mycelia
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were collected by filtration through Whatman filter paper, and washed with distilled
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water. Genomic DNA was isolated by using the Fungal DNA Kit (Omega Bio-Tek, Inc.
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Norcross, GA, USA) according to the manufacturer’s instructions. The extracted DNA
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was quantified by electrophoresis in 0.8% (w/v) agarose gels with 1 × TAE buffer
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containing 0.1‰ GelRed Nucleic Acid Stain (10,000 ×, Biotium, Bay Area, Cal, USA).
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DNA was diluted to a concentration of 50 ng/µl for further use in PCR reactions.
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Fungal universal primers ITS1 (5’ –TCCGTAGGTGAACCTGCGG-3’) and ITS4
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(5’-TCCTCCGCTTATTGATATGC-3’) were used to amplify the ITS region of fungal
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rDNA. PCR reactions were carried out in a total volume of 30 µl consisting of 15 µl of
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Gotaq PCR Master Mix (Promega, USA), 1 µl of fungal genomic DNA, 1 µl of forward
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and reverse primer (10 µmol). PCR conditions were as follows: denaturation at 95 ºC for
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3 min, 35 cycles of denaturation at 95 ºC for 30 s, annealing at 55 ºC for 30 s, extension
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at 72 ºC for 1min, and final extension at 72 ºC for 7 min. Amplification products were
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purified and sequenced. Basic Local Alignment Search Tool (BLAST) was used to
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identify the closest affiliated sequence in the GenBank/NCBI dataset.
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2.6. Determination of mycotoxins
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A 50 g sub-sample of maize kernels was finely ground with a grinder until they could
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pass through a 29 mm mesh screen, or they were rendered into a paste and stored at 4 ºC 7
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in suitable glass container before determination of mycotoxins.
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2.6.1 Determination of AFB1
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AFB1 in the maize powder was detected by the HPLC (high performance liquid
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chromatography). The specific methodology and technology were refer to the method
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reported by Ding et al. (2015).
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2.6.2 Determination of DON
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Determination of DON in different maize samples was performed by HPLC according
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to the method reported by Cui et al. (2013) with minor modifications. Finely ground
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samples (5.0 g) were extracted with 25 ml of acetonitrile : water (84 : 16, v/v), and the
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mixtures were blended for 3 min at 10,000 rpm using a blender (IKA, ULTRA-TURRAX
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T25 digital, Germany). The extract was filtered through Whatman No.4 paper and 10 ml
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of the defatted extract was purified through BondElute column (Agilent, USA), according
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to the manufacturer’s instructions. 2 ml of purified extract was transferred to another tube
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and evaporated to dryness under a steam of nitrogen at 40 ºC. The dry residue was
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dissolved and mixed well in 1 ml of HPLC diluting solution (acetonitrile : methanol :
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water, 5 : 5 : 90, v/v/v), after centrifuged at 12,000 rpm for 6 min, and was taken up
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further for HPLC/UV analysis.
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HPLC analysis was performed by using Waters 2695 with a Waters 2478 UV detector
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(220 nm) according to the method reported by Cui et al. (2013).
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2.6.3 Determination of ZEN
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Determination of ZEN in different maize samples was performed by HPLC according
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to the method reported by Langseth, Ellingsen, Nymoen & Okland (1989) and Bakan,
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Melcion, Richard-Molard & Cahagnier (2002) with minor modifications. Finely ground
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samples (5.0 g) were extracted with 25 ml of methanol : water (80 : 20, v/v), and the
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mixtures were shaken for 30 min at 200 rpm. The extract was filtered through Whatman
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No. 4 paper and 5 ml of filtered extract was diluted into 25 ml with deionized water.
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Then the dilution was passed through immunoaffinity columns (ToxinFast Columns, Cat.
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No. HCM0525, Huaan Magnech Biotech, Beijing, China) with a flow rate of one droplet
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per second, and was eluted with 2 ml of methanol into glass tubes. The purified extract
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was quantified by HPLC-FD.
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HPLC analysis was performed by using Waters 2695 coupled to a Waters 2475
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fluorescence detector (λexc 360 nm; λem 440 nm) and post-column derivation system,
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and an Agilent TC-C18 column. The mobile phase (acetonitrile : water, 60 : 40) was
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pumped at a flow rate of 0.5 ml/min. ZEN (Sigma-Aldrich, St. Louis, MO, USA) was
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used as standards. Quantification of ZEN levels was performed by the measurement of
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peak area compared with the standards solutions used for the calibration curve. LOD
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(limit of detection) was 1 µg/kg samples.
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2.6.3 Determination of FB1
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Determination of FB1 in different maize samples was performed by HPLC according to
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the method reported by González Pereyra et al. (2008) with minor modifications. Finely
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ground samples (5.0 g) were extracted with 20 ml of methanol : water (3 : 1, v/v), and the
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mixtures were shaken for 30 min at 200 rpm. The extract was filtered through Whatman
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No. 4 paper. 10 ml of the filtered extract was passed through immunoaffinity columns
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(ToxinFast Columns, Cat. No. HCM0825, Huaan Magnech Biotech, Beijing, China) with
194
a flow rate of one droplet per second, and was eluted with 4 ml of 0.5% acetic acid in
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methanol. The eluate was evaporated to dryness, redissolved in acetonitrile : water (1 : 1,
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v/v) and analysed for FB1 by HPLC using the methodology proposed by Shephard,
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Sydenham, Thiel & Gelderblom (1990) and modified by Doko, Rapior, Visconti &
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Schjoth (1995). An aliquot (50 μl) of this solution was derivated with 200 μl of o-
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phthaldialdehyde (OPA). The OPA solution was obtained by adding 5 ml of 0.1 mol/l
200
sodium tetraborate and 50 μl of 2-mercaptoethanol to 1 ml of methanol containing 40 mg
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of OPA. The FB1 OPA derivatives (20 μl solution) were analyzed using Waters 2695
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coupled to a Waters 2475 fluorescence detector (λexc 335 nm; λem 440 nm), and an
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Agilent TC-C18 column. The mobile phase (methanol : 0.1mol/l sodium dihydrogen
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phosphate) was pumped at flow rate of 0.7 ml/min. FB1 (Sigma-Aldrich, St. Louis, MO,
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USA) was used as standard. Quantification of FB1 levels was performed by the
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measurement of peak area compared with the standards solutions used for the calibration
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curve. LOD (limit of detection) was 1µg/kg samples.
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2.7. Statistical analysis
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The data were analyzed with the SAS (statistical analysis software) program using
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analysis of variance (ANOVA) for multiple comparisons followed by the Turkey test.
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The mycotoxins concentrations were expressed as percentage and mean ± RSD.
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3. Results and discussion
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3.1. Water activity (aw) and moisture content in pre- and post-nature drying maize
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Table 2 showed that the aw of pre-drying maize was higher than 0.90 except some
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samples (No. 6, 8, 18, 20) and the average value of aw was 0.917. Except some samples
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(No. 3, 9, 10, 11, 13, 14, 15), aw of post-drying maize was lower than 0.70 which was
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below the minimum range (0.70-0.80) required for the growth A. flavus (Hocking, 2007)
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and the minimum range (0.80-0.90) required for aflatoxins production (Passone, Rosso,
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& Etcheverry, 2012). Furthermore, the moisture contents of most maize kernels were
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under the standard of storage maize in China (≤14%) and were confirmed safe for long-
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term storage (Magan & Aldred, 2007a, b).
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3.2. Distribution and variation of mycobiota in pre- and post-nature drying maize
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As shown in Fig. 1, the percentage of kernels infected by fungi was significantly
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higher in pre-nature drying maize than in post-nature drying maize. For the former, the
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average percentages of kernels infected by fungi on PDA medium and DG18 medium
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were 90.76% and 86.52%, respectively. For post-nature drying maize, the average
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percentages of kernels infected by fungi on PDA and DG18 were 59.24% and 63.33%,
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respectively. In maize with high aw, the percentage of kernels infected by fungi was
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higher on PDA than on DG18. While for maize with low aw, the percentage of kernels
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infected by fungi was lower on PDA than on DG18. The results suggested DG18 is more
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adequate for the numeration of fungi in food with low aw than PDA and confirmed that
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DG18 is medium for xerophilic fungi (Samson, Hoekstra, & Frisvad, 2004; Pitt &
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Hocking, 2009).
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As shown in Table 3, in the maize kernels the following mycobiota were isolated on
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DG18: F. verticillioides (24.77%), F. graminearum (15.08%), Fusarium spp. (5.54%), A.
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flavus (4.93%), A. niger (7.51%), Penicillium spp. (3.86%), Eupenicilliium sp. (1.82%),
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Alternaria sp. (3.57%), Trichoderma sp. (2.20%). Of the Fusarium, F. verticillioides and
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F. graminearum were predominant and the most important because of their known
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toxigenic potential. F. verticillioides and F. graminearum were the predominant fungal
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species detected in maize, and were well-known fumonisin-producing species (Thiel et
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al., 1991) and deoxynivalenol-producing species (Snijders, 1990), respectively. This
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result is in accordance with the finding of Bakan, Melcion, Richard-Molard & Cahagnier
243
(2002).
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The results showed that fungal genera occurring in pre-nature drying maize kernels
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were Fusarium, Aspergillus, Alternaria and Penicillium, similar with many other studies
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(Streit et al., 2012). Fewer fungi were recovered from the post-nature dried maize kernels
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than pre-nature drying ones. Except for F. graminearum, the percentages of kernels
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infected by fungal species in pre-nature drying maize were higher than that in post-nature
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dried maize. The results suggested that the value of aw significantly influenced the
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percentages of kernels infected by fungi, especially Aspergillus spp. Of the Aspergillus, A.
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flavus was the dominant fungi isolated from maize kernels. Many studies have reported
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that high temperature and drought stress directly favor the growth, conidiation, dispersal
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of A. flavus (Cotty & Jaime-Garcia, 2007; Payne & Widstrom, 1992). Abdel-Hadi,
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Schmidt-Heydt, Parra, Geisen & Magan (2012) summarized the optimum aw for the
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growth of A. flavus is 0.90-0.99, while the sporulation, germination, and growth of F.
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verticillioides are optimized at 25-30 ºC and aw 0.70-0.80 (Vittorio, Andrea, & Paola,
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2009). Furthermore, F. verticillioides has been reported as a species that infects all the
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stages of plant development, infecting the roots, stalks and kernels (Munkvold, McGee,
259
& Carlton, 1997). So the contamination of F. verticillioides was higher than that of A.
260
flavus in the maize samples.
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In pre-nature drying maize kernels, the predominant fungi species was F.
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verticillioides, and the percentage of kernels infected by F. verticillioides was higher than
263
27%. The result was in accordance with the earlier published works (Kedera, Plattner, &
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Desjardins, 1999). The Fusarium species produces large quantities of microconidia,
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which most probably allow them to disseminate and easily contaminate maize ears in the
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field. In post-nature drying maize kernels, the predominant fungi species was F.
267
graminearum, the percentage of kernels infected by it was higher than 24%. The result
268
suggested that the growth of F. graminearum is rapid and easily contaminated maize
269
kernels during nature drying process. This means that mycotoxins (DON and ZEN)
270
produced by the Fusarium species should be emphasized after harvest. The result is
271
inconsistent with other researchers who considered that DON was mainly produced
272
before harvest (Bensassi, Zaied, Abid, Hajlaoui, & Bacha, 2010; Bottalico & Perrone,
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2002).
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3.3. Occurrence of major mycotoxins in pre- and post-nature drying maize kernels
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The mycotoxins levels in pre- and post-nature drying maize kernels are presented in
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Table 5. FB1 and DON were the major mycotoxins present in the samples. All pre- and
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post-nature drying maize kernels tested were found to contain FB1, with the mean level of
278
132.8 μg/kg and 100.2 μg/kg, respectively. FB1 concentrations ranged from 16.5 to 315.9
279
μg/kg for pre-nature drying maize and from 17.2 to 220.6 μg/kg for post-nature drying
280
maize, which are lower than the recommended standard of US Food and Drug
281
Administration (FDA) (2000 μg/kg). All post-nature maize kernels tested were found to
282
contain DON with the mean level of 1581.2 μg/kg, which is significantly higher than the
283
limit standard (1000 μg/kg) of China and US FDA. While only 7 of 22 pre-nature maize
284
kernels contaminated with DON ranged from 50.7 to 776.6 μg/kg. This result suggested
285
that DON could be produced during the nature drying process. The results of FB1 and
286
DON contamination are also in accordance with the results of the mycological analysis,
287
because F. verticillioides and F. graminearum, which were the predominant fungal
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species detected, are well-known fumonisin-producing species (Thiel et al., 1991) and
289
DON-producing species, respectively.
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The frequencies of AFB1 and ZEN were low in all the samples. For pre-nature
291
drying maize kernels, only one sample (No. 18) was found to contain AFB1 with the
292
concentration of 148.4 μg/kg, which is more than 7-folds of the Chinese standard limit of
293
20μg/kg. For post-nature drying maize kernels, AFB1 was undetectable and the
294
contamination rates of A. flavus on DG18 medium in all the samples except No. 4, 5 and
295
18 were zero or lower than 5%. The result mainly be attributed to low aw (< 0.70, Table 2)
296
of most samples which was below the minimum range of 0.70-0.80 established for the
297
growth of A. flavus (Hocking, 2007) and the minimum range of 0.80-0.90 for aflatoxin
298
production (Passone et al., 2010, 2012). Furthermore, the mean temperature ranged from
299
9-27 ºC and the RH ranged from 49 to 73% in the collection location in the autumn. Thus,
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the temperature was below 32-33 ºC considered to be the optimum temperature for the
301
growth of A. flavus by Hocking (2007), the RH was lower than 83-85% considered to
302
favor the growth of A. flavus (Oyeka, 2004). Liu, Gao & Yu (2006) obtained the similar
303
results. They found that the average content in maize kernels of Liaoning province in
304
China was found to be very low (0.99 μg/kg) although almost all samples collected
305
contained aflatoxins.
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For pre-nature drying maize kernels, three samples were found to contain ZEN
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ranging from 60.5 to 147.6 μg/kg, which all exceed the Chinese regulatory limit of 60
308
μg/kg. For post-nature drying maize kernels, also three samples were found to contain
309
ZEN ranging from 40.7 to 1056.8 μg/kg, ZEN levels in two samples heavily exceed the
310
Chinese regulatory limit. The occurrence of ZEN in this study was lower than that of a 14
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previous publication based on investigation in North Asia (Binder, Tan, Chin, Handl, &
312
Richard, 2007). In that report, ZEN occurrence was detected in 47.0% for different
313
commodities, feeds and feed ingredients with a much higher average concentration of
314
494.0 μg/kg. Recent studies by Li et al. (2014) for the presence of AFB1, OTA, DON and
315
ZEN in cereal and oil products from the Yangtze Delta region showed that ZEN was the
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major contaminants in the region and maize products had the highest incidences of ZEN
317
contaminations with the incidence at 35.7%, which is significantly higher than the
318
occurrence of ZEN in this study. A similar study by Ji, Xu, Liu, Yin, & Shi (2014) on
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wheat samples from Jiangsu province of Yangtze Delta region harvested during 2010-12
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showed 74% and 12.8% contamination with DON and ZEN, respectively. These results
321
show that ZEN has become a serious safety problem in cereal although the incidence of
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ZEN contaminations in North China Plain is significantly lower than that in Yangtze
323
Delta region.
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4. Conclusions
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This study demonstrated the distribution and variation of fungi and four major
326
mycotoxins in pre- and post-nature drying maize samples collected from North China
327
Plain. The percentage of maize kernels infected by fungi was significantly higher in pre-
328
nature drying samples than that in post-nature drying samples except for F. graminearum,
329
which increase from 6.06% to 24.09%. In maize, Fusarium, Aspergillus, Penicillium,
330
Eupenicillium, Alternaria and Trichoderma were main genera. F. verticillioides and F.
331
graminearum were the predominant fungal species, followed by A. niger and A. flavus.
332
We found that aw played a significant role on the occurrence of fungi. FB1 and DON were
333
the major mycotoxins presented in the samples, followed by ZEN and AFB1. The DON
15
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contaminations in post-nature drying maize kernels were significantly higher than that in
335
pre-nature drying maize. The results of mycotoxins contamination are in accordance with
336
the results of the mycological analysis.
337
Acknowledgments
338
We gratefully acknowledge the financial support of National Key Research and
339
Development Program of China (2016YFD0400105), the National Program of China
340
Basic Science and Technology Research (2013FY113400), the National Natural Science
341
Foundation of China (31571938) and Fundamental Research Funds for Central Non-
342
profit Scientific Institution. The funders had no role in the study design, data collection
343
and analysis, decision to publish, or preparation of the manuscript.
344 345
Conflicts of Interest
346
The authors declare no conflict of interest.
347
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Table 1 The detailed samples information of maize Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
City
Province
Heze
Shandong
Liaocheng
Shandong
Zibo
Shandong
Xingtai
Hebei
Handan
Hebei
Langfang
Hebei
Nanyang
Henan
Linyi
Shandong
475
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Table 2 Water (aw) and moisture content of pre- and post-nature drying maize Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Average
aw Post-drying
Pre-drying
0.973±0.003 0.983±0.004 0.944±0.004 0.975±0.004 0.917±0.004 0.537±0.034 0.923±0.001 0.765±0.002 0.988±0.002 0.986±0.001 0.987±0.003 0.964±0.002 0.975±0.003 0.984±0.004 0.904±0.002 0.914±0.003 0.973±0.001 0.822±0.001 0.928±0.001 0.783±0.002 0.978±0.001 0.962±0.003
0.695±0.004 0.731±0.035 0.602±0.001 0.676±0.003 0.680±0.004 0.460±0.002 0.513±0.018 0.560±0.023 0.912±0.003 0.749±0.039 0.765±0.023 0.651±0.003 0.734±0.001 0.864±0.002 0.848±0.002 0.699±0.004 0.622±0.003 0.699±0.035 0.683±0.003 0.633±0.002 0.423±0.004 0.554±0.045
22.39±0.56 25.48±0.23 19.06±0.45 26.10±0.78 18.98±1.01 9.85±0.34 18.79±1.32 13.22±0.86 31.63±0.23 30.25±1.24 31.32±0.63 24.3±0.57 24.78±0.43 29.34±0.79 18.77±0.88 18.29±0.54 24.3±1.00 13.99±0.35 17.46±0.28 13.37±0.48 27.63±0.53 27.77±0.43
12.73±0.78 12.05±0.98 9.98±1.2 12.33±1.01 10.78±0.68 8.85±0.24 9.81±0.02 10.47±0.40 17.79±0.71 12.14±0.35 13.18±0.64 12.22±0.47 14.86±0.81 15.32±0.32 15.59±0.53 12.00±0.37 10.73±0.24 12.44±0.64 12.18±0.42 11.38±0.28 9.85±0.19 10.19±0.46
0.917
0.671
22.14
12.13
Pre-drying
478
24
MC(%) Post-drying
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479 480
Table 3 List of fungal species from pre- and post-nature drying maize and there isolation frequencies on DG18
Samples Pre Post 2 Pre Post Pre 3 Post 4 Pre Post Pre 5 Post 6 Pre Post Pre 7 Post Pre 8 Post 9 Pre Post 10 Pre Post 11 Pre Post 12 Pre Post 13 Pre Post 14 Pre Post 15 Pre Post 16 Pre Post 17 Pre Post 18 Pre Post 19 Pre Post 20 Pre Post 21 Pre Post 22 Pre Post Pre Av Post Total 1
Fva 33.33 33.33 40.00 60.00 6.67 13.33 70.00 16.67 56.67 ND ND ND 30.00 73.33 10.00 43.33 ND 30.00 6.67 26.67 6.67 3.33 66.67 ND 40.00 6.67 23.33 3.33 30.00 10.00 36.67 6.67 36.67 33.33 26.67 43.33 6.67 56.67 ND 23.33 26.67 3.33 46.67 3.33 27.27 22.27 24.77
Fusarium Fgb 6.67 33.33 3.33 16.67 ND 16.67 ND 33.33 ND 70.00 ND 3.33 ND 3.33 ND ND 20 53.33 46.67 20 ND 36.67 ND 6.67 ND 30.00 30.00 60.00 ND 60 6.67 36.67 6.67 ND 6.67 6.67 ND 20 ND 6.67 3.33 10 3.33 6.67 6.06 24.09 15.08
Fsc ND 3.33 43.33 10 ND 20 ND 3.33 ND ND 3.33 ND ND ND 10 ND ND ND 3.33 ND ND ND 3.33 ND ND ND 23.33 10 10 10 23.33 3.33 23.33 3.57 ND 6.67 ND ND ND ND 10 ND 20 ND 7.88 3.19 5.54
Aspergillus Afd Ane 3.33 36.67 3.33 ND ND 6.67 ND 3.33 ND 13.33 ND 3.33 ND 3.33 23.33 ND 3.33 6.67 6.67 3.33 6.67 10 ND 6.67 ND 33.33 ND ND 13.33 3.33 ND ND 10.00 ND ND 3.33 3.33 6.67 3.33 ND 3.33 6.67 ND ND ND ND ND ND ND ND ND ND 3.33 6.67 3.33 ND 6.67 26.67 ND ND 6.67 ND ND 6.67 6.67 16.67 3.57 3.57 10.00 16.67 6.67 ND 43.33 40 3.33 10 40.00 50 3.33 6.67 ND ND ND ND ND ND ND ND 7.27 12.88 2.59 2.13 4.93 7.51
% kernels infected by fungi Penicillium Eupenicillium Psf Esg 3.33 3.33 ND ND ND ND ND ND 3.33 ND 3.33 ND 3.33 ND ND 3.33 6.67 ND ND ND ND ND ND ND 3.33 3.33 10 3.33 6.67 3.33 3.33 ND ND ND ND ND 3.33 ND 3.33 ND 3.33 ND 3.33 ND 6.67 13.33 ND ND 3.33 23.33 50 ND 6.67 6.67 ND ND 13.33 6.67 ND ND ND ND 3.33 ND ND ND ND 3.57 6.67 ND ND ND 3.33 6.67 6.67 ND 3.33 3.33 6.67 ND ND ND ND ND 3.33 ND ND ND 3.64 3.18 4.09 0.47 3.86 1.82
25
Alternaria Ash ND 3.33 ND ND 26.67 ND 6.67 ND 6.67 ND 3.33 3.33 10 ND ND ND 3.33 3.33 10 3.33 43.33 ND 6.67 ND 6.67 ND ND 3.33 ND ND ND ND ND 3.57 ND 3.33 ND ND ND 6.67 3.33 ND ND ND 5.76 1.37 3.57
Trichoderma Tsi ND ND ND ND ND ND ND ND ND ND ND ND ND ND 16.67 3.33 ND ND ND ND ND ND 3.33 3.33 26.67 ND ND ND ND ND 10 ND ND ND 33.33 ND ND ND ND ND ND ND ND ND 4.09 0.30 2.20
Other 10 ND 3.45 3.57 25.00 5.56 ND 4.17 ND ND 36.36 33.33 14.29 3.57 36.67 6.25 37.50 ND ND 5.56 5.00 ND ND ND ND ND ND ND 6.67 ND 16.66 ND 3.33 ND ND ND ND ND 3.33 ND 10 ND 26.67 3.33 10.68 2.97 6.82
ACCEPTED MANUSCRIPT
481
a=Fusarium verticillioides, b=Fusarium graminearum, c=Fusarium sp., d=Aspergillus
482
flavus, e=Aspergillus niger, f=Penicillium sp., g=Eupenicillium sp., h=Alternaria sp.,
483
i=Trichoderma sp., Av=Average
484
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486 487 Samples 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Av
Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Total
Table 4 List of fungal species from pre- and post-nature drying maize and there isolation frequencies on PDA Fusarium Fva Fgb Fsc 36.67 3.33 30.00 30.00 53.33 ND 56.67 6.67 33.33 36.67 40.00 ND 63.33 10.00 13.33 10.00 23.33 ND 63.33 ND 13.33 33.33 40.00 ND 93.33 ND 6.67 30.00 6.67 ND 10.00 ND ND 3.33 ND ND 10.00 ND 6.67 80.00 ND ND 30.00 ND 13.33 40.00 ND 6.67 3.33 ND ND 6.67 36.67 ND ND 40.00 6.67 26.67 20.00 ND ND 3.33 ND 3.33 33.33 ND 46.67 ND 30.00 10.00 3.33 ND 53.33 3.33 10.00 46.67 33.33 ND 16.67 46.67 6.67 20.00 53.33 ND 63.33 ND ND 50.00 43.33 3.33 33.33 ND 26.67 33.33 13.33 3.33 60.00 ND 10.00 10.00 3.33 ND 6.67 ND ND 26.67 23.33 ND 10.00 26.67 3.33 26.67 13.33 ND 3.33 ND 6.67 16.67 10.00 6.67 53.33 ND 43.33 ND 10.00 3.33 ND 60.00 30.00 6.67 16.67 ND 32.42 9.09 13.18 24.85 21.67 1.06 28.64 15.38 7.12
Aspergillus Afd Ane ND 3.33 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 10.00 13.33 3.33 3.33 ND ND ND ND ND 10.00 ND ND 20.00 3.33 ND ND ND 6.67 3.33 0.00 13.33 13.33 ND ND 3.33 10.00 ND ND ND 3.33 ND 3.33 ND 3.33 ND ND ND ND ND ND ND ND ND ND ND ND ND ND 10.00 ND ND ND 43.33 13.33 6.67 ND 36.67 43.33 10.00 13.33 ND ND ND ND ND ND ND ND 6.21 5.61 1.06 0.91 3.64 3.26
% kernels infected by fungi Penicillium Eupenicillium Psf Esg 16.67 ND ND ND ND ND ND ND ND ND 3.33 ND ND ND 3.33 ND ND ND ND ND 16.67 ND 6.67 ND ND ND 3.33 ND ND 3.33 ND ND 6.67 ND ND ND ND ND ND ND ND 6.67 3.33 ND 3.33 ND 3.33 ND 13.33 13.33 3.33 ND ND 3.33 3.33 ND 6.67 ND ND ND ND ND 3.33 ND 3.33 ND ND ND ND ND ND ND ND ND ND ND 3.33 6.67 6.67 ND ND ND ND ND ND ND ND ND 3.18 1.52 1.82 0.00 2.50 0.76
27
Alternaria Ash ND ND ND ND ND 3.33 ND ND ND 3.33 ND ND ND ND ND ND 20.00 0.00 13.33 3.33 30.00 ND ND 6.67 ND ND ND ND ND ND 3.33 ND ND ND ND 3.33 ND ND ND 3.33 ND ND ND ND 3.03 1.06 2.05
Trichoderma Tsi ND ND ND ND ND ND 20.00 ND ND 46.67 33.33 ND ND ND 26.67 ND ND 23.33 ND ND ND ND ND ND ND ND 6.67 ND ND ND 36.67 ND ND ND 76.67 36.67 3.33 30.00 ND ND ND ND ND ND 9.24 6.21 7.73
Other 10.00 6.67 3.33 3.33 10.00 ND 3.33 6.67 ND ND 10.00 3.33 3.33 13.33 6.67 16.67 ND ND 3.33 ND 13.33 ND 3.33 3.33 3.33 10.00 16.67 ND 30.00 ND ND ND 26.67 3.33 ND 3.33 ND ND ND ND 3.33 ND 10.00 13.33 7.12 3.79 5.45
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a=Fusarium verticillioides, b=Fusarium graminearum, c=Fusarium sp., d=Aspergillus
489
flavus, e=Aspergillus niger, f=Penicillium sp., g=Eupenicillium sp., h=Alternaria sp.,
490
i=Trichoderma sp. Av=Average
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492 493 Samples 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Mean
Table 5 Mycotoxins levels (µg/kg) in samples from pre- and post-nature drying maize. AFB1 prepost148.4±22.0 -
Zen pre141.1±37.0 60.5±12.4 147.6±17.6 15.9
DON
post788.3±46.3 1056.8±267.9 40.7±8.3 85.7
pre279.8±69.8 226.5±45.4 102.1±27.6 107.6±38.8 776.6±98.7 232.7±73.2 50.7±12.3 80.7
494
29
post1503.7±504.1 210.7±64.7 4244.3±356.8 476.6±102.4 9843.3±780.4 8.7±4.7 11.0±8.9 5.8±6.5 792.2±356.4 818.4±367.8 771.9±459.2 47.4±7.0 938.3±278.5 6287.5±503.7 1873.0±620.6 4968.1±236.4 59.7±17.7 87.0±25.9 72.9±30.2 32.4±18.9 1627.6±423.5 105.7±32.3 1581.2
FB1 pre152.5±30.1 35.6 ±2.9 16.5 ±1.7 260.9 ±29.2 70.2±2.0 32.6 ±3.2 29.7 ±1.6 68.3 ±4.5 41.2 ±1.8 56.2 ±3.4 36.0 ±2.2 52.7 ±2.0 238.0 ±5.0 57.1 ±2.7 43.9±2.3 196.9 ±2.5 315.9 ±52.2 268.3 ±83.3 264.1 ±150.1 156.8 ±35.2 303.5 ±82.2 224.7 ±69.0 132.8
post162.9 ±47.6 195.6 ±85.5 33.0 ±3.9 86.2 ±4.7 32.6 ±1.6 17.2 ±1.9 162.7 ±24.2 46.5 ±15.5 52.7 ±9.3 135.1 ±33.3 23.6 ±7.9 40.2 ±6.4 101.5 ±3.3 73.3 ±4.7 154.7±23.2 35.3 ±116.4 220.6 ±18.3 167.7 ±27.3 164.4 ±61.3 128.1 ±26.3 133.3 ±65.1 36.6 ±12.2 100.2
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Figure Legends Figure 1. Percentages of kernels infected by fungi in pre- and post-drying maize kernels.
30