Study of Fungal Colonization of Wheat Kernels in Syria with a Focus on Fusarium Species

Wheat is one of the main crops in Mediterranean countries, and its cultivation has an important role in the Syrian economy. In Syria, Fusarium head blight (FHB) has not been reported so far. Mycological analysis of 48 samples of wheat kernels collected from cultivation areas with different climatic conditions were performed in 2009 and 2010. Fungal isolates were identified at the genus level morphologically; Fusarium species were characterized morphologically and by species-specific PCR. The most frequent fungal genera found were Alternaria spp. and Cladosporium spp., with frequencies of 24.7% and 8.1%, respectively, while the frequency of Fusarium spp. was 1.5% of kernels. Most frequent Fusarium species were F. tricinctum (30% of all Fusarium isolates), F. culmorum (18%), F. equiseti (14%) and F. graminearum (13%). The mycotoxin production potential of selected Fusarium isolates was assessed by HPLC-MS analysis of rice cultures; chemotyping by PCR was carried out for comparison. All six F. graminearum strains tested produced small amounts (<3 mg/kg) of nivalenol (NIV). All ten F. culmorum strains tested produced large amounts of trichothecenes (>100 mg/kg); four strains produced NIV and six strains produced deoxynivalenol (DON) and 3-acetyl-deoxynivalenol (3Ac-DON). PCR chemotyping lead to an oversimplified picture, because all 3Ac-DON chemotype strains produced more DON than 3Ac-DON; furthermore, the strongest NIV producers produced significant amounts of DON. All tested strains of F. culmorum, F. graminearum, F. pseudograminearum (two strains) and most F. equiseti strains (five of six strains) produced zearalenone. Grains of durum wheat were more frequently colonized by Fusarium spp. than grains of soft wheat. Incidence of Fusarium spp. in irrigated fields was higher than in rainfed fields. The incidence of Fusarium strains producing mycotoxins raises concerns about the risk of Fusarium head blight to Syria and its consequences for public health.


Introduction
Durum wheat constitutes the largest part of the staple food in the southern Mediterranean countries [1]. In Syria, wheat cultivation covers 83% of the cultivated area and has a central role in the diet [2]. There are five distinct agro-climatic zones in Syria based on rainfall ( Figure 1) [3]. Wheat is present in all these areas. Rainfed wheat is concentrated in high rainfall zones 1 and 2, while irrigation is necessary in zones 3, 4 and 5. The largest zones are 5, 1 and 2, covering 53%, 42% and 40% of the total cultivated land, respectively [4].
Syria produces both durum and soft wheat over the winter season. Soft wheat is cultivated mainly in irrigated areas, durum wheat in rainfed areas. Depending on the rainfall, rainfed wheat yields are highly unstable, with the average ranging from less than 0.5 tons per ha in a drought year to over 1.7 tons per ha in a year of good rainfall. Yields are more stable in irrigated areas, with the national average ranging from 3.0 to 4.4 tons per ha [2]. Syrian agricultural policy resulted in an increase in cultivation of wheat in the irrigated land from 229,000 ha in 1988 to 800,000 ha in 2003 and 1.9 million ha in 2005 [4].
The seed infection by Fusarium pathogens is a great risk for wheat cultivation. Fusarium species are a widespread pathogenic fungi, which can cause Fusarium head blight (FHB) and Fusarium crown rot in wheat [5]. Fusarium crown rot is known to occur in Syria; Fusarium isolates recovered from the subcrown internode of durum wheat were identified as F. avenaceum, F. culmorum, F. equiseti, F. graminearum and F. poae [6]. FHB or scab, which is a more dangerous and important disease, has not been reported in Syria yet.
The distribution of Fusarium species in wheat and their dominance over other fungi is affected by climatic conditions (temperature, humidity, etc.), agricultural practices (soil tillage, crop rotation, nitrogen fertilizers, pesticide treatment, etc.) and cultivar susceptibility [4]. FHB causes high yield losses [4]; in addition, colonization of wheat with Fusarium species can cause the contamination of grain with toxic fungal secondary metabolites (mycotoxins) that are recognized as health hazards for both human and farm animals [8].
Toxigenic Fusarium species produce several mycotoxins, such as trichothecenes A and B, zearalenone, moniliformin, depsipeptides and fusaric acid. Most attention in the analysis of FHB-afflicted wheat grains has so far been devoted to deoxynivalenol (DON), acetylated forms of DON (3Ac-DON and 15Ac-DON), nivalenol (NIV), fusarenon X (Fus X) and zearalenone (ZEN) [7]. The knowledge of the occurrence of Fusarium species in different growing areas helps to predict the mycotoxin content of harvested grain. The maximum allowed limits for mycotoxin levels in food have been established for the protection of the consumer. For instance, limits for DON and ZEN in food exist in Europe (EU-regulation 1881/2006).
In addition to Fusarium spp. in FHB, other fungi, e.g., Alternaria spp., Cladosporium spp., Epicoccum spp. and Rhizopus spp., infect wheat grain in the field, causing quality loss, due to undesirable color and odor. In Syria, little information is available on fungi associated with durum and soft wheat grain and their distribution in the main production area.
The aims of the present research were (a) to study the diversity of fungal species colonizing wheat kernels from different provinces of Syria; (b) to determine the incidence of mycotoxin-producing Fusarium species; (c) to estimate the potential of selected Fusarium isolates to produce mycotoxins; (d) to compare infection frequencies of durum and soft wheat by Fusarium spp. and (e) to compare irrigated and rainfed fields regarding Fusarium spp. incidence.

Results and Discussion
In the surveys carried out in 2009 and 2010 on durum and soft wheat grains collected from different provinces in Syria, 17 different genera of fungi were identified in surface-disinfected kernels. Alternaria spp. and Cladosporium spp. were the most frequent, with an isolation frequency of 24.7% and 8.1%, respectively (Figure 2). High kernel infection with Alternaria and Cladosporium was detected in the samples collected from all six provinces investigated (Table 1). These saprophytic fungi are known to cause grey or black discoloration of heads and seeds, resulting in sooty molds, black points or smudge, and under certain conditions, they may produce mycotoxins. The prevalence of Alternaria and Cladosporium among fungal genera was in agreement with a study from Iran [9]. Storage fungal genera, Penicillium and Aspergillus, were isolated at frequencies of 5.5% and 2.4%, respectively ( Figure 2).  Fusarium spp. was present in 62.5% of all samples (Table 2) with a frequency of 2.4% (minimum 0.25%, maximum 25%). Fusarium species were mostly isolated from samples collected in the Daraa (agro-climatic zone 1, 2 and 3) and Al-Hassakeh (on the border between agro-climatic zones 1 and 2) areas, with frequencies of 2.4% and 1.9%, respectively, while the lowest relative frequency (0.4%) was in Deir Ezzor, a very dry region (agro-climatic zone 5; Figure 3). The differences in the incidence of Fusarium spp. among provinces can likely be accounted for by differences in their climate. F. tricinctum (26), F. culmorum (7), F. verticillioides (6), 100% 62.5% The incidence of Fusarium species causing FHB was likely underestimated, since severely infected and shriveled kernels, which are very light in weight, are expelled with the chaff during combined harvesting [10]. FHB has not been reported in Syria yet.
Promising sources of resistance to FHB have recently been identified in Syrian durum landraces [11,12]. Because durum wheat is more susceptible to FHB than soft wheat [13], we compared the incidence of Fusarium spp. in durum and soft wheat cultivars in Syria (Figure 4). Durum wheat was more often colonized by Fusarium spp. (frequency 2%) than soft wheat (frequency 1%). Colonization rates of durum and soft wheat cultivars by other fungal genera were comparable; the total colonization rates by all fungi were similar, too ( Figure 4). High humidity during flowering is one of the key factors promoting FHB [5]. The trend toward higher irrigation rates after the drought waves, which hit Syria recently, might therefore have increased the risk of FHB. We studied this question by comparing the incidence of fungi in kernel samples collected from irrigated and rainfed fields. Irrigated fields had a higher incidence of Fusarium spp. than rainfed fields (2% vs. 1%). Colonization of grains by other fungal genera appeared slightly higher in irrigated fields, too, and the same trend was reflected by the total colonization by all fungal genera ( Figure 5). A total of 163 Fusarium isolates were morphologically identified to the species level. Species-specific PCR was used to verify the assignments of selected isolates. Genomic DNA of strains putatively identified as F. culmorum (11 strains), F. graminearum (six strains), F. equiseti (nine strains), F. proliferatum (four strains) and F. verticillioides (five strains) and two isolates of F. pseudograminearum were used in this analysis. The products of DNA amplification with species-specific primers (see Experimental Section) were about 570 bp for F. culmorum, 400 bp for F. equiseti, 450 bp for F. graminearum, 585 bp for F. proliferatum, 578 bp for F. verticillioides and 523 bp F. pseudograminearum. These sizes correspond to published values for species-specific PCR products, confirming the morphological identification.
Our study indicates the dominance of F. tricinctum in Syrian wheat. This species is not considered as one of the main causal agents of FHB, but it was reported with a high incidence in some areas and under certain climatic conditions [14]. F. verticillioides has rarely been found in wheat [14]; Fusarium isolates similar to "F. moniliforme" (a now abandoned taxonomic name; the taxon was split into different species) were described in Syrian wheat kernels [15]. Table 2 shows the occurrence of Fusarium species in wheat samples from different provinces. F. culmorum was not detected in samples from Aleppo and Idlib provinces, while it was isolated from all other provinces. In the time period covered by our survey, some Fusarium species were limited to specific regions, such as F. graminearum, to Damascus and Deir Ezzor and F. pseudograminearum to Deir Ezzor. F. tricinctum was dominant in all provinces. Wheat samples from Daraa and Al-Hassakeh regions were most contaminated; F. tricinctum, F. equiseti and F. culmorum were identified in both regions. These results are in line with the report of El-Khalifeh et al. [1], who found inhomogeneous distribution of Fusarium spp. among Syrian provinces, too.
Comparison of the data obtained from the two years showed that the incidence of Fusarium spp. remained similar, but the colonization of grain with other fungal genera, particularly Alternaria, fluctuated to a large extent between the two years ( Table 1).
The widespread presence of agents of FHB in Syria is worrying, because the legal limits for mycotoxin content in food commodities are not established in this country. We therefore assessed the potential of randomly selected Fusarium isolates from Syrian wheat to produce mycotoxins by chemical analysis of rice cultures. As a preliminary assessment of trichothecene production, chemotypes of the strains selected for mycotoxin analysis were assessed by PCR.
In six out of 11 F. culmorum strains, using the primers directed to Tri3 (Tri3F1325/Tri3R1679), an amplification product of about 350 bp was obtained, as expected for 3Ac-DON chemotypes [16]. Five strains, amplified by the Tri7F340/Tri7R965 primers (Tri7 gene), generated a 625 bp fragment, expected for NIV producers. The 700-bp fragment specific for the 15Ac-DON chemotype was not found in any of the tested strains. The situation is similar to Italy [17] and to other European countries, in particular England, where 3Ac-DON and NIV chemotypes dominated over the 15Ac-DON chemotype [18], and Luxemburg, where 3Ac-DON and NIV chemotypes made up 53.2% and 46.8% of all F. culmorum isolates, respectively [19].
All six F. graminearum strains were tested using primers for Tri12 produced amplicons of 840 bp, as expected for NIV chemotype [20]. None of the strains produced amplicons of 670 or 410 bp, expected for 15Ac-DON and 3Ac-DON chemotypes, respectively.
Nine F. equiseti strains were tested for the presence of the trichodiene synthase gene involved in trichothecene synthesis [21]. The presence of a 658 bp amplification product revealed the potential for trichothecene production in seven of these strains.
Mycotoxin production by ten F. culmorum strains, six F. graminearum, two F. pseudograminearum and six F. equiseti strains in rice cultures was determined by HPLC-MS/MS. The results are shown in Table 3. Four out of ten F. culmorum strains produced nivalenol/fusarenon X, while six, classified as the 3Ac-DON chemotype by PCR, produced high DON amounts, smaller amounts of 3Ac-DON and yet smaller amounts of 15Ac-DON. This behavior is common in strains belonging to the 3Ac-DON chemotype [22], because acetyltrichothecene esterase (a product of the gene, Tri8) is present in all three chemotypes; merely its activities towards C3 and C15 positions differ [23]. Similar to our results, ten F. culmorum strains belonging to the 3Ac-DON chemotype from France produced large amounts of DON [24]. The fact that only two among these strains produced detectable amounts of 15Ac-DON can be explained by the overall 10-times lower trichothecene amounts produced (wheat grain cultures were used instead of rice cultures). In Norway, Germany, Denmark and Hungary, all of the strains that belonged to chemotype 3Ac-DON according to PCR produced DON, too [18,25]. Co-production of DON and NIV was reported less frequently; it has been described for a few European isolates of F. culmorum [25] and in a recent study [26] for all F. graminearum isolates of the DON chemotype. We hypothesize that the co-production of DON and NIV was overlooked in studies that used culture conditions less conductive to trichothecene production and/or less sensitive analytical methods. Our results showed that chemotyping by PCR generates an oversimplified picture of trichothecene productivity in Fusarium spp. Because 3Ac-DON chemotype strains produced more DON than 3Ac-DON, differentiation between strong and weak DON producers would be more useful than distinction between 3Ac-DON and 15Ac-DON producers. Most NIV producers in our work also produced Fus X, which is not surprising, because Fus X is 4-acetyl-NIV. NIV producers contain active trichothecene-4-acetylase, a product of the gene, Tri7 [27].
All six investigated F. graminearum strains belonged to the NIV chemotype; the PCR results were confirmed by chemical analysis of rice cultures. Similar results were reported for another area of the Middle-East/Iran [28], where the majority (46/57) of F. graminearum isolated from cereals possessed the NIV chemotype. The dominance of the 15Ac-DON chemotype of F. graminearum was reported for the Netherlands [29], England and Wales [18], Italy [30] and the USA [31], while the 3Ac-DON chemotype dominated in western Russia and Finland [32].
NIV production by F. equiseti was mentioned by several authors [7,33], while the production of DON by this species was reported less often [34]. Two F. equiseti strains in our work produced NIV (Table 3). A few F. equiseti strains originally appeared to produce DON, but a re-investigation did not confirm the result, indicating that F. equiseti does not contribute to the contamination of Syrian wheat with DON. Both F. pseudograminearum isolates produced DON in line with previous reports, e.g., by Monds et al. [35].
All investigated cultures of F. culmorum, F. graminearum and F. pseudograminearum and most strains of F. equiseti produced ZEN; the largest amounts were produced by F. pseudograminearum and F. equiseti. All F. culmorum strains were pathogenic on wheat (data not shown).
The study revealed that numerous toxigenic Fusarium species occurred in Syrian wheat. Grain from areas with higher rainfall was more often colonized with Fusarium and other fungal genera than grain from dry areas. Durum wheat was more susceptible to colonization than soft wheat.

Fungal Isolation
During 2009 and 2010, 48 grain samples of 34 durum and 14 soft wheat were collected from six Syrian regions distributed in five agro-climatic zones (Figure 1) [3]. Four hundred kernels (except for a single sample from Daraa from year 2009 consisting of only 100 seeds, due to insect damage), selected randomly from each sample, were disinfected in a sodium hypochlorite solution with 2% available chlorine for 2 min, rinsed with sterile water, dried on sterile filter paper, placed in petri dishes containing potato dextrose agar (PDA, Difco, Detroit, MI, USA) supplemented with neomycin and streptomycin sulfate (100 mg/L and 200 mg/L, respectively) and incubated at 22 °C in darkness for 7 days [36].
Micromorphology of fungal isolates was examined by light microscopy [37]. All Fusarium isolates were sub-cultured on water agar (2% of Bacto agar, Difco) using the single spore technique. Pure cultures of Fusarium spp. were grown at 22 °C (12 h photoperiod) for 10 days on carnation leaf piece agar (CLA) to produce macroconidia of uniform size and form and on PDA for the morphology of the colony [38].
The frequency of each fungal genus was calculated as the percentage of the total number of kernels.
F. graminearum chemotypes were identified using a multiplex version of another chemotype-specific test [30]. The primers, designed in the region of the Tri12 gene encoding an efflux pump for trichothecenes, distinguish among three chemotypes for B trichothecene. One primer for each pair is common to all chemotypes (12CON), while the other is specific for the 15Ac-DON chemotype (12-15F), 3Ac-DON chemotype (12-3F) and the NIV chemotype (12NF) [20]. Primers Tox5/1 and Tox5/2, derived from the DNA-sequence of the trichodiene synthase gene (Tri5), were used to test the ability of F. equiseti strains to produce trichothecenes [21].

Mycotoxin Detection
Rice media were prepared by autoclaving 50 g polished rice with 70 mL water. Rice cultures were inoculated with 3 agar plugs (1 × 1 cm) overgrown with Fusarium strains and incubated at 24 °C for 4 weeks. The samples were frozen at −20°C, freeze dried for 4 days and milled.
Rice cultures were extracted and defatted, as previously described [45]; the residue corresponding to 4 g rice was dissolved in 1 mL methanol/water (1:1, v/v). The analytes were separated on a polar-modified reverse-phase HPLC column (Polaris C18-Ether, 100 × 2 mm, 3 µm particle size; Agilent, Darmstadt, Germany) kept at 40 °C with a methanol-water gradient elution (10% to 98% in 7 min, followed by washing and equilibration steps) at a flow rate of 0.2 mL/min. Mass spectrometry detection was carried out as described [45] using mass transitions for 3Ac-DON, 15Ac-DON, Fux X and ZEN, described by Klötzel et al. [46], and for DON and NIV, described by Rasmussen et al. [47].

Conclusions
The mycological survey of wheat kernels from Syria revealed prevalent colonization of the grain with Alternaria spp. and the presence of storage fungi (e.g., Penicillium spp., Aspergillus spp.) and plant pathogenic Fusarium spp. in most samples. All Fusarium species isolated from Syrian wheat kernels are potential causal agents of FHB. PCR chemotyping and HPLC-MS analysis of rice cultures of selected Fusarium isolates demonstrated their potential to produce trichothecenes B and zearalenone and raised concerns about the risk of mycotoxin accumulation in Syrian wheat grain.
The incidence of Fusarium species in wheat grain appears low, but it has to be monitored, given the changes in agricultural policies in Syria. For example, higher irrigation rates used after the drought waves, which recently hit Syria, may increase the risk of FHB by causing a high humidity during anthesis. The increasing role of maize in crop rotation is another factor likely to increase the incidence of FHB in Syria [5].
The Syrian agricultural policy should provide guidance to agronomic practices to avoid the spread of FHB. The presence of mycotoxigenic Fusarium species in all wheat-growing areas in Syria raises concern about mycotoxin exposure via food grains and indicates that national legislature for the control of grain quality is indispensable.