Impact of Alternaria spp . and Alternaria Toxins on Quality of Spelt Wheat

There is an increasing consumer demand for alternative cereals nowadays. Spelt wheat (Triticum aestivum ssp. Spelta) appears to be a future source for the agriculture and food sector. Alternaria spp. infections might become a serious danger to the worldwide grain industry, resulting in yield losses and reduction of end-use quality, with potential harmful effect of Alternaria toxins on human and animal health. This paper presents the first assessment of the impact of Alternaria infection and its toxins to quality of spelt wheat. The results showed that fungal contamination significantly reduced both trade and technological quality parameters. Volume weight, thousand kernel weight and wet gluten were significantly decreased, while protein content was significantly higher in Alternaria inoculated treatments. Although with slight decrease, falling number was not significantly affected by fungal contamination. The most negative impact of alternariol (AOH) was registered on volume weight and thousand kernel weight (-0.847; -0.898), while highly significant positive correlation was found between AOH and protein content (0.758). Alternaria spp. additionally destroyed spelt gluten structure, resulting in reduction of dough energy and baking quality with no significant influence of mycotoxins (AOH and AME) on technological quality parameters.

Considering the fact that spelt is usually cultivated in low input system, with reduced use pesticides, continuous monitoring of pathogens and their impact on food safety and quality is of high importance.The latest literature overview showed that the predominant mycobiota on spelt wheat are Fusarium spp.(Hudec & Lacko-Bartošova, 2012) and Alternaria spp.(Kurowski, Damszel, & Wysocka, 2012), which are reducing the yield, but there is no data about impact of fungal contamination on technological parameters of spelt quality and food safety.
This research was carried out to assess the influence of Alternaria spp. to trade and technological quality of spelt wheat and to evaluate the correlation between Alternaria toxins and quality parameters.

Field Experiment and Artificial Inoculation
The filed trial was set up in randomized complete block design with four replicates in mid-October 2013.Each plot was consisted of 10 rows, 10 cm apart and 5 m long (the harvested area was 5 m 2 ) with seedling density of 500 seeds m 2 .Inoculation of three spelt wheat genotypes (Nirvana, Austria and Ostro) was performed on the full flowering stage by spraying spikes with 200 mL suspension of Alternaria spp.infection material (in a concentration of 1 × 10 6 /mL).In parallel, fungicide treatment (prochloraz 267g/L + tebuconazol 133g/L) in a concentration of 0.2% was applied.
Alternaria spp.isolates, derived from the contaminated kernels of spelt wheat in northeastern Serbia, were previously identified by PCR assays.The sequence of the primers, PCR reaction mixture, and the conditions used for detecting were identical to those described by Bensassi et al. (2009).
Multiplication of isolates was done on a potato dextrose agar followed with 14 days incubation at 25 °C in the dark.The inoculum was prepared by mixing infectious material in a blender with the addition of distilled water.Filtered suspension was adjusted to a concentration of 1 × 10 6 infective particles mL -1 using a haemocytometer.Inoculated spikes were immediately covered with polyethylene bags.After 24 h the bags were removed.Inoculation treatments were replicated after two weeks.In the full ripeness stage spikes were cut by hands and dehulled using laboratory tresher MDF1, RePietro, Gaggiano, Italy.

Alternaria Mycotoxins Determination
Determination of alternariol (AOH) and alternariol monomethyl ether (AME) was carried out on a 1260 series HPLC system with a DAD detector (Agilent Technologies, USA).Milled and homogenized spelt samples were extracted with acetonitrile 4% KCl (9:1, 75 mL) by shaking on a rotary mechanical shaker (185 rpm) at room temperature (22-24 °C) for 30 min followed by the addition of 1 N HCl (15 mL).After filtration, 45 mL of the filtrate (equal to 7.5 g of wheat sample) was purified with 90 mL of 0.05 M lead acetate.The filtration is repeated and 75 mL filtrate was extracted three times with 20 mL of dichloromethane.The organic phases were joined, evaporated to dryness, and dissolved in 1 mL of methanol for AOH and AME analyses by HPLC.The analytical column was Agilent Eclipse XDB-C18 (4.6 × 50 mm, USA) thermostated at 40 °C.Injection volume was 5 µL.A methanol/water mobile phase was used in a gradient regime (solvent A:H20 with 385 mg L -1 amoniumacetate, solvent B:methanol) at a flow rate of 0.5 mL min -1 .Initially mixture of the mobile phase was held 0.5 min, successively elevating the concentration of methanol linearly to 100% within 9.5 min.By maintaining of these conditions for 3.5 min, the concentration of methanol was converted back to the initial composition.The acquisition of chromatograms was completed after 20 min.The detector operated at an excitation wavelength of 257 nm with quantification limit of 1.25 µg mL -1 for both examinated Alaternaria toxins.Detection and quantification of the toxins were carried out by the Software Agilent Chem Station for LC 3D Systems Rev.B.02.01SR1 (260).

Standard Trade Technological Quality Parameters
Samples were analyze by standard methods for assessing trading and technological wheat quality: crude protein content (CP) in grain dry matter (NIT analyzer ''Infratec 1241''), wet gluten content (WG) in grain dry matter (ICC Standard No. 106/2) and gluten index (GI) using the apparatus Glutomatic Perten (ICC Standard No. 155), jas.ccsenet.falling num weight).

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The conten in the rang µg/kg).D between t confirmed contamina   Alternaria spp.influenced significant reduction of volume weight (VW) and thousand kernel weight (TKW) in all inoculated samples with exception in Ostro (Table 2).This was expected since kernels affected by Alternaria spp.may become shriveled, directly resulting the reduction of VW and TKW.On the other hand, protein content (CP) was significantly higher in Alternaria spp.inoculated spelt genotypes with high significant differences between treatments at all three genotypes.The similar findings reported (Šarić, Stojanović, Škrinjar, & Menkovska, 2008;Malaker, Mian, Bhuiyan, Reza, & Mannan, 2009) in evaluation of the impact of Alternaria spp. on the same quality parameters of common wheat.
Levels of wet gluten were variable depending on treatment with statistically significant differences between genotypes with exception at genotype Austria, indicating that Alternaria inoculation additionally decreased gluten properties.Falling number was not significantly affected by Alternaria spp.treatment, showing levels over 350 s at all three genotypes.Nevertheless, inoculation influenced degradation of starch and higher alpha-amylase activity and falling number was slightly decreased.Note.Different letters in the column show statistically significant values at p ≤ 0.05, according to Tukey's HSD; VW: volume weight; TKW: thousand kernels weight; CP: crude protein content in the grain dry matter; FN: falling number; WG: wet gluten content in the grain dry matter; GI: gluten index.
Abovementioned results were additionally affirmed by correlations between Alternaria toxins and assessed quality parameters (Table 3), where the statistically significant negative correlation was found between Alternaria toxins content and VW and TKW.AOH the most negatively effected VW and TKW (-0.847; -0.898), while highly significant positive correlation coefficient was detected between AOH and crude protein content (0.758).

Technological Quality Parameters
The ANOVA test showed statistically high significance of impact all sources of variation on examined parameters, indicating that extensographic parameters are genotype characteristic, but also indicators of dough quality (Table 4).Note.Different letters in the column show statistically significant values at p ≤ 0.05, according to Tukey's HSD test.
Alternaria spp.influenced reduction of dough energy and thus baking quality as it is shown in Table 4.The highest and statistically significant energy is detected at Nirvana genotype in both treated with fungicides (92 cm 2 ) and inoculated samples (85 cm 2 ), while Ostro has the lowest energy in the treated and inoculated samples at the levels of 24 cm 2 and 29 cm 2 , respectively.Resistance and extensibility were also significantly lower in Alternaria spp.inoculated variants.Our results are in correlation with conclusions of Antes et al. (2001) who assessed Aspergillus and Penicillium species and concluded that fungal contamination have reduced dough resistance and extensibility influencing low bread volume.In contrary, Prange et al. (2005) reported that that Fusarium infection did not negatively influence baking quality highlighting concerns that fungal contamination and their mycotoxins is not evidenced through baking quality analysis which might have considerable impact on food safety.
Mixolab results confirmed that Alternaria spp.infection markedly demages both protein and starch quliaties of spelt wheat.The average value of C2, representing the protein weakening, was lower in artificially inoculated variants for all genotypes, with exception in Ostro.Our results confirmed findings of Papoušková et al. (2011) and Capouchová et al. (2017) where Fusarium spp.significantly affected protein structure at wheat grains, with a slightly better resistance of spelt wheat.
According to correlation analysis presented in Table 5, there were no statistically significant correlations between the mycotoxin content (AOH and AME) and the technological quality parameters.Note.*no correlation was statistically significant.

Principal Component Analysis (PCA)
The similarity of observed samples is indicated by the close vicinity of the points which are representing them in the PCA graphic.The variables are presented by vectors in factor space, while the correlation between two variables is indicated by the angle between vectors (the high correlation corresponds to small angle between vectors) (Krulj et al., 2016).Having this in mind, it can be observed that high positive correlation exists between AME and AOH content (r = 0.990, p < 0.001), and also the strong negative correlation to trade quality parameters VW and TKW.The statistically significant correlations between trade quality parameters were not observed.WG was negatively correlated to rheological parameters: extension, resistant and ration (r = -0.887;r = -0.823and r = In terms of food safety, continuous monitoring of Alternaria contamination must be of high priority, in both filed and storage facilities of spelt wheat, particularly taking in consideration that spelt wheat is commonly grown in low input systems with limited pesticides use.In the case of a strong infection, herewith stimulated by artificial inoculation, Alternaria toxins are strongly correlated with reduction of trade quality, potentially providing risks for human and animal consumption.

Table 1 .
Content of Alternaria mycotoxins in inoculated spelt wheat kernels and treated with fungicide a Note.Different letters in the column show statistically significant values at p ≤ 0.05, according to Tukey's HSD test; AOH-alternariol, AME-alternariol monomethyl ether.

Table 2 .
Trade quality parameters of spelt wheat genotypes

Table 3 .
Correlation between mycotoxin content and the trade quality parameters of spelt wheat genotypes Note. *Correlation is statistically significant at p < 0.05 level, **Correlation is statistically significant at p < 0.10 level; AOH: alternariol; AME: alternariol monomethyl ether; VW: volume weight; TKW: thousand kernels weight; CP: crude protein content in the grain dry matter; FN: falling number; WG: wet gluten content in the grain dry matter; GI: gluten index.

Table 4 .
Extensograph parameters and mixolab characteristics of spelt flour from different genotypes inoculated with Alternaria spp.and treated with fungicides

Table 5 .
Correlation between the mycotoxin content and the rheological quality parameters