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Ergot sclerotia and ergot alkaloids occurrence in wheat and rye grains produced in Croatia

Ergot alkaloids (EAs) are mycotoxins produced by several species of fungi of the genus Claviceps, among which Claviceps purpurea is the most widespread in Europe. This species has been found in many economically important cereal grains, such as rye, wheat, triticale, barley, millet and oats. The distribution of EAs contamination has a sporadic incidence with many factors involved in its occurrence, greatly varying between fungal strains, geographic regions, host plants and regional/local weather conditions, at the same time emphasizing that cool, damp weather favours ergot by enhancing the germination of sclerotia. The aim of this study was to investigate the occurrence of ergot sclerotia and EAs in wheat and rye grain samples (n = 64) taken during the year 2021 from Croatian cereal producers located in the Central and Eastern parts of Croatia. In two of the rye samples the presence of ergot sclerotia was detected in the amount of 259 mg/kg and 536 mg/kg, whereas none of the wheat samples tested positive for ergot sclerotia. A higher contamination with EAs was determined in the rye samples (18% contaminated, max 167.4 µg/kg), while a lower frequency of contamination, with only one positive sample (1.9 %), was determined in wheat (68.5 µg/kg). The results pointed to a low-level of EAs contamination of wheat and rye cultivated by Croatian producers during the whole investigative period. However, although a low percentage of the positive tested rye samples with EAs was determined, the content of ergot sclerotia in two of the samples was higher than is allowed by legislation for foodstuffs. Because the levels of these mycotoxins and ergot sclerotia content can vary depending on a number of factors, further research of them is required over a longer period of time and under different cereal cultivation and processing conditions.


Introduction
Ergot alkaloids (EAs) are mycotoxins produced by several species of fungi of the genus Claviceps. Among them, Claviceps purpurea is the most widespread in Europe, whereas other species mostly occur in tropical and subtropical areas. This species has been found in more than 400 monocotyledonous plants, grasses and many economically important cereal grains, such as rye, wheat, triticale, barley, millet and oats (Haarmann et al., 2009;EFSA, 2017). All these cereals represent an important part of the daily human diet and are widely consumed by the population, including infants, children, adolescents, and the elderly, and therefore have to be safe (Agriopoulou, 2021). Today over 50 EAs are known, among which ergometrine, ergotamine, ergosine, ergocristine, ergocryptine (mixture of α-and β-isomers), ergocornine and their corresponding -ine epimers, and specifically monitored in European countries (EFSA, 2012).
The entire life cycle of Claviceps purpurea is complex and starts when windborne ascospores land on the featherlike stigmas of susceptible wild, forage grasses in spring (Richard, 2007;Miedaner and Geiger, 2015). The fungus gains entry into the host plant from sclerotia that are present in the soil. The infecting fungal elements (ascospores) are ejected forcibly but also are assisted in gaining access to the host plant by the wind and splashes of rain. Ascospores infect the seed heads of plants during the flowering period, and replace the developing fungal tissue with an alkaloid wintering body, recognized as ergot, an ergot body or sclerotia. The sclerotia are brown to purple-black in colour and contain EAs (Krska and Crews, 2008;Mulder et al., 2015). They can be harvested with the grain and if not eliminated before final processing and thus can end up in food or feed produced from contaminated grains (Richard, 2007).
Sclerotia have a negative impact on the quality of grasses and grains due to the presence of different classes of alkaloids, which are considered undesirable substances for human and animal health. However, advances in science and improvements in the application of agronomy and technology measures, such as milling and heat treatment that have proven effective in the reduction of ergot sclerotia, have eradicated the outbreak of major ergotism epidemics in recent decades (EFSA, 2005(EFSA, , 2012. On the other hand, EAs have been industrially produced and used as active pharmaceutical drugs in medical applications, predominantly in obstetrics and in the treatment of migraine headaches (Haarmann et al., 2009; EFSA, 2012). Also, it is known that controls put in place by millers and grain processors include processes such as physical separation and cleaning through visual supervision, and separation using optical sorters to remove discoloured and misshapen grains also removes the sclerotia.
The physiological effects of EAs have been recognised through the consumption of contaminated cereals, flour and bread. Contaminated feed with toxic levels of EAs has been found to affect the reproductive cycles of pigs, poultry and cattle, causing terminated pregnancies (Coufal-Majewski et al., 2016). They also have been shown to impact digestive systems, resulting in under-performance in weight-gain of farm animals for meat production (Klotz, 2015). When consuming small amounts of contaminated cereals, indigestion occurs, whereas higher amounts cause ergotism, a disease that is manifested in hallucinations, pain and strong vasoconstrictions, which eventually lead to dry gangrene and limb death. In the worst case, kidney and heart failure can occur, causing death. Cases of domestic animal poisoning have been reported in cattle, horses, sheep, pigs and poultry (Nicholson, 2012).
Recently it has been shown that EAs are more common in foods and feed containing rye and wheat (Agriopoulou, 2021). Taking into account this evidence, the toxicity of EAs, the lack of research on ergot sclerotia and EA occurrence in Croatia, and the newly prescribed maximum levels (MLs) recently defined by the European legislation (Regulation (EU) 2021/1399), the aim of this study was to investigate the occurrence of ergot sclerotia and EA contents in wheat and rye grain samples taken from Croatian cereal producers during the 2021 harvest season.

Sampling and sample preparation
During 2021, a total of 64 samples of wheat (n=53) and rye (n=11) were collected from cereal producers located in central and eastern Croatia. Sampling and sample preparation were performed in accordance with Commission Regulation (EC) No 401/2006, which lays down the methods of sampling of the official control of the levels of mycotoxins in foodstuffs, and Commission Regulation (EC) No 152/2009, which lays down the methods of sampling and analyses for the official control of feed. The samples were stored in a cool and dry place. After the determination of ergot sclerotia content, samples were used for further ELISA analysis of EAs. Prepared test portions were ground into a fine powder with a particle size of 1.0 mm using an analytical mill (Cylotec 1093, Tecator, Sweden), and then stored at 4ºC until EA analyse.

Visual method for the determination of ergot sclerotia
Ergot sclerotia were determined according to IAG method-A4 "Method for the Determination of Ergot (Claviceps purpurea Tul.) in Animal Feedingstuff" (IAG, 2008). Qualitative determination was performed macroscopically on ergot sclerotia fragments. Quantification was performed by selecting and weighing the ergot sclerotia fragments per kilogram of sample. Ergot sclerotia were identified based on their characteristic features. The identification was facilitated by comparison with reference material (from previous proficiency testing materials) and the existing descriptions. Ergot sclerotia are elongated with a length of up to several centimetres, dark violet to black in colour. The shape is similar to cereal kernels. They only consist of fungal hyphae. The material identified as ergot was selected and weighed. The amount of ergot fragments in mg/kg of the sample was calculated using the following formula:

ELISA performance
Extraction procedure and ELISA test were performed according to the manufacturer's instructions with the Ergot Alkaloids ELISA kit (Randox, Crumlin, United Kingdom) which determines the total content of ten EAs. For each 2.5 g sample of milled cereals, 25 mL extraction solution from the ELISA kit was added, then shaken vigorously on the vortex for 1 min and rolled "head over head" for 15 mins. The content was then centrifuged at 1500 rpm for 2 mins and the extracted samples were diluted with a diluted washing buffer at 1:4 (200 µL + 800 µL) for wheat samples and at 1:9 (100 µL + 900 µL) for rye samples. Samples were mixed thoroughly and prepared for application on an ELISA microtitre plate. In the plate wells, pipetting of standards, samples, quality control and conjugation, and all incubation and washing steps were performed according to manufacturer's instructions. Any colour reaction was stopped by the addition of 100 µL stop solution per well. After the last step in which the colour changed from blue to yellow, the optical density was measured at 450 nm within 10 mins. The ELISA assay was performed using a Chemwell 2910 automated analyser (Awareness Technology, Inc., USA).
All chemicals and solvents used during the extraction process were of analytical grade. Quality control of the implemented ELISA method was performed using the reference QC material of rye flour T22180QC (Fapas, York, England) with an assigned value in total 10 EAs, ranging from 266-572 µg/kg, and a mean value of 419 µg/kg. On this QC material, trueness was determined by analysing six replicates of this material, and repeatability was determined using the same steps on two other occasions in the same analytical conditions and also in six replicates per analysis.

Calculation and statistical analysis of EAs
The total concentration of EAs (µg/ kg) was calculated from a six-point calibration curve using a mathematical interpolation. The results of EA sample concentrations were multiplied by a dilution factor of 50 for wheat and 100 for rye. The obtained values were further corrected for the mean value of recovery determined by the kit manufacturer's instructions through validation at three levels of fortification (50, 75 and 150 µg/ kg for wheat and 125, 250 and 500 µg/kg for rye).
Statistical analysis was performed using the Statistica Software Ver. 10.0 (StatSoft Inc. 1984 to 2011, USA), with the statistical significance level set at 95% (P=0.05).

Results and Discussion
As data on the ergot sclerotia and EAs presence in different cereals are very scarce for Croatia, this study investigated their occurrence in wheat and rye, as these cereals are most commonly contaminated with EAs in other countries (Agriopoulou, 2021). The European Commission recommends the monitoring of EAs in food and feed (2012/154/EU) for Member States, requiring them to gather reliable data on the year-to-year variations of these mycotoxins in cereals and cereal products intended for human consumption, and animal consumption. It is recommended that at least the following six main EAs and corresponding epimers (12 in total) be tested: ergocristine, ergocristinine, ergotamine, ergotamine, ergocryptine, ergocryptinine, ergometrine, ergometrinin, ergozine, ergozinine, ergocornine, and ergocorninine. According to the same recommendation, Member States should also determine, where possible, the content of ergot sclerotia in a sample, to improve the knowledge of the relationship between the presence of ergot sclerotia and the concentrations of individual EAs. In this study, the total EA content was monitored in unprocessed wheat and rye produced in 2021 by Croatian producers, so the investigated samples were not cleaned in an industrial ergot sclerotia removal process. The ELISA method used for EAs determination was validated by the ELISA kit manufacturer, and prior to the determination of EA concentrations in the samples in this study, trueness and repeatability were analysed/checked in the laboratory with CRM material. The results of validation and quality control of the implemented ELISA method for determination of EA concentrations are shown in Table 1. Based on the all validation and quality control results, the implemented ELISA method was recognised as suitable for the efficient quantitative determination of EA concentrations in the sampled cereals.
The results of the obtained ergot sclerotia particle content and total EA concentrations in wheat and rye samples obtained in this study are shown in Table  2. Two rye samples exceeded the ML for the content of sclerotia (259 mg/kg and 536 mg/kg) for foodstuffs (200 mg/kg; Regulation (EC) 1881/2006), but were below the ML defined for feedstuffs (1000 mg/kg; Directive 2002/32/EC). No ergot sclerotia were found in any of the wheat samples.
EA contamination was detected in two rye samples from which the ergot sclerotia were isolated (Figure 1). EA presence was determined in total in a low percentage of the analysed cereals (mean 4.7%), with a determined low number of all positives   In earlier European studies of EAs in cereals, as summarized by the European Food Safety Authority (EFSA, 2017), the profile and concentration of individual EAs varies considerably in different grains and batches of grain. The occurrence of EAs was investigated on 4,528 food samples collected between 2011 and 2016, with almost 70% sampled between 2014 and 2015 in 15 different European countries, with more than 50% coming from the Netherlands and around 28% from Germany. Among the EAs, ergotamine, ergocristine and ergosine were quantified in about 15% of samples, while ergometrinine and ergocorninine were only reported in just 6% and 8% of samples, respectively. Overall, in the food samples, only 11% of the analytical results were quantified. This general result is in accordance with the results of this study, although some samples of the large European study had significantly EA concentrations than reported here. In the same report by EFSA, the study on a total of 654 feed samples collected in five European countries during the same period showed that in more than half of these samples, not a single EA was found above the limit of quantification (LOQ) of the implemented analytical method (n = 352).
In other studies, the occurrence of EAs have also been reported. Malysheva et al. (2014) reported high rates of EA contamination in rye-and wheatcontaining foods in northern Europe. Rye-containing foods were found to have 1121 µg/kg and wheat-containing foods were found to have 591 µg/kg EAs, respectively. In a Swiss study, 16 The period of rainfall and mainly the period of flowering are the two critical periods in which the fungus parasitises the seed heads of living plants (Guo et al., 2016). It was concluded that EAs could be detected in grains even when ergot bodies were removed by a hand-cleaning procedure (Shi et al., 2019). The sclerotia are harvested together with the cereals or grass and can thus lead to contamination of cereal-based food and feed products with EAs (Storm et al., 2008). Moreover, during the milling of cereals, sclerotia are not easily separated from the healthy grains, but instead they are fragmented and can be transferred to the finished product such as flour (Pitt and Miller, 2017).
Cleaning protocols become even more unreliable when fungal sclerotia are produced in dry climates, given the similar size to grains, making them difficult to remove (Krska and Crews, 2008). It was found that the cleaning and processing of cereals can remove up to 82% of sclerotia (Agriopoulou, 2021), meaning they are not completely eliminated and the problem of EA contamination of cereals can still be detected at high levels (EFSA, 2005). For example, an EA concentration of 7255 mg/kg was reported in rye flour from Germany (Krska and Crews, 2008). Agriopoulou (2021) reported that it is known that ergot sclerotia can be removed from cereals by grain cleaning machines based on photocells, though this is not standard in all countries and milling companies and the integrated system of a management's approach that includes all the individual control strategies is important to mitigate EAs in cereals and cereal-based foods.
All the mentioned studies show that the EA contamination has a sporadic incidence. It is clear therefore that many factors are involved in EA occurrence, and that their concentrations vary greatly between fungal strains, geographic regions, host plants and regional/ local weather, all of which show that cool, damp weather favours ergot by promoting the germination of sclerotia.

Conclusions
During the study period, ergot sclerotia and the detected EA contents indicated a low-level of contamination of wheat and rye cultivated by Croatian producers. Since EA levels can vary depending on a number of factors, further research of these mycotoxins and ergot sclerotia content is required over a longer period of time and under different cereal cultivation and processing conditions. Additional research should focus on the impact of geographic regions, host plants and regional/local weather, as well as the technological processing procedures (grinding, heat treatment, etc.) on EA concentrations in the final product. These are the most important factors involved in EA occurrence in cereals and byproducts. The ultimate goal is to ensure the health safety of food and feed in terms of the occurrence of these mycotoxins and their toxic impacts on human and animal health.