How to grow ergot fungus on rye bread

1. Introduction

Mycotoxins are fungal toxin metabolites produced by a wide array of fungi. Several species of Aspergillus, Penicillium and Fusarium genera are responsible for the production of the mycotoxins of most concern (e.g., aflatoxins, ochratoxins and fumonisins among others). However, the genus Claviceps, which is a plant pathogen, is able to produce a group of mycotoxins, ergot alkaloids (EAs), whose effects have been known since the Middle Age (ergotism) [1]. Among the several species of Claviceps, the most important is the Claviceps purpurea, which is known to infect more than 400 plant species of monocotyledonous plants, grasses and economically important cereal grains such as rye, wheat, triticale, barley, millet and oat [2]. The fungal ascospores are windborne; they land, attach and germinate on the pistil surface of the plant during anthesis, initiating host-pathogen interaction that leads to a sclerotia production [3]. Sclerotia are the fruiting structure that achieve maturation after five weeks of fungal infection and contain alkaloids [3]. The presence of sclerotia has a double effect, on one hand sclerotia have a negative implication reducing the quality of grasses and grains because of the presence of different classes of alkaloids, being considered undesirable substances for animal and human health; on the other hand, because of the pharmacological effects, EAs have been industrially produced and used as active pharmaceutical drugs in medical applications predominantly used in obstetrics at the beginning of the 20th century, and in the treatment of migraine headaches [2,4]. EAs toxicity properties are known and have long since been characterised, past episodes of Claviceps purpurea-associated intoxication have been registered worldwide but more recent outbreaks of human poisoning by grain crops contaminated with sclerotia in developing country countries indicate that ergotism is still a disease of public health importance [5].

In 2012, upon a European Commission request, the European Food Safety Authority (EFSA) delivered a scientific opinion EAs in food and feed [4] where the establishment of a group acute reference dose (ARfD) of 1 μg/kg body weight (bw) and a group tolerable daily intake (TDI) of 0.6 μg/kg bw per day for the sum of the EAs were reported. These health-based guidance values (HBGVs) are based on the conclusions of the Contaminant Panel that defined the vasoconstrictive effect on tail muscular atrophy in rats as the critical effect for hazard characterisation [4].

With the aim to protect human and animal health, European Union (EU) measures are set to keep contaminants at levels that are toxicologically acceptable with maximum limits at defined levels, which are reasonably achievable by following good agricultural, fishery and manufacturing practices and taking into account the risk related to the food consumption [6]. In the case of EAs it happened that the scarce information available on the occurrence and on the correlation between sclerotia presence and EAs content did not allow the setting of appropriate limits. Hence, the EU published a recommendation [7] to ask to the member states and professional stakeholder organizations to produce data on ergot alkaloids including occurrence data and specific information on the relationship between the presence of ergot sclerotia and the level of individual EAs in food and feed, and to report to EFSA their findings. Presently, EAs are not regulated as such, but ergot sclerotia content is set by the Commission Regulation (EC) No. 1881/2006 in unprocessed cereals (with the exception of corn and rice) used for humans [6], the amount of ergot in food being restricted to 0.05% at a maximum. In addition, rye ergot (Claviceps purpurea) in feed materials and compound feed containing unground cereals is also restricted to a maximum content of 1000 mg/kg relative to moisture content of 12% [8]. The setting of appropriate and achievable maximum levels is in the pipeline of the European Commission and should be based on the sum of 12 EAs (ergometrine, ergosine, ergocornine α-ergocryptine, ergotamine and ergocristine and reletive epimers, ergometrinine, ergosinine, ergocorninine, α-ergocryptinine, ergotaminine and ergocristinine).

Liquid chromatographic (LC) methods with fluorometric or mass spectrometric detector (FLD or MS/MS) are available for the analysis of EAs with limit of quantification/detection (LOQ/LOD) in agreement with those to be achieved for the individual epimers as requested in the framework of the proposed maximum limits, 10 µg/kg for cereal grains, 4 µg/kg for cereal milling products and 2 µg/kg for final cereal based consumer products. Ranges of LOQ/LOD between 0.01-0.5 μg/kg for HPLC-FLD or 0.15-2.78 μg/kg for LC-MS/MS have been achieved and reported in literature [9,10,11,12]. The chromatographic run length needed for the separation of the 12 EAs and sample preparations represent an analytical constrain for the setting of HPLC-FLD methods [13]. More easily, LC-MS/MS methods guarantee the determination of the 12, usually with positive mode electrospray ionization (ESI+). Since epimerization is hard to control during sample extraction and analysis, it is always the best option to analyse both -ine and -inine form and specify the EAs content as sum. Enzyme-linked immunosorbent assay (ELISA) techniques have been developed and represent an option for a rapid and low-priced screening of EAs in agricultural crops and grain flour. However, being less specific and less accurate than LC-FLD or LC-MS/MS methods, they are less attractive than dipstick type tests, which represent the preferable option for testing EAs presence in the field.

Occurrence data on EAs has increased in the last five to eight years and EFSA could deliver a risk assessment opinion thanks to the availability of analytical results derived from food samples collected between 2011 and 2016 in 15 different European countries. More than 50% of the data came from the Netherlands and around the 28% from Germany, Italy being very scarcely represented [14]. From a general overview of the EFSA opinion, the highest levels of EAs in food were found in rye and rye-containing commodities, in particular the “rye milling products” category showed an upper bound-lower bound (LB-UB) range of 198-239 μg/kg. Among processed food, the highest levels of EAs were found in “mixed wheat and rye bread and rolls” (33-82 μg/kg), “rye bread and rolls” and “rye flakes” with ranges of LB-UB of 29-67 μg/kg and 35-83 μg/kg, respectively [14].

The aim of this study was to make available an EAs survey on grain and grain-based products namely, wheat and rye flours and bread from the Italian market. An LC-MS/MS method for the determination of the six principal alkaloids (ergometrine, ergosine, ergocornine, α-ergocriptine, ergotamine and ergocristine) and the relative epimers, -inine forms (ergometrinine, ergosinine, ergocorninine, α-ergocryptinine, ergotaminine and ergocristinine) was used for the monitoring of wheat- and rye-based products, after performance characteristics verification.

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