Penicillium taxonomy is not easy for the inexperienced, and compared to Aspergillus it is a more diverse genus, in terms of numbers of species and range of habitats [32]. At present, P. verrucosum and P. nordicum are the only OTA producers known and accepted in this genus, despite some reports on OTA production by other species [33,34]. Penicilliumcasei and P. mediolanense are synonyms for P. verrucosum and P. nordicum, respectively [34]. Nevertheless, different examples of incorrect citations of some Penicillium spp. producing OTA (e.g., P. cyclopium, P. viridicatum, P. chrysogenum) have been recently listed [23,34]. It is worth bearing in mind that in the last century, OTA producers in this genus were classified as P. viridicatum for many years. Main species concepts for P. viridicatum, P. verrucosum and P. nordicum are summarized in Table 1.

Penicillium verrucosum and P. nordicum have common morphological characteristics such as very similar colony diameters on many culture media or rough stipes (Figure 1). These are slow growing species of the subgenus Penicillium, which is by far the most difficult taxonomically, both because there are numerous species and because apparent differences between species are small. Many species classified in this subgenus are morphologically similar, and identification using traditional morphological techniques remains difficult [32]. Recently, Frisvad and Samson [33] pointed out that a polyphasic approach, including a combination of DNA sequences, extrolite production and other phenotypical characters, is necessary to classify and identify species of Penicillium subgenus Penicillium. These authors keyed a total of 58 species in this subgenus. Many of these species are very common, being associated mainly with stored foods. The OTA producing species are placed in the series Verrucosa of the section Viridicata [33].

The series Verrucosa is monophyletic based on the phylogenetic analysis of partial β-tubulin sequences of strains representing all accepted species in this subgenus. This series includes two subclades, one consisting of the non OTA producing species P. thymicola and the other subclade consisting of strains of the two OTA producing species, P. verrucosum and two genetic groups of P. nordicum strains [42]. A similar genetic profile among these species was detected using CO1 (mitochondrial cytochrome c oxidase 1) DNA barcoding [43]. In this study, P. verrucosum and three strains of P. nordicum had identical CO1 barcodes, whereas four P. nordicum strains showed their own unique barcode. Currently, P. viridicatum is placed in the series Viridicata of the section Viridicata [33]. This series also includes other incorrectly cited OTA producing species such as P. aurantiogriseum, P. cyclopium or P. polonicum. Some years before, Larsen et al. [35] had described two distinct groups of OTA producing Penicillium strains based mainly on differences in secondary metabolite profiles. Some strains grouped with the ex-type culture of P. nordicum ATCC 44219 were classified as P. nordicum. Other strains grouped with the type culture of P. verrucosum NRRL 965 were classified as P. verrucosum.

These species are ecologically different. P. nordicum is generally recovered from meat and cheese products whereas P. verrucosum is recovered mainly from plant-derived material. Besides, most of the isolates of this latter species have a characteristic dark brown reverse color on Yeast Extract Sucrose agar (YES), whereas almost all the P. nordicum strains show a pale, creamy or dull yellow reverse color in this culture medium [33,35]. The colony pattern of P. viridicatum is different on YES (Figure 1). Among other differences, Frisvad and Samson [33] considered P. verrucosum among the species always negative (no reaction) or occasionally producing a yellow reaction for the Ehrlich test and P. nordicum among the species with a yellow reaction. These colored reactions are related to the production of some alkaloids. Castellá et al [44] confirmed these two groups of OTA producing strains in the genus Penicillium by Random Amplified Polymorphic DNA (RAPD) (Figure 2) and Amplified Fragment Length Polymorphism (AFLP) analyses, which are useful to differentiate these two species. However, the analysis of the ITS-5.8S rRNA gene sequences of these strains was not able to discriminate these two groups, indicating a close phylogenetic relationship between P. verrucosum and P. nordicum.

Penicillum verrucosum is an important ochratoxigenic species because it is the major producer of OTA in cereals such as wheat, barley, oats and rye, in temperate and cold climates. This species is the main source of OTA contamination in cereals associated to the porcine and avian nephropathy detected in temperate and cold countries such as Denmark, Sweden, Canada or the United States [45,46]. An association of OTA with the human Balkan endemic nephropathy also has been suggested, but to date the etiology of this disease remains unresolved and controversial [47,48]. Contamination of animal feeds with OTA may result in the presence of residues in edible offal and blood products, whereas the OTA contamination in meat, milk and eggs is negligible. However, higher concentrations of OTA may occur in certain local specialties such as blood puddings and sausages prepared with pig blood serum [49]. At present, maximum levels for OTA in meat and meat products are not established in the European Community. However, the consideration of setting a maximum level for OTA for edible offal and blood products is under discussion. In Denmark, since 1978, the contamination of pig meat with OTA has been assessed indirectly by the inspection of pigs’ kidneys for the presence of macroscopic lesions of porcine nephropathy [50]. Nephritis is a common cause of condemnation of pig kidneys in Great Britain, but there are few studies of OTA in cases of porcine nephropathy identified at slaughter in other countries [51]. In France, the first national monitoring program showed that pigs are clearly exposed to OTA and monitoring of pork products and of feed for swine is necessary. Swine, like poultry, are exposed to OTA through their feed, which is composed of cereals such as barley, maize, oats and wheat that are susceptible to contamination by this mycotoxin [52]. In a recent study [53], more than 80% of the combine harvested samples (including rye, barley and wheat, among others) contained P. verrucosum, showing that much grain was contaminated prior to drying and storage. These authors pointed out that the early contamination with this species is a latent risk of OTA production if the grain is not handled properly after harvest.

It is well known that P. verrucosum is much more frequently found on cereals in countries where they occasionally have OTA problems as in the North European countries compared with South Europe where levels of OTA generally seem to be lower or they are not detected [54]. Recently, the occurrence of P. verrucosum on feedstuff and retail wheat flours purchased in the Spanish market was determined [55,56]. This species was the only OTA producing Penicillium species detected in these substrates. Although the occurrence and abundance of P. verrucosum producing species were moderately low in this study, these results confirmed the potential risk of OTA and CIT production in these products and the occurrence of P. verrucosum in South European countries.

Penicillium nordicum is normally present in the air and on the surface of hams in dry-cured ham manufacturing plants in Italy [57]. Recently, both species were also detected on the surface of sausages from northern Italy [58]. In this study, approximately 45% of these samples were positive for the presence of OTA. However, this toxin was not identified inside the dry meat. These authors [58] concluded that the presence of OTA on the surface of sausage constitutes a health risk when moulds are not removed from casings. Penicillium verrucosum has been also isolated from Speck [59] and Istrian dried ham [60].

In contrast, the OTA producing species P. nordicum and P. verrucosum were not isolated during some studies of the mycobiota of the processing areas of North European meat products, such as fermented sausage, liver pâté [61] and smoked dry-cured ham and dry-cured lamb leg [62]. Mould growth is not accepted on most types of North European meat products and is considered as both an economic and aesthetic problem for the producers. At the moment, neither of the two species, P. nordicum or P. verrucosum, have been cited from Spanish dry-cured meat products such as Iberian ham [63] or fermented meat sausages [64].