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Two similar Penicillium isolates could not be identified as previously described species in a survey of orchard apples from Tunisia for patulin-producing fungi. These isolates are described as novel species using multilocus DNA sequence analysis of partial β-tubulin, calmodulin and nuclear ribosomal internal transcribed spacer regions; and morphological, physiological and biochemical characteristics. The isolates were considered negative for patulin production since the IDH gene fragment was not detected and the compound detected at the same retention time of patulin (14.9 min) showed a different UV spectrum using U-HPLC/UV-DAD. In terms of phylogeny, the two isolates clustered with Penicillium section Ramosa and are closely related to Penicillium chroogomphum, Penicillium lenticrescens and Penicillium soppii. Furthermore, their macro- and micromorphological traits differed from these species. Hence, the isolates represent a novel species in Penicillium section Ramosa and the name Penicillium tunisiense sp. nov. is proposed, with the type strain MUM 17.62 (=ITEM 17445).

We read with interest in your journal that Penicillium expansum strains S3, S31 and S87 (Rharmitt et al., 2016) were considered negative for possession of the isoepoxydon dehydrogenase (idh) gene for patulin (PAT) production and positive for PAT production. This led to speculation that an alternative pathway was involved in patulin production. However, the authors did not provide the diode array (DA) UV spectra of the HPLC peaks assigned to PAT, which assist in confirming its identification. UV spectra could usefully be provided in papers when DA analysis has been performed especially for unusual results such as these: At least a statement to say whether the spectra were identical, or not, to the PAT standard is required. One of the current authors (RRMP) reported for the first time the PCR test to assess mycotoxigenic fungi using the idh gene coupled with PAT detection by (a) TLC (Paterson et al., 2000; Paterson et al., 2003) and (b) HPLC UV (Paterson et al., 2003). idh negative and PAT positive strains were not detected. Similarly, Luque et al. (2011) did not detected this combination using micellar electrokinetic capillary electrophoresis, and HPLC-MS for PAT detection. The principle was employed in reverse transcription real-time PCR for idh (De Clercq et al., 2016). The current authors found that a compound which has (a) the same retention time as PAT, but (b) a different UV spectrum using Ultra HPLC DA detection (UHPLC DAD) in a Penicillium strain (Fig. 1), during recent screening work to detect patulin from penicillia isolated from Tunisian apples. Hence, the strain was negative for PAT detection. This was confirmed by the strain being idh negative. Incidentally, the chromatogram for P. expansum (S13) in Rharmitt et al. (2016) had a co-eluting metabolite with PAT as deduced by the peak having a distinct shoulder. The UV spectrum of this sample would probably be different from pure PAT although this was unreported. Perrone et al. (2017) recommended MS and NMR to establish correct ochratoxin A (OTA) production in species not associated conventionally with this mycotoxin. The questionable data were from HPLC-FLD (fluorescence detection) where other fluorescent compounds with the same retention time could be misidentified as OTA. Few laboratories may be able to afford MS and NMR and all methods need assessing on a cost-benefit basis to indicate what is suitable for most laboratories (Paterson et al., 2018). Rharmitt et al. (2016) employed HPLC DAD but the analytical potential of the technique was unfulfilled. Furthermore, Rharmitt et al. (2016) suggested the primers they employed may not have bound to the target DNA hence the negative idh result, which also undermines their premise of a different pathway for PAT. Fig. 1. UHPLC chromatograms and UV spectra for the (i) idh negative and patulin negative Penicillium strain (MUM 17.62) and (ii) patulin (PAT) standard. A, B are the chromatogram and UV spectrum of the PAT standard respectively. C is the false PAT positive chromatogram and D is the PAT negative UV spe...