A new species of Penicillium section Ramosa from Tunisian Apples

One of the limiting factors that influence the fruits economic chain value is the relatively short shelf-life period caused by fungal infections. The symptoms of fungal infection may be observed at different times but many fungi may remain dormant for varying periods until post-harvest favorable conditions become available for their development. In a mycotoxin contamination survey of apples from markets in Tunisia, 54 Penicillium strains were isolated. However, two isolates could not be assigned to any described species based on morphological and molecular phylogenetic analyses. The aim of this study was the characterisation and description of this new putative species. For morphological analyses, MUM 17.62 and MUM 17.80 were inoculated in triplicate in CYA, YES, G25N, CSN and MEA media and incubated in the dark at 25 ºC for 7 days. CYA plates were also incubated at 30ºC and 37ºC. Colony size was measured and for microscopy analysis fungi grown in MEA was used. Multilocus sequence analysis (MLSA) was performed through comparison of partial β-tubulin (benA), calmodulin (cmd) and nuclear ribosomal internal transcribed spacer (ITS) region with sequences available in GenBank derived from type strains of Penicillium species. All the sequences were aligned and phylogenetic trees were assembled using MEGA. For MUM 17.62 and MUM 17.80 morphologically, the colonies growth was very restricted in the different media. No growth was observed on CYA at 30 ºC and 37 ºC. The strains show slight differences in green colour. Both present velutionous, sulcate and irregular colonies in MEA. Microscopically, the conidiophores are biverticillate and conidia ellipsoidal. MLSA revealed that the two strains belong to Penicillium section Ramosa. Fingerprinting using the M13 microsatellite showed that the two strains are not clones and analysis of the isoepoxydon dehydrogenase (idh) gene revealed that they lack the ability to produce patulin. In summary, in terms of multigene phylogeny the two strains are closely related to P. lenticrescens, P. chroogomphum and P. soppii of the section Ramosa however they well-circumscribe a sp. nov. coined as Penicillium tunisinus.info:eu-repo/semantics/publishedVersio

LC-MS/MS methodology for simultaneous determination of patulin and citrinin in urine and plasma applied to a pilot study in colorectal cancer patients

Biomarker-driven research has been proposed as a successful method to assess the exposure of individuals to xenobiotics, including mycotoxins, through estimation of their metabolites in biological fluids. A methodology to determine patulin (PAT) and citrinin (CIT) in human urine and plasma using liquid chromatography coupled to tandem mass spectrometry was developed and validated in the present study. Selectivity/specificity, linearity, limit of detection and quantification, apparent recovery, intraday- and interday-precision and measurement uncertainty were investigated for validation purposes. Finally, the method was used to analyze human urine (n = 100) and plasma (n = 100) case-control samples, where 50 samples originated from colorectal cancer patients and 50 from age/sex-matched controls. This case-control study revealed that PAT was not detected in urine samples, however occurred in 25% of the analysed plasma samples with an average concentration of 11.62 ± 6.67 ng/mL in the positive samples. CIT was found in urine samples (74%) and plasma samples (36%) with average concentrations in the positive samples of 0.45 ± 0.24 ng/mL and 0.49 ± 0.2 ng/mL respectively. No statistically significant difference of PAT and CIT concentration among colorectal cancer and control patients (p > 0.05) was observed.Highlights: Development and validation of an LC-MS/MS method to determine patulin and citrinin in biological fluids; Patulin was detected in plasma (25%) for the first time; Citrinin was present in 74% of Tunisian urine samples; Citrinin was present in 36% of Tunisian plasma samples.The authors acknowledge the support of the Tunisian Ministry of Education and Scientific Research, the Toxicology and Environment Research Laboratory LR12SP07 (CAMUR, Tunis, Tunisia) and MYTOXSOUTH (https://mytoxsouth.org) (Ghent University and Global Minds International Thematic Network). Dr. Arnau Vidal expresses thanks to The Research Foundation Flanders(Wetenschappelijk Oderzoek- Vlaanderen, FWO) for the postdoctoral grant (12Y1719N).info:eu-repo/semantics/publishedVersio

Penicillium species identification and new insights on mycotoxins in food commodities (apples, chilli and cheese)

The 10th International Palestinian Conference of Laboratory Medicine and The 15th Arab Conference of Clinical BiologyAmong certain groups of filamentous fungi that produce mycotoxins, relevant contaminates in food, the genus Penicillium is of great importance. Penicillium is ubiquitous in nature and inevitable, although it can be controlled from the field to the fork. Mycotoxins are fungal secondary metabolites that cause sickness or death in people when ingested, inhaled, and/or absorbed. Major mycotoxins associated with common penicillia are: Ochratoxin A (P. verrucosum and P. nordicum), patulin (P. expansum), citrinin (P. expansum), cyclopazonic acid (P. camemberti), penicillic acid (P. radicicola) and secalonic acid D, F (P. griseofulvum). Penicillia identification is time-consuming and sounder polyphasic identification, which includes phenotypic and genotypic approaches, is recommended. However, in many laboratories, the standard character for identification is still morphology. Taking this into account, results from Penicillium species isolated from Tunisian apples, Chilean traditional chilli (Merkén), Italian cheeses and their mycotoxin profiles (patulin and ochratoxin A) will be presented in this work. For morphological analyses, isolates were inoculated in triplicate in different media. Fungi grown in MEA for colony and microscopy analyses were used. Multilocus sequence analysis was performed through comparison of partial -tubulin, calmodulin and ITS with sequences available in GenBank. Specific primers for genes involved in the mycotoxins pathways were used for PCR amplification. After extraction the mycotoxins were quantified using HPLC-FLD (fluorescence detection). From Tunisian apples isolates, a novel species Penicillium tunisiense of section Ramosa is proposed. This is not a patulin producer with the idh gene negative in contrast with the other dominant P. expansum isolates. In addition, ochratoxigenic strains P. verrucosum and P. crustosum were isolated from chilli and cheese samples, respectively, and characterised with genes involved this mycotoxin production. Our findings show that mycotoxigenic Penicillium strains, as food contaminants, remain an important field of study and more knowledge needs to be learned.info:eu-repo/semantics/publishedVersio

Penicillia diversity from food identified polyphasically, including mycotoxin production

Portuguese Foundation for Science and Technology (FCT). It was under the scope of the strategic funding of the UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01–0145-FEDER-006684) and the BioTecNorte operation (NORTE-01–0145-FEDER-000004), funded by the European Regional Development Fund through Norte2020—Programa Operacional Regional do Norte (Portugal