Alessia Lorè

Development of a microcantilever-based immunosensing method for mycotoxin detection

Mycotoxins, such as aflatoxins and ochratoxin A, are presently considered as the most important chronic dietary risk factor, more than food additives or pesticide residues. Therefore, the serious health and economic consequences of mycotoxin contamination have created the need for rapid, sensitive, and reliable techniques to detect such dangerous molecules within foodstuffs. We here report on the development of an innovative immunosensing method for mycotoxin detection, based on antibody-immobilized microcantilever resonators, a promising label free biosensing technique. A considerable part of the work is devoted to show the effect on microcantilever resonance frequency of the composition of the incubation buffer, as well as of the washing and drying procedure. We show the feasibility of using microcantilever resonator arrays to effectively identify total aflatoxins and ochratoxin A, at low concentrations (3 ng/mL and less than 6 ng/mL, respectively), with relatively low uncertainty (about 10%) and good reproducibility for the same target concentration. Furthermore, the developed immunosensing method shows a limited cross-reactivity to different mycotoxins, paving the way to a highly specific technique, able to identify different mycotoxins in the sample. To our knowledge, this work represents the first example in literature of successfully immunodetection of low concentrations of multiple mycotoxins by microcantilever resonator arrays.

Ochratoxigenic Black Species of Aspergilli in Grape Fruits of Northern Italy Identified by an Improved PCR-RFLP Procedure

A collection of 356 isolates of Aspergillus spp. collected during 2006 and 2007 from grapevines in northern Italy were identified through Internal Transcribed Spacer based Restriction Fragment Length Polymorphism (ITS-RFLP) and tested for ochratoxin A (OTA) production. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, HinfI and RsaI, distinguished five different RFLPs. From each pattern, three samples were sequenced and the nucleotide sequences showed different species corresponding to Aspergillus niger, A. carbonarius, A. tubingensis, A. japonicus and A. aculeatus. By comparing the sequences of the ITS regions, also the uniseriate species A. japonicus and A. aculeatus could be differentiated by HinfI digestion of the ITS products. Among the aspergilli, A. niger was the major species associated with grapes during 2006 (57.4%), while A. carbonarius was the major species during 2007 (46.6%). All the strains of Aspergillus were tested for their ability to produce OTA on Yeast extract sucrose medium (YES), as it was tested as an optimal substrate for the evaluation of OTA production by black aspergilli. Out of 356 isolates, 63 (17.7%) isolates produced OTA ranging from 0.05 to 3.0 µg mL−1. Most of the ochratoxigenic isolates were A. carbonarius (46) in both years, but also some strains of A. tubingensis (11) and A. japonicus (6) produced lower amounts of OTA.

Specific PCR primers for the detection of isolates of Aspergillus carbonarius producing ochratoxin A on grapevine

Aspergillus carbonarius is the main fungus responsible for ochratoxin A (OTA) production on grapes and in wine. Fungal polyketide synthases are involved in OTA biosynthesis in Aspergillus species. The ketosynthase (KS) domain of an A. carbonarius gene encoding polyketide synthase (pks) was isolated, cloned and sequenced. The nucleotide sequence showed high similarity to KS domains isolated from other Aspergillus species. The sequence was used to design a new set of primers in order to identify potential producers of OTA from among isolates of A. carbonarius. The primers amplified all the A. carbonarius strains tested specifically, and did not amplify any other species of Aspergillus or Penicillium normally found on grapes or involved in OTA biosynthesis. Further, gene expression was related to OTA production. Both gene transcription and levels of the mycotoxin were higher when A. carbonarius was grown on YES medium at 15°C and 30°C, whereas a low transcription and mycotoxin presence were observed when the fungus was grown on PDB medium at the same temperatures. No transcription or OTA production were observed when the fungus was grown on either YES or PDB at 10°C. The primers will be useful for the detection of ochratoxigenic strains of A. carbonarius both in vineyards and during wine production.

First Report of Penicillium griseofulvum Causing Blue Mold on Stored Apples in Italy (Piedmont)

In northern Italy, blue mold can occur generally on apples after 3 months of storage under controlled atmospheres. The mold can be caused by Penicillium griseofulvum Dierckx (synonym P. urticae Bainier). During 2008, several postharvest fruit rots were observed on apples (cv. Golden Delicious) after 180 to 240 days of storage at 1°C. Approximately 8% of the fruits showed blue mold. Apples had been cultivated in Aosta (Aosta Valley Region) and Lagnasco (Piedmont Region). Infected fruits showed soft, watery, brown spots enlarging rapidly at 20°C. There was a distinct margin between soft rotted flesh and firm healthy tissues. Under high humidity, masses of blue-green spores formed on the surface of the lesion. Apple fruit excisions from the margin between the healthy and diseased tissues were plated on potato dextrose agar (PDA), pH 5.6. The recovered fungus produced abundant mycelium and conidia, with the colonies attaining a diameter of 2.0 to 2.4 cm after 7 days at 20 ± 2°C on PDA. Colonies were mostly yellow-green, with a yellowish-to-orange brown underside. Conidiophores were mononematous or loosely synnematous, hyaline, with branches strongly divergent. Phialides were cylindrical with a very short neck. Conidia were ellipsoidal, sometimes subglobose, 2.5 to 3.5 × 2.2 to 2.5 μm, hyaline to greenish. Preliminary morphological identification of the fungus (2) was confirmed by PCR using genomic DNA extracted from mycelia of pure cultures. Two sequences, obtained through the amplification of ribosomal region ITS1-5.8S-ITS2 (1), were blast searched in GenBank and showed 99% sequence coverage and 99% similarity to ribosomal sequences of P. griseofulvum. Two sequences were deposited in GenBank with Accession Nos. HQ012498 (a strain from Aosta Valley) and HQ012499 (a strain from the Piedmont Region). Pathogenicity was tested on 20 ripe fruits each of four apple cultivars (Golden Delicious, Red Chief, Granny Smith, and Royal Gala). Fruits were surface sterilized with 1% sodium hypochlorite. Conidial suspensions (30 μl of 105 conidia/ml) of the fungus were placed on artificial wounds generated on the apple surface. Control fruits were treated with sterile water. Seven days after inoculation, the symptoms were reproduced on the four cultivars and P. griseofulvum was reisolated on PDA from the inoculated fruits of all four cultivars. Control fruits were symptomless. An analysis using high-performance liquid chromatography with diode array of the rotting tissues associated with inoculated fruits of all four cultivars (4) confirmed, as in the case of other strains of P. griseofulvum, the production of the mycotoxin patulin (12.1 to 44.4 mg kg-1). Previously, P. griseofulvum was reported on apple in other countries such as the United States (3), Japan, Egypt, and Brazil. To our knowledge, this is the first report of P. griseofulvum on apples during storage in Italy. References: (1) R. Nilsson et al. FEMS Microbiol. Lett. 296:97, 2009. (2) R. A. Samson and J. L. Pitt. Integration of Modern Taxonomic Methods for Penicillium and Aspergillus Classification. Harwood Academic Publishers, Singapore, 2001. (3) P. G. Sanderson and R. A. Spotts. Phytopathology 85:103, 1995. (4) D. Spadaro et al. Food Addit. Contam. B 1:134, 2008.