Early detection of Aspergillus carbonarius and A. niger on table grapes: a tool for quality improvement

Aspergillus carbonarius and A. niger aggregate are the main fungal contaminants of table grapes. Besides their ability to cause black rot, they can produce ochratoxin A (OTA), a mycotoxin that has attracted increasing attention worldwide. The objective of this work was to set up a simple and rapid molecular method for the early detection of both fungi in table grapes before fungal development becomes evident. Polymerase chain reaction (PCR)-based assays were developed by designing species-specific primers based on the polyketide synthases (PKSS) sequences of A. carbonarius and A. niger that have recently been demonstrated to be involved in OTA biosynthesis. Three table grape varieties (Red globe, Crimson seedless, and Italia) were inoculated with A. carbonarius and A. niger aggregate strains producing OTA. The extracted DNA from control (non-inoculated) and inoculated grapes was amplified by PCR using ACPKS2F-ACPKS2R for A. carbonarius and ANPKS5-ANPKS6 for A. niger aggregate. Both primers allowed a clear detection, even in symptomless samples. PCR-based methods are considered to be a good alternative to traditional diagnostic means for the early detection of fungi in complex matrix for their high specificity and sensitivity. The results obtained could be useful for the definition of a ‘quality label’ for tested grapes to improve the safety measures taken to guarantee the production of fresh table grapes.

[1]  C. Vázquez,et al.  Specific detection of Aspergillus carbonarius by SYBR Green and TaqMan quantitative PCR assays based on the multicopy ITS2 region of the rRNA gene. , 2009, FEMS microbiology letters.

[2]  H. W. Platt,et al.  QuantifyingVerticillium dahliae in soils collected from potato fields using a competitive PCR assay , 2002, American Journal of Potato Research.

[3]  A. Hocking,et al.  Fungi and mycotoxins in vineyards and grape products. , 2007, International journal of food microbiology.

[4]  A. Atoui,et al.  Targeting a polyketide synthase gene for Aspergillus carbonarius quantification and ochratoxin A assessment in grapes using real-time PCR. , 2007, International journal of food microbiology.

[5]  Ali Atoui,et al.  Approche de la mycotoxinogénèse chez Aspergillus ochraceus et Aspergillus carbonarius : études moléculaire et physiologique , 2006 .

[6]  A. Lichter,et al.  Occurrence of ochratoxin A producing fungi in wine and table grapes in Israel. , 2006, International journal of food microbiology.

[7]  A. Hocking,et al.  Survival and growth of Aspergillus carbonarius on wine grapes before harvest. , 2006, Journal of food microbiology.

[8]  Zofia Kozakiewicz,et al.  Ochratoxin A in grapes and grape-derived products , 2006 .

[9]  N. Martí Ochratoxin A and ochratoxigenic modulds in grapes, must and wine. Ecophysiological studies , 2006 .

[10]  C. Vázquez,et al.  PCR detection assays for the ochratoxin-producing Aspergillus carbonarius and Aspergillus ochraceus species. , 2005, International journal of food microbiology.

[11]  P. Battilani,et al.  Ochratoxin A production by Aspergillus carbonarius on some grape varieties grown in Italy , 2004 .

[12]  P. Battilani,et al.  Occurrence of ochratoxin A-producing fungi in grapes grown in Italy. , 2003, Journal of food protection.

[13]  J. Flaherty,et al.  Multiplex polymerase chain reaction assay for the differential detection of trichothecene- and fumonisin-producing species of Fusarium in cornmeal. , 2002, Journal of food protection.

[14]  D. Ginzinger Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. , 2002, Experimental hematology.

[15]  Thomas D. Schmittgen,et al.  Real-Time Quantitative PCR , 2002 .

[16]  Fanrong Kong,et al.  Identification of Aspergillus fumigatus and Related Species by Nested PCR Targeting Ribosomal DNA Internal Transcribed Spacer Regions , 2001, Journal of Clinical Microbiology.

[17]  John I. Pitt,et al.  Integration of modern taxonomic methods for Penicillium and Aspergillus classification , 2000 .

[18]  A. Burt,et al.  The Evolutionary Biology and Population Genetics Underlying Fungal Strain Typing , 1999, Clinical Microbiology Reviews.

[19]  C. Petropoulos,et al.  Advances in quantitative PCR technology: 5' nuclease assays. , 1998, Current opinion in biotechnology.

[20]  M. Castegnaro,et al.  Occurrence and significance of ochratoxin A in food. , 1998, Food additives and contaminants.

[21]  H. Ishiwata,et al.  Comparison of official methods for 'readily oxidizable substances' in propionic acid as a food additive. , 1996, Food additives and contaminants.

[22]  R. Singh,et al.  Molecular Methods in Plant Pathology , 1995 .

[23]  K. Raper,et al.  The genus Aspergillus , 1966 .

[24]  L. Fourie,et al.  Ochratoxin A, a Toxic Metabolite produced by Aspergillus ochraceus Wilh. , 1965, Nature.

[25]  G. Martin The Genus Aspergillus. , 1946, Science.