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Title: Use of a rep-PCR system to predict species in the Aspergillus section Nigri

Author
item PALENCIA, EDWIN - University Of Georgia
item Klich, Maren
item Glenn, Anthony - Tony
item Bacon, Charles

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/7/2009
Publication Date: 10/1/2009
Citation: Palencia, E.R., Klich, M.A., Glenn, A.E., Bacon, C.W. 2009. Use of a rep-PCR system to predict species in the Aspergillus section Nigri. Journal of Microbiological Methods. 79:1-7.

Interpretive Summary: Several species of black-spored fungi belongs to the genus Aspergillus, which are referred to as the Aspergillus section Nigra. These fungi are commonly found on many produce, especially peanuts and corn, where most are pathogen and some isolates produce the highly carcinogenic toxin ochratoxin A. However, this section consists of several species that are difficult to determine using conventional microscopy. They are referred to as cryptic and must be identified at the level of molecular determinations. We scrutinized this group of fungi using molecular technique, referred to as barcoding, due to its simplicity and reduce expense and rapidity. We report in this paper the successful determination of the reported species with this technique, for the first time present a preliminary screening of identified species found on corn and peanuts as endophytes. The method is highly reproducible, quick, convenient, and cheaper than conventional molecular techniques.

Technical Abstract: The Aspergillus niger aggregate within the A. section Nigri, is a group of black-spored aspergilli which taxonomy has been elusive. REP-PCR has become a rapid and cost-effective method for genotyping fungi and bacteria. In the present study, we evaluated the discriminatory power of a semi-automated rep-PCR barcoding system to distinguish morphotypic species and compare the results with the sequencing data obtained from ITS and partial calmodulin regions. For this purpose, 23 morphotyped black-spored Aspergillus species were used to create the A. section Nigri library with this barcoding system. The dendrograms, which were based on Pearson correlation coefficients, illustrated four different clustered groups: two A. niger aggregate cluster (named I.A and I.B), the heterogeneous A. carbonarius cluster (II), and the uniseriate cluster (III). The rep-PCR showed higher resolution than the internal transcribed spacer (ITS) rDNA and the partial calmodulin gene sequencing procedures. Additionally, 34 unknown field isolates, collected from different locations in the United States, were tested with this automated system. The results indicated that, at 95% similarity threshold, only 12% of the field isolates were similar to one of the genotypes included in the A. section Nigri library, whereas at 90% similarity threshold, about 64% of the field isolates matched genotypes in the reference library. Based on these results, this system has the potential for use as a highly analytical and reproducible tool for identifying the black-spored aspergilli.