A PCR ITS-RFLP method for identifying fungal contamination of date palm (Phoenix dactylifera L.) tissue cultures

Abstract

Morphological and molecular procedures have been used in the study to characterize the most abundant fungal contaminants of date palm tissue cultures. Aspergillus species were found to be the most frequently isolated species, followed by the species of Alternaria and Penicillium. Internal Transcribed Spacer (ITS) products of the predominant fungi were analyzed by PCR amplification with ITS1 and ITS4 primers. The results of BLAST search of the ITS sequences revealed the identity of the fungal species. Three species were found to belong to the genus Aspergillus as Aspergillus clavatus, Aspergillus niger and Aspergillus terreus, while, two species were found for the genus Alternaria with Alternaria alternata and Alternaria citri, and two species for the genus of Penicillium with Penicillium expansum and Penicillium glabrum. The restriction fragment length polymorphism (RFLP) of the ITS amplicons was used to discriminate between fungal species and provides an alternative method to sequencing ITS products; both restriction endonucleases EcoRI and SmaI were used to digest ITS products, and three types of fragment patterns were detected: A type (one undigested fragment), B type (two fragments type) and C type (three fragments type). This is the first study which used ITS sequence and ITS-RFLP techniques as a rapid and reliable procedure for identification of date palm fungal contamination in the laboratories of tissue cultures in Iraq.