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.
Highlights
Date palm is one of the most popular fruit trees in Middle Eastern countries
The results of isolation and identification of contaminated date palm tissue cultures according to the morphological features (Elliot, 1917; Ellis, 1971) indicated that the genus Aspergillus was the most predominant fungal genera to be associated with date palm tissue cultures, with a total percent of isolation reaching 49.8% (Table 1), followed by the genera: Alternaria and Penicillium while the genera Cladosporium and Epicoccum were isolated at very low frequencies
For the genus Aspergillus, three different fragments were seen as follow: 531, 575 and 580 bp, while two different fragments of sizes 305 and 150 bp were seen with genus Alternaria, two fragments of sizes 580 and 600 bp were seen within the genus Penicillium
Summary
Date palm is one of the most popular fruit trees in Middle Eastern countries. Total world production of dates was estimated to be more than 7.5 million metric tons in 2009, from a total harvested area of 1.3 million ha. In Iraq, many publications reported on the methodology (or applicability) of tissue culture of elite date palm cultivars, including Sherafy, AlSayer, Hilawi, Khasab, Um Al-Dihin, Barhi, Kantar, Shwaythee, Breem, Alawaidy and Ashkar (Muhsen, 2006; Al-Khalifa et al, 2009; Al-Najm, 2009; Jasim et al, 2009; Al-Meer and Yassen, 2010). Date palm explants are exposed to microbial infection at all stages of tissue culture; contamination coming with the explants themselves, or occurring during the propagation procedures (Al-Mussawi, 2010). A survey of fungal contamination of different date palm cultivars during embryogenic callus production, revealed that the fungi Aspergillus niger, Aspergillus clavatus and Alternaria alternata were the most abundant contaminants of six different date palm cultivars, including Um Al-Dihin, Shwaythee, Breem, Barhi, Hilawi and Al-Sayer (Hameed and Abass, 2006)
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