MOLECULAR STUDY ON CLADOSPORIUM SPECIES ISOLATED FROM AIR OF CAIRO, USING UNIVERSALLY PRIMED-PCR (UP) AND INTERNAL TRANSCRIBED SPACER (ITS) PCR TECHNIQUES

Abstract

Universally Primed-PCR (UP-PCR) and Internal Transcribed Spacer-PCR (ITS-PCR) based genomic fingerprinting techniques are considered a good methods that rely on specifically targeted primers. These techniques, which analyse the rDNA, have been shown to be relatively robust and discriminatory. This study was designed to investigate and characterize the molecular variation among Cladosporium strains collected at different sites in Cairo by using two different fingerprinting methods, Universally Primed- PCR (UP- PCR) and Internal Transcribed Spacer (UP- PCR) technique. The Cladosporium isolates investigated were isolated from air of Cairo by settle plate method. The samples were then purified and identified by using culture based techniques, microscopical methods, and biochemical reactions followed by confirmation in the regional center for mycology and biotechnology (RCMB). Molecular fingerprinting, and genetic similarities among Cladosporium species populations depending on microsatellites-polymerase chain reaction (ITS-PCR). Primers used are ITS4, and ITS5. PCR products were digested with 3 restriction enzymes and separated by agarose electrophoresis. Restriction patterns generated by Cfo I, Msp I and Rsa I. In addition, we have applied the Universally Primed PCR (UP-PCR) technique using two primers L21 and Fok1.The current work showed prominent discriminatory power given by amplification of internal transcribes spacers PCR regions followed by restriction with Cfo I enzyme than other endonucleases. moreover, Fok1 primer revealed minor variability among Cladosporium strains using UP-PCR genotyping technique. primer pair, a 700–800bp fragment was successfully amplified from all the isolates, while no PCR amplification was observed in negative controls. In addition, these were in the same line with that of Dean et al. , who analyzed the genera Stachybotrys, Penicillium, Aspergillus , and Cladosporium in order to identify and characterize by simple ITS method, in which each organism underwent ITS-PCR that amplified ribosomal sequences generating products from 550 to 600 bp followed by enzymatic digestion with Eco RI, Hae III, Msp I, and Hinf I, and show that using this combination of restriction enzymes enables the identification of these fungal organisms at the species level. Results of the current investigation showed moderate genetic similarity between Cladosporium isolates ranged from 50-70 % for inter-specific and 70-100 % for intra-specific comparisons. The present results indicate that, the dendrogram constructed with ITS-PCR digested with Msp I revealed that all isolates of C. cladosporioides and almost C. herbarum were grouped into a major 9 cluster delimited from other Cladosporium species comprising four molecular groups with genetic dissimilarity 30%. These results were similar to that of Kawasaki et al. (1993) that was conducted on Cladosporium carrionii and classified the 38 isolates into 4 mtDNA types (Type I to Type IV) based on the restriction patterns with Msp I, Sau3A I and Hae III. ITS-PCR digested with Rsa I revealed that the genetic similarity between Cladosporium species isolates ranged from 20 to 42 % for inter-specific and 42 to 100 % for intraspecific comparisons. The application of UPGMA clustering produced 3 large clusters within the population with a branched–off at genetic similarity of GS=20 %, each consisting of several sub clusters (phenons). Dendrogram of the ITS-PCR