Molecular Characterization of Different Species of Aspergillus nidulans Group

The present study deals with the phylogenetic analysis of A. nidulans group and assay of how much A. aegyptiacus belongs to A. nidulans group or A. versicolor group. The revealed results can be summarized as follows: 1The eight fungal isolates used in this investigation were identified as two isolates of Aspergillus aegyptiacus (AUMC 6122 & 3603),E. nidulans (AUMC152), E. rugulosa (AUMC164), E. variecolor (AUMC165), A.caespitious (AUMC4289), A. sydwoii (AUMC 4221), A.versicolor (AUMC 92),by Assiut University Mycological Center (AUMC) whichwere isolated during the present study; using dilution Plate method from different sites in Fayoum Governorate and were employed in the present study. 2The best mycelial growth for subsequence studies was obtained on liquid Czapek's-Dox medium supplemented with 1 g/l yeast extract under dark conditions at 28°C for one week. In absence of yeast extract, the fungi grow faintly after two weeks of incubation under the same conditions. 3Large quantities of DNA were extracted from the eight isolates of Aspergillus spp. DNA yields appeared as clear, intense and sharp bands using CTAB extraction method. 4RAPD analysis was used to investigate the polymorphism for all isolates under study using six arbitrary primers A1, A2, A3, B1, B2 and B4. The six random primers recorded variation between the four isolates. The total number ofbands was (142) for all six primers. Primer (A1) produced the highest number of bands (32), while the lowest number of bands was (18)produced by primer (B2).The size of the amplified bands varied with the different primers. The largest band was at 3,000-bp, which was amplified by primer B4 of isolate No. 2 (E. variecolor), while the smallest one was at 100 bp.amplified by primer B1of isolate No. 3 (E. rugulosa). 5The degree of similarity was determined and dendrogram was established for the Aspergillus isolates using UPGMA program. The Aspergillus isolates were collected into two main clusters (Cluster I, Cluster II). Cluster II included the two isolates of Aspergillus aegyptiacus only while Cluster I was splitted into two subclusters (Cluster A, Cluster B). Cluster A, included: E. nidulans; E. rugulosa and Cluster B, included only E. variecolor 6The results obtained by partial sequencing of ITS gene showed only one unique band range from 250270bp in ITS1 region and 320-350 bp in ITS2 region. 7Purification and sequencing of the PCR products for the isolates under investigation were performed in Genetic Analyzer Unit, Egypt. DNA sequences were obtained using Dye Terminator Cycle Sequencing. Blast program (www.ncbi.nlm.gov/blast) which was used to assess DNA similarities of eight isolates sequenced with already recorded sequences on NCBI Genebank. Multiple sequence alignment and molecular phylogeny were performed using BioEdit software, the phylogenetic tree was displayed using the TREEVIEW program (Mega4 and ClastalW2) & Conclusion 8Based on the length of ITS1 of the eight Aspergillus spp. could be divided into two clusters. The first cluster consisted of E. nidulans, E. variecolor and E. rugulosa at value 1.2 and subcluster consisted of E. nidulans, E. variecolor at value 0.5. The second cluster included the two isolates of Aspergillus aegyptiacus (AUMC 6122 & 3603),A. sydowii, A.versicolor and A. caespitiousat value 2.4 and subcluster included two isolates of Aspergillus aegyptiacus (AUMC 6122 & 3603) and A. sydowii, A. versicolor at value 1.7. Depicting the genetic relationship among Aspergillus spp. revealed that E. nidulans is closely related to E. variecolor and two isolates of Aspergillus aegyptiacus (AUMC 6122 & 3603) closely related to A. sydowii, A. versicolor in phylogeny. 9Based on the length of ITS2 sequences, the five Aspergillus spp. could be divided into two clusters. The first cluster consisted of Emericella species and one isolate of Aspergillus aegyptiacus (AUMC 3603) at value 0.58; the subcluster at value 0.25 included E. nidulans, E. variecolor and E. rugulosa. The second cluster included only one isolate of Aspergillus aegyptiacus (AUMC 6122) at value 0.75. Depicting the genetic relationship among Emericella species revealed that E. variecolor and E. rugulosa are closely related at value 0.1 in phylogeny. 10-The above results lend further support to the recommendation that A.aegyptiacus is closely related to A. versicolor group, rather than A. nidulans group.