MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF A SOIL-DERIVED Aspergillus niger STRAIN - Nayya Waseem Dar; Muhammad Anjum Zia; Muhammad Shahid; Bushra Akhtar (Journal of Animal and Plant Sciences, 2026)
Aspergillus niger is a filamentous fungus of great importance to the industry and biotechnology. A detailed strain-level characterization of A. niger isolated from soil was carried out in this study, which entailed the integration of morphological, biochemical, molecular and in silico analyses. Morphology showed black conidial heads together with biseriate phialides. Supportive evidence is given by enzymatic assays and carbohydrate fermentation tests. The molecular identification was performed by PCR amplification of the Internal Transcribed Spacer (ITS) region, with primers ITS1 5’-TCCGTAGGTGAACCTGCGG-3’ and ITS4 5’-TCCTCCGCTTATTGATATGC-3’ which amplify a region of about 570 bp. Sanger sequencing generated a high-quality consensus sequence (Phred score > 40) which was deposited in NCBI GenBank database (Accession No. PV056011 and PV056012 forward and reverse respectively). Comparisons made by BLASTn with the reference A. niger showed homology of 99-100%. Phylogenetic analysis was done in MEGA12 using the neighbor-joining method with 1000 bootstrap replications. In silico PCR AmplifX confirmed the specificity of the Primer, producing one unique primer pair that amplified a 570 bp-length product with 57% GC content. To characterize genomic features of the strain, RNAfold was used to predict stable secondary structure with a minimum free energy of -213.10 kcal/mol, EMBOSS to determine the GC content, MEME Suite to determine identified three conserved motifs and DnaSP analysis revealed minimal genetic differentiation (Fst = -0.00338) and high gene flow (Nm = 40.67) among populations. Haplotype network analysis with PopART revealed seven different haplotypes, the most prevalent being Hap_3 located in the center of the network. It is a combination of the processes that provides a useful base for the precise identification, characterization of fungus and the biotechnological potential of isolates for enzyme production and organic acid synthesis.
Keywords: journal article; Molecular identification; Phylogenetic analysis; In silico PCR; RNAfold; Motif discovery; Haplotype