A highly efficient electrophoretic method for discrimination between two Neoscytalidium species using a specific fungal internal transcribed spacer (ITS) fragment

Abstract

Neoscytalidium (or N.) dimidiatum and N. novaehollandiae are two aggressive plant pathogenic species that affect several agricultural crops. Early detection and identification of these fungi are of critical importance to bring about the effective minimization to the threat they pose to the infected plants. Herein, two species of Neoscytalidium were rapidly discriminated by utilizing the rRNA internal transcribed (ITS4-5.8S-ITS5) PCR primers. A total of 100 isolates of Neoscytalidium species, which were isolated from Iraqi canker-infected fig trees, were included in this study. Two discrete electrophoretic PCR bands were observed in Neoscytalidium isolates-A-variants were about 546 bp, while B-variants were about 993 bp in length. The comprehensive phylogenetic analysis of both DNA variants revealed that A-variants resided between N. novaehollandiae and N. hyalinum, while B-variants were closely related to N. dimidiatum. Furthermore, the highly specific re-constructed tree of both electrophoretic variants demonstrated that B-variants share a high similarity with N. novaehollandiae. Additionally, the secondary structures for both variants were predicted computationally to reveal the structural patterns that each variant follows. In conclusion, a small rRNA locus comprising 22 nucleotides that differs in the two variants is potentially responsible for this species-specific classification. The main divergence in the amplified loci led to the classification of these fungal variants into two main species, namely N. dimidiatum and N. novaehollandiae, demonstrating that the amplification by ITS4-ITS5 rRNA fragment is a beneficial strategy that can be employed for the assessment of Neoscytalidium diversity in the natural ecosystems.

Identification of Candida Species Using 26S Ribosomal RNA Gene Sequencing in Patients with Periodontitis

“…This feature could be attributed to the specific characteristics that kept in ribosomal sequences (10). Moreover, the cost of undergoing such 26S rRNA-based speciation procedure is so low as compared with other costly techniques, such as those relied on DNA hybridization, or multiplex PCR detection (31).…”

“…were studied by generating a specific comprehensive tree for Candida spp. The present 26S ribosomal-based comprehensive tree was generated according to a neighbor-joining based method (10). All the included Candida species were annotated by a cladogram-made parameters.…”

“…In addition to the common utilization of 16S rRNA-based amplification, the conserved sequences within the ITS4-ITS5 rRNA gene are recognized, and amplification of the variable regions leads to unique signatures that are utilized for identification of the particular fungus to the species level (10). Several ribosomal-based sequencing studies have been used for the study of fungal taxonomy and phylogeny.…”

The Gly152Val mutation possibly confers resistance to beta-lactam antibiotics in ovine <i>Staphylococcus aureus</i> isolates

“…A specific comprehensive bacterial tree was constructed in which the observed bacterial variants were compared with their neighbor homologous bacterial sequences, using a neighbor joining method described by Al-Shuhaib et al (2018a). Next, the blast results of the observed variants were combined and aligned together using Clustal Omega-based tools.…”

Determination the Lipase Activity of Staphylococcus sp. Strain Isolated from Clinical Specimens

“…A specific comprehensive bacterial tree was constructed in this study according to the protocol described by 10 . The observed bacterial variants were compared by reference sequences neighbor homologous by means of NCBI-BLASTn server 11 .…”