Ulva sp.

Noor, Sadia, Begum, Mst. Nadira, Rony, Satyajit Roy, Uddin, Mohammad Zashim, Sohrab, Md. Hossain & Mazid, Md. Abdul, 2024, Bioactivity and chemical screening of endophytic fungi associated with the seaweed Ulva sp. of the Bay of Bengal, Bangladesh, Botanica Marina (Warsaw, Poland) 67 (2), pp. 115-129 : 116

2.3 Identification of endophytic fungi associated with Ulva sp.

Endophytic fungi were identified taxonomically based on macroscopic and microscopic morphological characterization and molecular identification. The morphological characteristics of each isolate, including the rate of growth, mycelium depth and hyphal orientation, colour, texture, elevation, margin, diameter and form of the colony, as well as spore view, were observed after 3, 6, 9 and 12 days. For molecular identification of fungi, genomic DNA was extracted and the entire Internal Transcribed Spacer region ( ITS 1, 5.8S, ITS 2) was amplified and sequenced ( Martin and Rygiewicz 2005). At first, a colony from 4 to 7 days of pure culture of each fungus was selected, scratched with a sterile surgical blade and ground to make powder with liquid nitrogen using a pestle and mortar.Then,DNA was isolated using the Maxwell ® 16 LEV Plant DNA Kit ( AS 1420, Promega, USA) ( Cappuccino and Sherman 1996). DNase-free RNase (30 min at 37 ° C) was used to remove RNA contamination from the isolated DNA, and this was then stored at −20 ° C for further analysis. Then, the ITS region from the isolated DNA was amplified by PCR ( Raja et al. 2017), followed by nucleotide sequencing of the amplicons by the Sanger dideoxy sequencing method.

Raw sequence data was processed using BioEdit 7.2 and then compared to those available in the rRNA/ ITS database of the National Center for Biotechnology Information ( NCBI) GenBank. The nucleotide sequence of each isolate of the present study was tested for its relatedness with similar sequences using the Basic Local Alignment Search Tool ( BLAST).Based on total score, query cover and percent identity, the selected sequences were grouped into distinct clades by constructing the phylogenetic tree. The phylogenetic tree was constructed using the Maximum Likelihood method and the Tamura-Nei model (Tamura and Nei 1993) conducted in MEGA-X software ( Kumar et al. 2018). The support of each node was assessed using a bootstrap technique with 1000 iterations, and the tree was scaled with branch lengths denoting the number of substitutions per site. A subculture of each isolate has been deposited at the BCSIR Laboratories, Dhaka, Bangladesh.