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Isolation and Identification of Mycorrhizal Fungus from an Epiphytic Orchid (rhynchostylis Retusa L. Bl.)

MS Bhuiyan
Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh

MM Hossain
Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh

Kazi Shakhawath Hossain
Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh

Mohammad Nurul Islam
Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh

Abstract/ Executive Summary:

Isolation and identification of mycorrhizal fungi from the roots of Rhynchostylis retusa indicated that the cultural and microscopic features, namely colony appearance, colony colour, the diameter of vegetative hyphae, presence of monilioid cells, right-angle branching pattern of the fungal endophyte corroborated the identity of the fungus Rhizoctonia like anamorphs of Ceratobasidium species. Fungal identity was further confirmed through sequencing and analysis of internal transcribed spacer (ITS) sequences of the nuclear ribosomal DNA (nrDNA). ITS sequence of the isolate RhY10A6 (accession no. MN120903.1) showed > 99% similarity with several isolates of the teleomorphic fungus Ceratobasidium sp. in NCBI mega blast search. The phylogenetic analysis is based on the maximum parsimony method, this orchid mycorrhizal fungus is clustered with several isolates of Ceratobasidium sp. or Rhizoctonia like fungi. It showed near-distant relation with Ceratobasidium ramicola (GeneBank accession no. NR_138368.1) which is an orchid mycorrhizal fungus. Therefore, molecular characterization validated the morphological data. The techniques established in this orchid will facilitate to isolate and accurate identification of the mycorrhizal fungus.

Keywords: Isolation, Orchid mycorrhiza, IFS sequence, Rhynchostylis retusa

Location: Kaptai forest of Rangamati district of Chattogram, Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Fungal Disease, Orchid

Objectives:

To accurate identification of root-associated mycorrhizal fungus in R. retusa following morpho-molecular techniques.

Methodology:

Fresh roots of Rhynchostylis retusa growing naturally in the Kaptai forest of Rangamati district of Chattogram, Bangladesh were collected in air-tight plastic bags during May-June (rainy season) and November-December (winter season) and used within 24 hrs for isolation of fungal symbionts. The root samples were taken randomly from five plants, washed with running tap water, cut thin transverse sections at different root portions, stained with lactophenol cotton blue, and observed under a microscope to ensure the presence of fungal colonization. The incidence of fungal colonization and formation of pelotons in the root sections was calculated by the following formula (Hossain 2019): Number of cells colonized 20x microscope field view/Total number of cells 20x microscope field view × 100. The roots showing the presence of fungal pelotons were rinsed with distilled water and surface sterilized by 0.1% HgCl₂ for 6 - 10 mins and then washed 3 times with double sterile distilled water. Final disinfection was done by dipping them in 70% ethanol for 30 sec and washed 3 times with double sterile distilled water. The roots were then aseptically cut into sections approximately 2 mm thickness and placed in 9 cm Petri dishes containing potato dextrose agar (PDA) medium. The plates were incubated in the dark at 27°C until fungal hyphae were visibly growing from root specimens onto the medium. The fungi growing from the inner portion of the root section were considered probable mycorrhizal fungi. Pure cultures were obtained by transferring hyphal tips onto a fresh PDA medium. The nature of fungal growth, colony surface, and reverse colors was recorded at young and mature stages. The hyphal diameter and dimensions of monilioid cells were measured by Nikon E600 light microscope (Nikon, Tokyo, Japan) by mounting the mycelium in lactophenol triglycero-cotton blue on glass slides. Growth rates were determined according to Currah et al. (1987) by inoculating uniform bits of mycelium at the middle of PDA plates. Radial increments in colony size were measured at 48 hrs intervals over two weeks. Growth rates were represented by averages based on three replications. For determination of nuclei number in vegetative and monilioid cells, a small portion of the mycelial mat was fixed in 2% formaldehyde for 2 min on a glass slide and rinsed with distilled water for 1 min, followed by staining with gold antifade reagent with diamidino-2-phenylindole (ProLong® DAPI, Invitrogen Ltd., Eugene, OR, USA) for 10 min, and destained with distilled water for 2 min. A drop of 50% glycerin was placed over the stained specimen and covered with a coverslip. Micrographs were taken using a Nikon E600 microscope equipped with a fluorescence accessory with a mercury lamp. The fungal isolates were inoculated on sterilized potato dextrose agar medium (50 ml/250 ml flask) and incubated at 28°C in an incubator (New Brunswick Scientific, Edison, NJ) for two weeks. DNA has been extracted from 15 days old cultures according to the protocol described by Liu et al. (2000). The amplicons of ITS1, 5.8S ribosomal RNA, and ITS2 were achieved using ITS1 (5’ TCC GTAGGTGAACCTGCGG) and ITS4 primers (5’GCTGCGTTCATCGATGC) (White et al. 1990). The PCR reaction was performed in 50 µl reaction buffer containing 5 µl of dNTP mixture, 5 µl of 10×PCR buffer, 1 µl of each primer (10 pmol), 0.4 µl of Taq polymerase 1U, 2 µl of genomic DNA, and 35.6 µl of MiliQ water. The PCR cycling condition comprised of an initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 45 sec and extension at 72°C for 7 min. The amplified PCR products were purified and concentrated. The nucleotide sequences of the amplicons were generated at McLab, California, USA on a 3730 DNA Analyzer (Thermo Fisher Scientific, USA). A consensus sequence was generated from the forward and reverse sequences by using BioEdit 7.2.6 software (Hall 1999). The consensus sequence was deposited in the NCBI GenBank (Accession no. MN120903.1) The consensus sequence was placed into the web-based Basic Local Alignment Search Tool (BLAST) of NCBI GenBank (https://blast.ncbi.nlm.nih.gov/) to find out similar species/isolates. The phylogenetic relationships were established based on the maximum parsimony tree by using an analysis program of the MEGA 6.0 software (Tamura et al. 2013). In this analysis of Bootstrap replications, the number was 1000.

Source of Information: Bangladesh J. Bot. 50(1): 85-91, 2021 (March)

Article Link (Online Journal): DOI: https://doi.org/10.3329/bjb.v50i1.52675

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The molecular and morphological approaches established for isolation and identification mycorrhizal fungi from R. retusa will facilitate elucidate the diversity and variability of the species of mycorrhizal fungi. In this way, new species of fungi could be described and the mycorrhizal fungi could be used for conservation purposes

Knowledge Management: Journal