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Isolation, Identification and Molecular Characterization of Fungal Laboratory Contaminants (2017--2018)

S. Ahmed
Biotechnology Division, BARI, Joydebpur, Gazipur

M.R. Kabir and
Biotechnology Division, BARI, Joydebpur, Gazipur

M.A.Y. Akhond
Biotechnology Division, BARI, Joydebpur, Gazipur

Abstract/ Executive Summary:

Twenty fungal contaminants were isolated from various plant tissue cultures and characterized at the molecular level. The fungi were found to be from 13 genera. Six fungi were from Aspergillus genus, two from Curvularia genus, and two were from Purpureocillium genus. The other ten fungal species were identified from ten different genera. Some could have importance in the field of medicine and some could be used as biocontrol agents and warrants further investigation.

Keywords: Microbial contaminants, Molecular characterization, Fungi

Location: Biotechnology Division, BARI, Joydebpur, Gazipur

Start Date: 00-00-2017

End Date: 00-00-2018

Program Area: Variety and Species

Commodity: Fungal Disease

Objectives:

The present experiment was proposed for the identification and molecular characterization of the microbial contaminants in the Biotechnology laboratory of BARI.

Methodology:

Twenty fungal contaminants were collected from contaminated vials of different tissue culture experiments in the laboratory of the Biotechnology Division, BARI, Joydebpur. Fungi were cultured in PDA (Potato Dextrose Agar media, Harold, 1998) media and incubated at 28-30°C in the dark. The genomic DNA was extracted from mycelia and/or spores of all cultured fungi by using a plant DNA extraction kit following the manufacturer’s instructions. The ITS region for 18s rDNA was PCR amplified using the universal primer pair ITS-1 and ITS-4. The amplified fragments were sequenced and later aligned using Genius software. The consensus sequences were compared with the existing Genbank database using BLAST (basic local alignment search tool) and 98-100% similarities were considered to infer the identity of the isolates.

Source of Information: Annual Research Report 2017 2018, Biotechnology Division, BARI, Joydebpur, Gazipur

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

From PCR amplification of ITS region, twenty fungal isolates obtained from different tissue culture contaminations in the biotechnology laboratory yielded a single fragment of approximately 500 bp. BLAST search using the Genbank database showed that the fungal isolates were from 13 different genus. Six isolates (7, 8, 14, 16, 18 and 20) shared similarities with six species of aerobic mold fungus Aspergillus (A. versicolor, A. niger, A. ruber, A. whitfieldii, A. sp. and A. sclerotiorum respectively) which commonly grows in sugar/salt and oxygen rich as well as nutrient depleted environments like damp walls. Aspergillus is also used as microbial fermentation. Therefore, damp indoor walls and alcohol may be the possible sources of these contaminants in tissue culture. Two isolates (3 and 13) were identified as two species of another mold fungus Curvularia genus (C. americana and C. geniculata) which is a facultative pathogen of many plant species and common saprobe in soil. Isolate 1 and 19 showed similarities with a saprobic fungus Purpureocillium (P. lilacinum, and P. sp) which is found in soil, nematodes, rhizosphere of crops and can be used as biocontrol agent for controlling plant-parasitic nematodes. So these are possible contaminants from root or bulb explants. The other ten isolates were similar with ten different genera. Isolate 2 was similar to the false smut fungus Ustilaginoidea virens. Isolate 4 was resembling a marine-derived fungus Talaromyces tratensis, which may be contaminated from indoor air and can be used as a biocontrol agent against pathogenic fungi like Alternaria, Curvularia, Bipolaris, Fusarium. A saprobic plant pathogenic fungus causing dieback, Eutypella scoparia was identified from Isolate 5 where human sputum, dead plant tissue, or explants from soil may be a potential sources of contamination. Isolate 6 was related to Cladosporium para halotolerans, found in hypersaline environments. From BLAST search a saprobic fungus Tritirachium dependens, was found to be comparable with Isolate 9. Endophytic or saprobic fungus Dothideomycetes sp. was recognized from Isolate 10. Isolate 11 was associated with skin fungus Toxicocladosporium irritans. Isolate 12 showed similarity with Arthrinium malaysianum. This fungus commonly occurs as endophyte or saprobe on different plant parts and produces industrially important enzymes (Mukharjee et al. 2016). A soil-borne pathogenic fungus Fusarium equiseti, causing wilt and damping-off diseases of crops was found in Isolate 15. Isolate 17 was related to Penicillium ramusculum found in soil, decaying vegetation air and common contaminants on various substances. Penicillium is important in biotechnology, food, medicine, biodeterioration and other fields.

Knowledge Management: Report/Proceedings