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Lab in Mycology
PG & Research department of Biotechnology and Microbiology
National College, Tiruchirappalli, Tamil Nadu- 620001, India
1
Isolation and characterization of Aspergillus luchuensis strain NCT376 sampled as
an epiphyte from a beard lichen (Usnea sp.) growing on a tree bark in Ooty, Tamil
Nadu, India
Background
The beard lichen Usnea sp. was sampled during a field visit to Ooty on September 13th
2022. The sample were collected in a zip lock cover and transported to the laboratory
within 24h. About 3cm length of the lichen sample were cut and washed off the debris
using distilled water. With an objective of isolating endophyte, the sample was treated in
3% sodium hypochlorite for 1 minutes, ethanol for 30 seconds and washed in sterile
distilled water for 1minute. The sample was imprinted over the first potato dextrose agar
(PDA) plate (negative control for endophyte) and vertically inoculated in the second PDA
plate. The plates were incubated in mold’s lab condition for 3 days. An un-inoculated PDA
act as a positive control.
Result
Fig. 1: Source of collection of the beard lichen and the epiphytic fungal growth in the
mother plates.
Lab in Mycology
PG & Research department of Biotechnology and Microbiology
National College, Tiruchirappalli, Tamil Nadu- 620001, India
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No growth was observed neither in the lichen inoculated plate nor the positive control.
But growth was observed in the imprinted first plate, revealing an epiphyte that resisted
surface sterilization processes (fig. 1).
The white hyphal biomass was transferred onto fresh PDA agar plates and incubated in
mold’s lab condition. The plate was observed every day and macro-morphological
features was documented every day. Black sporulation was observed from day two and
fills the plate within 5 days of incubation (fig. 2). Front and reverse plate photography
was documented. Reverse plate venation patterns were manipulated with power point
picture effect to throw light on venation patterns (fig. 2).
The macroscopic structure like colony features including diameter of the colony after 7
days and its texture, shape, color of conidia, mycelia, and reverse plate venation
resembled the phyla Ascomycota.
Fig. 2: Front and reverse plate morpho-changes document from day 2 through day 4.
On day 5, lactophenol cotton blue staining was performed from the pure culture. During
microscopic analysis, 4 morphological keys were focussed upon viz., 1. Mycelial
structures (400X), 2. Hyphal structures (400X), 3. Spore bearing (condiophore) structure
(1000X) and 4. Spore structure (1000X).
Lab in Mycology
PG & Research department of Biotechnology and Microbiology
National College, Tiruchirappalli, Tamil Nadu- 620001, India
3
Microscopic characteristics of conidial heads, stipes, color and length, vesicles shape and
seriation, metula covering, conidia size, shape and roughness resembled the genera
characteristics of Aspergillus sp. (fig 3).
Fig. 3: Microscopic characteristics of conidial heads, stipes, metula covering, conidia and
its spore size was displayed.
In order to preserve the structure of the condial head, slide culture technique was
adopted. A slide culture set-up within a petri plate containing non-absorbent cotton base
over which a glass slide was mounted on couple of ear bud sticks, was sterilized by
autoclaving (fig. 4).
Fig. 4: Slide culture set-up and the results of lactophenol cotton blue staining.
Lab in Mycology
PG & Research department of Biotechnology and Microbiology
National College, Tiruchirappalli, Tamil Nadu- 620001, India
4
The slide containing a cube of PDA block was inoculated on side with fungal spores and
incubated for 5 days under mold’s lab condition. Following incubation, the coverslip was
placed over a glass slide containing lactophenol cotton blue, and analysed in the
microscope (fig. 3).
Mold’s DNA was extracted using CTAB method and commercially out-sourced for Internal
Transcribed Spacer (ITS) gene sequencing. The sequences obtained were aligned using
BioEdit and similarity search was done through BLAAST analysis. During similarity
search analysis, rRNA/ITS database was selected to get a close similarity with vouchered
fungal species. The uncultured/environmental sample sequences were excluded from the
search results.
The ITS sequence generated in this study had 99.28% similarity with Aspergillus
luchuensis (GenBank acc. No.: NR_135449) sequenced by Hong et al (2013) [Hong, S.-B.,
Lee, M., Kim, D.-H., Varga, J., Frisvad, J. C., Perrone, G., Gomi, K., Yamada, O., Machida, M.,
Houbraken, J., & Samson, R. A. (2013). Aspergillus luchuensis, an industrially important
black aspergillus in east Asia. In K. McCluskey (Ed.), PLoS ONE (Vol. 8, Issue 5, p. e63769).
Public Library of Science (PLoS). https://doi.org/10.1371/journal.pone.0063769].
Based on the macro-, and micro-morphological and ITS gene features, 49 ITS gene
sequences vouchered Aspergillus species including Aspergillus luchuensis was obtained
from GenBank and subjected to phylogram construction. The ITS gene sequence
produced in this study was represent as NCT376 with red triangle in the phylogram (fig.
5). Among the 49 Aspergillus spp. Used in the phylogram, the ITS gene sequences of the
strain NCT376 sequenced in the present study precisely claded with Aspergillus
luchuensis (fig. 5). The ITS sequence of the present study could be accessed through a
GenBank accession number OQ586394.
The Aspergillus luchuensis isolated in this study is deposited in National College Culture
Collection Centre (ncccc) available for other user for research and academic purposes.
The culture can be requested here www.ncccc.in.
Lab in Mycology
PG & Research department of Biotechnology and Microbiology
National College, Tiruchirappalli, Tamil Nadu- 620001, India
5
Fig. 5: Evolutionary relationships of taxa was inferred using the Neighbor-Joining
method. The percentage of replicate trees in which the associated taxa clustered together
in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn
to scale, with branch lengths in the same units as those of the evolutionary distances used
to infer the phylogenetic tree. The evolutionary distances were computed using the
Maximum Composite Likelihood method and are in the units of the number of base
Lab in Mycology
PG & Research department of Biotechnology and Microbiology
National College, Tiruchirappalli, Tamil Nadu- 620001, India
6
substitutions per site. This analysis involved 49 nucleotide sequences. All ambiguous
positions were removed for each sequence pair (pairwise deletion option). There were a
total of 548 positions in the final dataset. The ITS gene sequence produced in this study
was represent as NCT376 with red triangle in the phylogram.
Taxonomic hierarchy of NCT376
Phylum: Ascomycota
Class: Eurotiomycetes
Order: Eurotiales
Family: Aspergillaceae
Genus: Aspergillus
Species: A. luchuensis
Authority: Inui (1901)
Strain: NCT376