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National College Culture Collection Centre
Department of Biotechnology and Microbiology
National College (Autonomous)
Tiruchirappalli- 620 001
India
1
Isolation and characterization of Aspergillus fumigatus NCT357 associated with post-
harvest decay of Okra (Abelmoschus esculentus)
Objective: To isolate and characterize a mold associated with a decaying vegetable (Okra)
Sampling: Decaying ladies finger, Okra (Abelmoschus esculentus) was sampled from
vegetable market in a polyethane bag and brought to mold’s lab at PG and Research
Department of Biotechnology and Microbiology, NCT within an hour.
Sample inoculation: The spoiled segment of Okra was sampled using a sterilize blade and
inoculated at the centre of potato dextrose agar (PDA) plate. An un-inoculated PDA plate
acted as control. The plates were incubated at Mold’s lab temperature for 5 days.
Observation: Following 5 days of incubation, 2 types of molds (green and black) colony
were observed (fig. 1). The black mold was also observed in control plate, hence discarded.
The green mold was further processed for characterization.
Fig. 1: Front and reverse mother plate picture after 5 days of incubation.
Sub-culturing: Thick green spore from the centre of the colonies were sampled using
sterile forceps and transferred to fresh PDA plates. The plates were sealed and incubated
National College Culture Collection Centre
Department of Biotechnology and Microbiology
National College (Autonomous)
Tiruchirappalli- 620 001
India
2
under mold lab temperature for 7 days. The front and reverse plate view of every day was
observed and documented. Light green color biomass was first 2 days of incubation (fig. 2).
Fig. 2: Front and reverse plate picture of fungal biomass after 2 days of incubation.
Following 5 days of incubation, the mycelia spread over the PDA was observed. Agar
venations were clearly evident (fig. 3).
Fig. 3: Front and reverse plate picture of fungal biomass after 5 days of incubation
National College Culture Collection Centre
Department of Biotechnology and Microbiology
National College (Autonomous)
Tiruchirappalli- 620 001
India
3
100% hyphal coverage with green sporulation was observed on 7th day. Changes in agar
venation pattern was observed (fig. 4).
Fig. 4: Front and reverse plate picture of fungal biomass after 2 days of incubation
Lactophenol cotton blue staining:
Grease free glass slides were taken and cleaned with 70% ethanol.
Few drops of lactophenol cotton blue (LCB) was added at the centre of the slide and a
bunch of hyphae from the centre of the mold colony was transferred to the LCB drops.
The hyphal biomass was dispersed in the LCB drops using sterile forceps.
The dispersed biomass was fixed was placing a cover slip.
The slides were observed under 40X and 100X magnification of the bright field
microscope
Observation:
The spore bearing structure and conidia resembles Aspergillus sp. The green conidia with
greenish phialides were observed (fig. 5). Spherical conidiospores were observed (fig. 6).
National College Culture Collection Centre
Department of Biotechnology and Microbiology
National College (Autonomous)
Tiruchirappalli- 620 001
India
4
Fig. 5: Lacto phenol cotton blue staining of conidiophore.
Molecular identification through ITS gene sequencing
Mold’s samples were commercially outsourced for sequencing bi-directionally the Internal
Transcribed Spacer gene using ITS1 and ITS4 primers. The ITS sequence produced in the
present study (641 bp) shared 99.53% sequence similarity with the ITS sequence of
Aspergillus fumigatus (GenBank acc. No.: MT576578).
Fig. 6: Spherical conidiospores.
National College Culture Collection Centre
Department of Biotechnology and Microbiology
National College (Autonomous)
Tiruchirappalli- 620 001
India
5
The Neighbor-Joining method in which the
associated taxa clustered together in the
bootstrap test (500 replicates) are shown
next to the branches. The evolutionary
distances were computed using the
Maximum Composite Likelihood method
and are in the units of the number of base
substitutions per site. This analysis involved
13 nucleotide sequences. There were a total
of 582 positions in the final dataset. The
sequence produced in this was represented
by NCT357 and Rhizopus microsporus
(Genbank acc. no.: OP380719) was used as
an out-group.
Strain and its sequence accession: The
pure culture of the present study was cryo-
preserved in 50% glycerol and submitted at
National College Culture Collection Centre
(NCCCC) (www.ncccc.in) and assigned a
number NCT357. The strain can be obtained on placing the request to NCCCC. The ITS
sequence was submitted to Genbank and can be globally accessed through the accession
number OP518290.
Taxonomical hierarchy of NCT357
Kingdom: Fungi
Phylum: Ascomycota
Class: Eurotiales
Order: Eurotiomycetes
National College Culture Collection Centre
Department of Biotechnology and Microbiology
National College (Autonomous)
Tiruchirappalli- 620 001
India
6
Family: Trichocomaceae
Genus: Aspergillus
Species: A. fumigatus
Strain: NCT357
Authority: Fresenius 1863