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Isolation and characterization of a fungicolous mold Cladosporium oxysporum strain NCT370
Objective: Is to isolate and identify a mold species from a puff ball
mushroom growing on a tree bark.
Sampling: A puff ball mushroom growing over a tree bark was sampled
in a sterile polyethane bag and transported within 2 hours of collection
to the Mold’s lab at PG and Research Department of Biotechnology and
Microbiology, National College, Trichy (fig. 1).
Inoculation: Powdery mass found on the surface of the puff ball was
transferred to the centre of a potato dextrose agar (PDA) plate under
laminar condition. The PDA plates were incubated under Mold’s lab
temperature until sporulation. An un-inoculated PDA plate, kept in open
condition in the laminar flow during the inoculation process, acted as a
control.
Observation: A powdery mass growth from the centre of plate progresses towards the edges and
100% of PDA surface was occupied on 3rd day (fig. 2a, b). No growth was observed in the control.
Fig. 2: Front (a) and reverse (b) plate picture taken after 72 hours of incubation
Sub-culture: To test the purity of the culture, a fragment of hyphae was transferred to a fresh PDA
plates and incubated under Mold’s lab temperature. The plates were observed every day.
Fig. 1: Puff ball mushroom sample
collected from a tree bark
a
b
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Observation: The growth was rapid from day one (fig. 3a, b).
Fig. 3: Front (a) and reverse (b) plate picture of pure culture taken after 24 hours of incubation
100% growth was observed on the day 2 (fig. 4a, b). Reverse plate reveals an orange pigmentation
pattern (fig. 4b).
Fig. 4: Front (a) and reverse (b) plate picture of pure culture taken after 48 hours of incubation.
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b
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b
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No colored spores were observed. After 5th day, powdery growth occupied the entire plate excerpt
from the centre (fig. 5a, b).
Fig. 5: Front (a) and reverse (b) plate picture of pure culture taken after 96 hours of incubation
Microscopic analysis: The mycelia was subjected to lactophenol cotton blue (LPCB) staining. The
slide was examined under 100X resolution.
Observation: Septal hyphae was observed (fig 6a, b). No sporulating structure was observed excerpt
a zygote like structure at the hyphal septate (fig. 7a) and pseudo-hyphae like growth resembling
spores in chains (fig. 7b).
Fig. 6: Observation of mycelia (a) and septal hyphae (b) under 100X objective.
Cladosporium like sporulation patterns were observed (fig. 8a) with oval shaped spores (fig. 8b).
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b
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b
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Fig. 7: Observation of gamete-like structure (a) and spore chain-like structure (b) under 100X
objective.
Fig. 8: Observation of spores in chains (a) and oval spore structure (b) under 100X objective.
Identification of the mold through DNA barcoding: Mold sample was commercially out-sourced
for sequencing the Internal Transcribed Spacer (ITS) gene using the primer pair ITS1 and ITS4. ITS
gene sequence generated in this study shared 99.04% similarity with Cladosporium oxysporum type
specimen CPC14371 (GenBank accession number NR_152267). A Neighbor-Joining tree is
constructed with 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
a
b
a
b
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tree. 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 11
nucleotide sequences. All
ambiguous positions were
removed for each sequence
pair (pairwise deletion
option). There were a total of
533 positions in the final
dataset. The ITS sequence of
this study is indicated with a
red triangle and NCT
accession number NCT370.
ITS sequence of Rhizopus
microspores isolated earlier
in our lab was used as an out-
group. The numbers prefix to
reference species name
indicate the respective
GenBank accession numbers.
Strain and its sequence accession: The C. oxysporum isolated in this study was cryo-preserved and
stored at National College Culture Collection Centre. The strain NCT370 is available to other users for
academic and research purposes. Request could be made at www.ncccc.in. The ITS sequence
produced in this study could be globally accessed through a GenBank accession number OP598878.
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Taxonomy of Cladosporium oxysporum NCT370
Kingdom: Fungi
Phylum: Ascomycota
Class: Dothideomycetes
Order: Cladosporiales
Family: Cladosporiaceae
Genus: Cladosporium
Species: C. oxysporum
Strain: NCT370
Authority: Berk. & M. A. Curtis (1868)