Isolation and identification of Mucor mucedo strain NCT358 from old cooked rice

Abstract

Objective is to isolate and characterize a mold associated with old cooked rice.

Full text

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Fig. 2: Moldy growth observed after 24hrs
incubation.
Isolation and identification of Mucor mucedo strain NCT358 from old cooked rice
Objective: To isolate and characterize a mold associated
with old cooked rice
Sampling: Four days old cooked rice sample (fig. 1) infested
with black mold was used as the sample. Sample was
covered with polyethane wrapper and bought to mold’s lab
at PG and Research Department of Biotechnology and
Microbiology, National College, Tiruchirappalli (NCT).
Sample inoculation: Using sterile nichrome needle
under laminar flow condition, a fragment of mold was
transferred into fresh potato dextrose agar (PDA)
plates. An un-inoculated PDA plate acted as control.
The plates were incubated at Mold’s lab temperature.
Observation:
 Widespread growth of single mold species was observed
after 24hrs of incubation (fig. 2).
 On day 3, 100% of PDA surface was covered with moldy
growth (fig. 3) without any agar venation pattern
observed at reverse side of the plate (fig. 4).
 On day 6 black color fruiting bodies were observed (fig.
5) without any agar venation patter in reverse plate (fig.
6).
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 and fixed using a cover slip.
The slides were observed under 40X and 100X magnification of the bright field microscope.
Fig. 1: Moldy growth in old rice sample

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Fig. 3: Moldy growth observed after 3days of
incubation.
Fig. 4: Reverse plate observed after 3days of
incubation.
Fig. 5: Growth with black fruiting body observed
after 6days of incubation.
Fig. 6: Reverse plate observed after 6days of
incubation.

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Fig. 7: Globose sporangia
Fig. 8: Sporangia after spore
release
Fig. 9: Sticky sporangial
spores along the hyphal
fragment
Fig. 10: Columellae
structure
Fig. 11: Spore structure observed under
oil immersion (100x magnification).
Fig. 12: Surface hyphal
growth in PDB.
Observation: No rhizoids were detected. The solitary globose sporangia
resembling Mucor sp. was clearly observed (fig. 7). The ruptured sporangium
resembled like an umbrella after the spore release (fig. 8). Sticky spore along
the sporangium and hyphae were observed (fig. 8, 9). An oval shaped
columellae was observed (fig. 10). Sporangiospores were oval in shape (fig.
11).
Broth characteristics
The hyphal fragments were transferred into potato dextrose broth (PDB)
and incubated in shaking incubator (100rpm) at mold’s lab temperature for
5 days. The growth of the mold was observed every day.
Observation: Surface hyphal growth was observed from day 1 (fig. 12).
Sporulation (light brown to black color) starts from day 2 (fig. 13) and rapidly
progresses in day 3 (fig. 14). Mature black spore structures are evident on day
4 (fig. 15).
100X

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Fig. 13: Sporulation on day 2 in PDB
Fig. 14: Sporulation on day 3 in PDB
Fig. 15: Sporulation on day 5 in PDB
Identification of molds through DNA barcoding
The mold sample was outsourced for commercial
sequencing for the internal transcribed spacer
(ITS) gene fragment using the ITS1 and ITS4
primer pairs.
The Neighbor-Joining tree with 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 substitutions per
site. This analysis involved 12 nucleotide
sequences. All ambiguous positions were removed
for each sequence pair (pairwise deletion option).
There were a total of 603 positions in the final
dataset.
The ITS sequence produced in this study was
indicated by NCT358 in the phylogram. Rhizopus

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microspores sequence (GanBank acc. no.: OP380719) was successfully delineated as an out-group in
the phylogram. The ITS sequence of the present study shared 98.74% similarity with Mucor mucedo
(GenBank acc. no.: MH854783) and can be globally accessed through the GenBank acc. no.:
OP546531.
Glycerol stocking and culture deposition:
The mold species was preserved in 50% glycerol stock and successfully revived in PDA plates. The
glycerol stocks were deposited in National College Culture Collection Centre (www.ncccc.in) and is
available for end users by placing an order from the above mentioned website.
Taxonomy of Mucor mucedo
Kingdom: Fungi
Phylum: Mucoromycota
Class: Mucoromycotina
Order: Mucorales
Family: Mucoraceae
Genus: Mucor
Species: M. mucedo
Strain: NCT358
Authority: Spatafora et al. 2016