Isolation and characterization of Rhizopus microsporus strain NCT339 from spoiled radish (Raphanus raphanistrum)

Abstract

Objective is to isolate and identify the mold growing in spoiled radish vegetable.

Full text

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Isolation and characterization of Rhizopus microsporus strain NCT339
from spoiled radish (Raphanus raphanistrum)
Objective: To isolate and identify the mold growing in spoiled radish vegetable.
Sampling: Spoiled radish were collected in sterile polyethylene bag from a vegetable market at
Trichy and transported to Mold’s lab at PG and Research Department of Biotechnology and
Microbiology, NCT within an hour.
Sample inoculation: The infested region of the radish is sampled with sterile cotton bud and
simple streaked into potato dextrose agar (PDA) (HiMedia). The plates were incubated in mold’s lab
at the room temperature.
Observation: Two types of molds were observed (black color and white color) (Fig. 1).
Fig. 1: Mixed culture obtained after 5 days of incubation. Front and reverse views were shown.
Subculture: The white mold was selected for sub-culturing. Using a dissection needle, white
mycelia was picked and transferred to the centre of a freshly prepared PDA plate. The plates were
incubated in mold’s lab at the room temperature.
Observation: Growth of mold covered 80% of the PDA plate on 3rd day (Fig. 2) and 100% on day 5.
(Fig. 3). No precise agar venation patterns were observed.

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Fig. 2: Pure culture of white mold growth obtained on 3rd day. Front and reverse views were
shown.
Fig. 3: Front and reverse plate picture (Day 5).
Lactophenol cotton blue staining
Glass slides were made grease free by treating with detergents and wiped with tissue papers
dipped in 70% ethanol.
Using a dropper or pipette, few drops of lactophenol cotton blue (LCB) were added at the centre
of the glass slide.

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Using a sterile forceps or through inoculation needle, fungal biomass is uprooted from the
media (care was taken to avoid agar pellets) and soaked in LCB drops in the slide.
The fungal hyphae was spread evenly in the LCB drops and covered with a clean cover-slip or
cello tapes were used to fix the stain. Care was taken to not trap any air bubbles between the
slide and cover slip or cello tapes.
The slide was examined under bright field microscope at 40X and 100X magnifications.
The objective was to observe and record four types of fungal morphology such as 1) Mycelial, 2)
Hyphae, 3) Spore bearing and 4) Spore structures.
Observation: Sporangium with spores was observed (fig. 4). The released spore clinging on to the
sporulating structure was also observed (fig. 5). The spore was oval shaped (fig. 5).
Fig. 4: Sporagium and sporangiospores stained with LCB.

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Fig. 5: Sporangiospores clinging on to the spore producing structure and spore morphology was
observed using LCB staining.
Broth morphology:
Using a sterile needle, fungal mycelia was transferred into potato dextrose broth (PDB) and the
culture was incubated in shaking incubator (@100 rpm) at 30⁰C for 5 days.
Observation: Matt like growth on the surface of the broth was observed following incubation (fig.
6). Blackish green spores were observed.
Fig. 6: Matt like growth on the surface of the broth.
Culture preservation
 The mold culture was preserved in 50% glycerol.
 5ml of sterile distilled water is mixed with 5ml of 100%
glycerol to make up a 10ml of 50% glycerol solution.
 The mixture was sterilized by autoclaving.
 1ml of 50% glycerol was transferred to a sterile 2ml screw cap
cryo-tube.

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 The fungal biomass was uprooted from the PDA and inoculated into sterile glycerol stock using
ethanol sterilized forceps.
 Few more forceps full of fungal biomass was transferred until the stock was cloudy or the
fungal biomass was clearly visible in the glycerol stock solution.
 The glycerol stock was stored in -80⁰C, overnight.
 Following overnight preservation, the mold hyphae was retrieved from the glycerol stock using
sterile needle and inoculated at the centre of fresh PDA plates to check for re-viability.
 Following 3 days of incubation, PDA plated was observed for pure culture growth.
 Two more glycerol stocks were made from the revived plates as mentioned above.
Observation: The plates were successfully revived and pure culture of the mold was confirmed. 3
glycerol stocks were made following above said steps for a mold species. 2 stocks were stored in -
80⁰C and 1 in -20⁰C. Hereafter, -20⁰C will be used for further assay.
Culture deposition
The mold culture was deposited to National College Culture Collection Centre (www.ncccc.in) and
an accession number was assigned as NCT339.
Molecular characterization
DNA isolation
DNA was isolated from the fungal biomass following below given protocol.
 0.75ml of 2% Cetyltrimethyl ammonium bromide (CTAB) [(Tris (pH-8.0)- 100 mM, EDTA (pH -
8.0)- 20 mM, NaCl-2M, β-mercaptol ethanol- 5%)] was taken in ‘v’ bottomed 1.5 ml micro
centrifuge tube.
 Using sterile forceps, mycelia biomass was transferred into the CTAB solution.
 The mycelia were grinned using micropestle (if required, initially 600µl of CTAB was added for
grinding and 400µl was added later to remove the mycelia attached in micropestle).
 The tube was incubated at 65⁰C in the water bath for one hour with vortexing at every 10
minutes interval.
 Following incubation, 0.75ml of chloroform: Isoamyl alcohol (24:1) mixture was added and
gently mixed by inverting tubes for several times.
 The tubes were centrifuged at 8000 RPM for 10 minutes.

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 0.5ml of supernatant was transferred into to a fresh 1.5 ml microfuge tube and DNA was
precipitated by adding 1ml (double the volume) of isopropanol or per chilled 100% ethanol.
 The tubes were centrifuged at 10,000 rpm for 10 minutes.
 The supernatant was discarded.
 The pellet was washed by adding 70% pre-chilled ethanol and gently mixed by inverting the
tubes several times.
 The tubes were centrifuged at 10,000 rpm for 10 minutes and the pellets were air dried at the
room temperature.
 Based on the pellet size, Tris-EDTA (TE) buffer [10 mM Tris, 1 mM Ethylenediaminetetraacetic
acid (EDTA)] was added and stored at -20⁰C until further analysis.
Agarose gel electrophoresis
DNA was checked in 1.2% agarose gel was prepared for electrophoresis using Ethidium bromide as
staining dye and bromophenol blue as tracking dye. 5 µl sample was using for loading. Following
positive confirmation, remaining DNA sample was slit into 3 tubes. One tube was used for
commercial outsourcing and remaining two was stored at -80⁰C.
ITS gene amplication and sequencing
The sample was commercially outsourced for Internal Transcribed Spacer gene amplification using
the primer pair, ITS1: TCCGTAGGTGAACCTGCGG and ITS4: CCTCCGCTTATTGATATGC and
sequenced at Bioserve Biotechnologies Pvt., Ltd., India.
Sequence analysis and species identification
BLAST analysis revealed that the ITS sequence of the present study shared 99.38% sequence
similarity with Rhizopus microsporus (GenBank Reference number: MT620751), isolated and
identified from rice wine.
Neighbor-Joining method tree is drawn to scale, with branch lengths in the same units as those of
the evolutionary distances used to infer the phylogenetic tree was show in figure 7. 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 674 positions in the final dataset. Evolutionary analyses were
conducted in MEGA11. The ITS sequence produced in the present study was indicated as NCT339 in
the phylogram (Fig. 7).

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Fig. 7: Phylogram constructed for ITS sequences confirmed the identification of the mold species as
Rhizopus microsporus.
ITS sequence produced in the present study could be accessed through the GenBank accession
number OP380719.