Morphological and Molecular Characterization Of Micromycetes Associated With Seeds of Selected Cotton (Gossypium Hirsutum L.) Varieties

Abstract

A total of 14 varieties (CB1-CB14) of cotton (Gossypium hirsutum L.) seeds were collected from Cotton Research, Training and Seed multiplication Farm, Sreepur, Gazipur to detect and identify the seed borne fungi by morphological and molecular techniques. The sequence results obtained using the ITS1 and ITS4 primers were compared with NCBI GenBank and BOL database using BLAST analysis. In the present investigation, a total of 29 fungal isolates were morphologically identified from different varieties of cotton seeds, of which 19 fungal isolates were identified by molecular techniques. Among the isolated fungi, Aspergillus subramanianii, A. toxicarius, A. wentii, Penicillium aculeatum, P. citrinum, Rhizomucor sp. and Meyerozyma guilliermondii have been reported as new records for Bangladesh. Bangladesh J. Plant Taxon. 29(2): 297-312, 2022 (December)

Full text

Bangladesh J. Plant Taxon. 29(2): 297-312, 2022 (December) DOI: https://doi.org/10.3329/bjpt.v29i2.63531
© 2022 Bangladesh Association of Plant Taxonomists
MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF
MICROMYCETES ASSOCIATED WITH SEEDS OF SELECTED COTTON
(GOSSYPIUM HIRSUTUM L.) VARIETIES
AMINA KHATUN, SHAMIM SHAMSI* AND MA BASHAR
Department of Botany, University of Dhaka, Dhaka 1000, Bangladesh
Keywords: Mycoflora; Cotton varieties; ITS; PCR amplification; Sequence analysis.
Abstract
A total of 14 varieties (CB1-CB14) of cotton (Gossypium hirsutum L.) seeds were
collected from Cotton Research, Training and Seed multiplication Farm, Sreepur,
Gazipur to detect and identify the seed borne fungi by morphological and molecular
techniques. The sequence results obtained using the ITS1 and ITS4 primers were
compared with NCBI GenBank and BOL database using BLAST analysis. In the present
investigation, a total of 29 fungal isolates were morphologically identified from different
varieties of cotton seeds, of which 19 fungal isolates were identified by molecular
techniques. Among the isolated fungi, Aspergillus subramanianii, A. toxicarius, A.
wentii, Penicillium aculeatum, P. citrinum, Rhizomucor sp. and Meyerozyma
guilliermondii have been reported as new records for Bangladesh.
Introduction
Cotton, unique among agricultural crops, provides food and fibre. Cotton is major natural
fibre crop and also provides us edible oil and seeds by-products for livestock food. Cotton is
cultivated in tropical and subtropical regions of more than seventy countries of the world, which
represents 2.5% of the all cultivated land. Cotton is the second important cash crop in Bangladesh
and it is also called white Gold.
Cotton is generally propagated by seeds and these are potential harbour of numerous micro-
fungi which may impair seed germination resulting in the production of abnormal seedlings
(Bateman and Kwasna, 1999; Khanzada et al., 2002). Most cotton diseases are transmitted through
seeds which in most cases affect the quality of the fibre and seeds. Seed diseases may cause seed
rot and damping-off of the seedlings reducing subsequently the number of stands. Various fungal
seed borne pathogens have been reported in the world which reduce germination percentage and
seedling vigour of cotton seeds (Jeyalakshmi et al., 1999; Eisa et al., 2007; Tomar et al., 2012).
In Bangladesh, so far, a total of 14 diseases of cotton were recorded of which 12 diseases are
caused by fungal pathogens (BARI, 1990). Majority of cotton diseases are seed-borne viz.,
Alternaria blight, bacterial blight, Fusarium wilt, Myrothecium blight, Cercospora blight,
Exserohilum blight etc. (BARI, 1990). In Bangladesh, Alternaria tenuis, Aspergillus flavus, A.
niger, A. fumigatus, Fusarium moniliforme and Rhizopus nigricans were reported to be
predominant in cotton seeds (Lutfunnessa and Shamsi, 2011). Aspergillus flavus, A. niger,
Curvularia lunata, Fusarium moniliforme var. subglutinans, F. sporotrichioides and Rhizoctonia
solani were found to be pathogenic for 3 varieties of Hill cotton (Gossypium arboreum) in
Bangladesh (Naznin and Shamsi, 2018).
*Corresponding author: E-mail: prof.shamsi@gmail.com

298 KHATUN et al.
The correct species name of a plant pathogenic fungi is most important for the development of
effective disease control management, quarantine purposes and as a basis for making decisions to
protect agricultural crops as well as other natural resources from fungal pathogens (Rossman and
Palm-Hernandez, 2008). So far, no molecular identification report is available regarding fungi
associated with cotton seeds in Bangladesh. Therefore, the present research work was undertaken
to find out the pathogenic fungi associated with different cotton varieties following morphological
as well as molecular identification.
Materials and Methods
Seed samples of CB1-14 were collected from Cotton Research, Training and Seed
multiplication Farm, Sreepur, Gazipur after harvesting and kept in clean glass jars, labeled
properly and preserved at room temperature for subsequent use.
Fungi associated with selected rice samples were isolated with following “Tissue planting
method” on PDA medium (CAB, 1968), “Blotter method” and “Paper Towel method” (ISTA,
1996). Morphological identification of the isolates was determined following the standard
literatures (Thom and Rapper, 1945; Rapper and Thom, 1949; Subramanian, 1971; Barnett and
Hunter, 1972; Benoit and Mathur, 1970; Booth, 1971; Ellis, 1971, 1976; Sutton, 1980). Molecular
identification was done following Amer et al. (2011) with some modification.
DNA extraction
For DNA extraction, the fungi were grown on PDA medium at 25± 2ºC for 15 days. With a
sterile spatula one gm fungal mycelia were taken in 1.5 ml Eppendorf tubes from the petri plates.
The mycelia were immediately grinded with a homogenizer machine in each Eppendorf with 400
µl sterile extraction buffers (200mM Tris- HCL, 250mM NaCl, 25mM EDTA, 0.5% SDS). Then 6
µl of 20 mg/ml RNase was added in each Eppendorf. The tubes were placed in 65ºC preheated
water bath for 10 minutes. The samples were taken from the water bath and cooled down to room
temperature. In each sample, 130 µl of 3M sodium acetate, pH 5.2 was added. Samples were
vortexed for 30s at maximum speed to mix well and incubated at -20º C for 10 minutes. The
samples were centrifuged at 13,000 rpm, 4º C for 15 minutes. The supernatants were transferred to
fresh tubes and an equal volume of isopropanol was added to each sample, mixed well and were
incubated at 4°C for one night. Samples were then centrifuged at 6000 rpm, 4ºC for 20 minutes.
White coloured pellet was formed. The supernatant was discarded and the pellet was washed with
700 μl of 70% ethanol in two times. The DNA pellets were then air dried in an oven at 40°C for at
least 10 min. The resultant DNA pellet was then resuspended in 100 μl of 1 x TE (10 mM Tris-
HCl, 1 mM EDTA) buffer (pH 8.0). The DNA was dissolved overnight at 4 ºC in the refrigerator.
PCR amplification
Molecular identification of the isolates was completed using the internal transcribed spacer
(ITS) region. PCR amplification was conducted using the ITS1 (5'-TCCGTAGGTGAACCTG
CGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') primers for the ITS gene. The PCR
was performed in 0.2 ml PCR tube with 25 reaction volume containing 2.00μl Template DNA,
12.5μl Master mix, 1.0μl Forward Primer, 1.0μl Reverse Primer and 8.5μl MilliQ H2O. Reaction
mixture was vortexed and centrifuged in a micro centrifuge. The PCR was introduced by an initial
denaturation step at 94ºC for 5 minutes following 35 cycles of 94, 54 and 72ºC each for 30 sec,
with a final extension step of 5 min at 72º C and ended with 4º C. PCR amplified products were
stored in – 20º C freezer for analysis by resolving in 1% agarose gel. The gel was prepared using
1.0g agarose powder containing 8μl ethidium bromide. Agarose gel electrophoresis was conducted
in 1× TAE buffer at 90 Volts and 300 mA for 60 minutes. Alongside the ITS reactions, one

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF MICROMYCETES 299
molecular weight marker 1kb DNA ladder was electrophoresed. DNA bands were then
photographed by a Gel Documentation system (model: DI-HD, UK).
Sequence analysis
The PCR amplified products were purified by alcohol precipitation and sequenced through
automated sequencer in Centre for Advanced Research in Sciences (CARS), University of Dhaka.
The obtained sequences were compared with already available sequences in the National Center
for Biotechnology Information (NCBI, Bethesda, MD, USA) using BLAST program
(http://blast.ncbi.nlm.nih.gov) to identify the genus and species of the isolates.
Results and Discussion
Morphological identification
Twenty-nine fungal species, representing 14 genera were found to be associated with 14
varieties of cotton seeds. The isolated fungi were Aspergillus aculeatus Lizuka, A. flavus Link, A.
fumigatus Fresenius, A. niger Van Tiegh, A. nidulans Eidam, A. subramanianii Visagie, Frisvad &
Samson, A. tamarii Kita G., A. toxicarius Murak, A. wentii Wehmer, Curvularia lunata (Wakker)
Boedijn, Colletotrichum gloeosporioides Penz & Sacc, C. gossypii Southw., Chaetomium
globosum Kunze., Fusarium moniliforme J. Shelden, F. nivale (Fr.) Sorauer, F. oxysporum
Schlechtendal, F. fujikuroi Nirenberg, F. solani (Mart.) Sacc., Lasiodiplodia theobromae (Pat.)
Griffon & Maubl., Meyerozyma guilliermondii (Wick.) Kurtzman & M. Suzuki., Mucor sp. P.
Micheli ex L., Penicillium aculeatum Raper & Fennell, Penicillium citrinum Thom, Rhizoctonia
solani Khun., Rhizopus stolonifer (Ehrenb.) Vuill., Rhizopus oryzae Went & Prins. Geerl.,
Rhizomucor sp. Lucet & Costantin, Syncephalastrum racemosum Cohn and Trichoderma viride
Pers.
Based on morphological characteristics, these 29 fungal isolates were identified provisionally.
In the present investigation, some fungal species were unable to identify up to species level based
on the morphological features only. Hence, molecular characterization of the fungal species was
performed for proper identification. For further confirmation of these 29 fungi, ITS sequence
based molecular analysis was performed and 19 were confirmed up to species level.
Key morphological features of the nineteen fungi identified by molecular analysis:
Aspergillus aculeatus Lizuka, Annls sci. nat. (Bot.), Ser. 5, 8: 240 (1867) (Fig. 1A)
Aspergillus aculeatus is a ubiquitous species that usually isolated from rotting fruits and soil.
Colonies effuse, brownish black. Mycelium well developed, septate, profusely branched and
hyaline. Cells are multinucleate. Conidiophores are very long, often with a foot cell, straight or
flexuous, swollen at the apex in to a spherical vesicle. Surface of vesicle is covered by closely
packed, more or less clavate shaped branches. Conidia catenulate, dry, usually globose, echinulate,
dark brown in colour.
Specimen examined: Six varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun 04,
07 May 2018.
Aspergillus flavus Link. (Fig. 1B)
Colony colour on PDA medium is grayish powdery and fast growing. Conidial heads are
yellow to green became brownish in edge. Conidiophores are less than 1.0 mm length and 10- 20
μm diameter, vesicle was glubose to subglubose. Conidia are glubose minutely accumulate and
measured 2.5-3.5 μm. Mycelia well developed, septate, hyaline and profusely branched.

300 KHATUN et al.
Conidiophores 300-600 µm long. Cells are multinucleate Vesicles 10 - 35 µm in diameter.
Sterigmata 8 - 14 × 3 - 5 µm.
Specimen examined: Fourteen varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun,
09, 11 September, 2017.
Aspergillus fumigatus Fresenius. Beitragezur Mykologie 3:81 (1863) (Fig. 1C)
Colonies flat, olivaceous green, mycelia well developed, septate. Cells are multinucleate.
Conidiophores are long, often with a foot cell, straight or flexuous, swollen at the apex into a
spherical vesicle. Surface of vesicle are covered by closely packed more or less clavate branches.
Conidia catenulate, dry, usually globose, echinulate and smooth. Colonies of the fungus produced
thousands of minute pale green conidia 2-3 µm.
Specimen examined: Isolated from fourteen varieties of cotton seeds (Gossypium hirsutum
L.) A. Khatun, 07 March 2017.
Aspergillus subramanianii Visagie, Frisvad & Samson: 66 (1877) (Fig. 1D)
Colonies yellow to yellow-orange, ochraceus or buff, powdery to granular. Conidial heads
radiate, later splitting into several columns. Conidiophores brownish, 1-1.9 µm long, rough
walled. Vesicles globose and phialides biseriate covering almost the entire surface of the vesicle.
Conidia spherical to sub spherical, 2.5-3.5 µm in diameter, smooth walled to finely roughen.
Sclerotia are pink to vinaceous-purple coloured, irregular shaped and up to 1 mm diam. It is a
species with rough walled stipes, biseriate conidial heads, yellow to ochre conidia and sclerotia
that do not turn black.
Specimen examined: Eight varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun
07, 12 December 2017.
Aspergillus tamarii Kita G, in Centralb. F. Bakt., 37, No. 17/21, pp. 433-452. (1913) (Fig. 1E)
Aspergillus tamarii belongs in Aspergillus Section Flavi, and resembles A. flavus and A.
parasiticus, but conidia colour is olive to brown and are larger, with thick, conspicuously
roughened walls. Colonies on Czapek’s solution agar spreading broadly at room temperature with
vegetative hyphae mostly submerged, fruiting areas at first colourless, then passing through
orange yellow shades to brown in old colonies. Not showing true green but often presenting a
suggestion of green that is transient and limited to areas of young heads; reverse uncoloured or
occasionally pinkish. On AFPA, it produces a deep brown reverse coloration, in contrast to the
orange yellow of A. flavus and A. parasiticus. Conidial heads varying greatly in size in the same
fruiting area, from more or less columnar but not completely globose and upto 30µ in diameter,
with radiating chains and columns of conidia. Conidiophores arising from submerged hyphae upto
1or 2mm. in length, colorless with walls becoming abruptly thinner at the base of the vesicle.
Vesicles globose to subglobose, 25 to 30µ in diameter. Sterigmata, in large heads. Conidia
ranging from more or less pyriform, through subglobose to globose, commonly ranging from 1.5-
3µ in diameter,
Specimen examined: Twelve varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun
04, 05 March 2018.
Aspergillus wentii Wehmer (Fig. 1F)
Aspergillus wentii is an asexual, filamentous, endosymbiotic fungus. It produces single-celled,
globose, conidia in unbranched, filamentous chains. Spores are smooth, colourless, and ellipsoidal,

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF MICROMYCETES 301
approximately 1–2 µm in diameter. Conidia are darker yellow to brown in colour when mature
and have a single wall. The elongating chains of conidia are dispersed through slightly pigmented,
vase-shaped structures known as phialides that are around 6-8 µm in diameter. The conidial head
or vesicle is yellow to darker coffee-coloured and 6.0–8.0 µm in diameter. The conidiophore can
grow anywhere, 3-5 millimeters in length, has a glassy or hyaline appearance and
although granular conidiophores have been found. It produces aerial hyphae, white or sometimes
yellow in colour that can grow to a few millimeters in length. Foot cells have dense walls and are
branched.
Specimen examined: Twelve varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun
02, 07 July 2017.
Aspergillus toxicarius Murakami, (1971) (Fig. 2A)
Aspergillus toxicarius belongs in Aspergillus Section Flavi, and resembles A. flavus,
but conidia of A. toxicarius are coloured olive to yellow, and are larger, with thick, conspicuously
roughened walls. Conidiophores are less than 1.2 mm length and 10- 20 μm in diameter, vesicle
was glubose to subglubose and 15-30 μm in diameter. Colonies effuse yellowish green. Mycelia
well developed, septate, hyaline and profusely branched. Conidiophores 10-18 µm long. Cells
are multinucleate. Vesicles 15 - 30 µm in diameter. Sterigmata 10-14 × 4-5 µm. Conidia
greenish, catenulate, globose or pyriform, smooth, 4 - 5 µm in diameter. Colonies spreading
broadly, dark cress green.
Specimen examined: Eight varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun
14, 16 October 2017.
Curvularia lunata (Wakker) Boedijn. [Cochliobolus linatus Nelson & Haasis]. Ellis MB, Mycol.
Pap. 106: 2-43, 1966. (Fig. 2B)
Colonies are effuse, brown, grey or black, hairy, cottony or velvety. Stromata rarely formed in
culture, colonies on PDA markedly zonate. Conidiophores are solitary, mostly unbranched,
straight or slightly undulating, mostly flexuous geniculate, mid brown, septate up to 250 µm.
Conidia are mostly 3-septate, dark brown, mostly curved, third cell from the base is broader and
darker than others, broader cells are mid brown and other cells paler, smooth, 20.5 - 31.78 × 8.5
– 13.5 µm.
Specimen examined: Seven varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun,
06, 07 October 2017.
Fusarium oxysporum Schlecht, Flora berol. 2: 139, (1824) (Fig. 2C)
Mycelium are delicate, white in color in the culture plate. Microconidia borne on simple
phialides arising laterally on the hyphae. Microconidia generally abundant, variable, oval,
ellipsoid, cylindrical, straight, 5-12 × 2.2-3.5 µm in size and macroconidia are thin walled,
generally 3-5 septate, fusoid-subulate and pointed at both ends; 3 septate 7-14 × 3-5 µm, 5 septate
35-60 × 3-5 µm. The most commonly found spores are 3 septate.
Specimen examined: One variety of cotton seeds (Gossypium hirsutum L.) A. Khatun 17
September 2017.
Fusarium moniliforme Sheldon 1904. Rep. Neb. Agric. Exp. Stn 17:23-32 (Fig. 2D)
They are extensive and cottony, white, often with some tinge of pink mycelium. Reverse
pinkish yellow. Mycelia are hyaline, profusely branched, septate. Conidiophores are hyaline, 0-2
septate. Phialides hyaline, 16 - 20 × 3 – 4 µm in diameter and conidia are hyaline, variable,

302 KHATUN et al.
Fig. 1. Conidiophore and conidia of different fungi associated with cotton seeds. A. Aspergillus aculeatus,
B. A. flavus, C. A. fumigatus, D. A. subramanianii, E. A. tamarii and F. A. wentii (Bar = 50 µm).

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF MICROMYCETES 303
Fig. 2. Conidiophore and conidia of different fungi associated with cotton seeds. A. Aspergillus toxicarius,
B. Curvularia lunata, C. Fusarium oxysporum, D. F. moniliforme, E. F. fujikuroi, and F. F. solani
(Bar = 50 µm).

304 KHATUN et al.
Fig. 3. Conidiophore and conidia of different fungi associated with cotton seeds. A. Lasiodiplodia
theobromae, B. Mucor sp., C. Penicillium citrinum, D. P. aculeatum, E. Rhizopus oryzae, F. Trichoderma
viride and G. Meyerozyma guilliermondii (Bar = 50 µm).
principally of two kinds. Microconidia and macroconidia. Microconidia hyaline, 1-celled, ovoid or
oblong, borne singly or in chains, 5 - 15 × 2 - 3 µm. Macroconidia hyaline, several-celled, slightly
curved or bent at the pointed ends, 3 - 5 septate, 3 septate conidia 25 - 35 × 3 - 4 µm, 5 septate
conidia 30 - 50 × 3 - 5 µm.

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF MICROMYCETES 305
Specimen examined: Three varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun
25, 29 July 2017.
Fusarium fujikuroi Gibberella fujikuroi (Sawada) Wollenw., (1931) (Fig. 2E)
Colonies are white, floccus to slightly felt. Conidia are hyaline, fusiform, ovate or clavate; one
or two celled, measured 26.7-73.6 × 8.1-17.1 μm. Mycelium sparse to densely floccose or felted.
Conidiophores hyaline, usually 0-2 septate.
Specimen examined: Three varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun
03, 05 September, 2017.
Fusarium solani (Mart.) Sacc., Michelia 2: 296, 1881, emend. Snyder & Hansen pro. parte, Am.
J. Bot. 26:740, 41. (Fig. 2F)
Cottony whitish mycelium was found at the coller parts of the plant and the surrounding soil
was infected wilt the pathogen. Growth rate is 3.2 cm; colony greyish-white and aerial mycelium
striate, sparse to dense and floccose. Microconidia develops abundantly in the fresh isolates after
2-3 days. They are formed from lateral conidiphores. Microconidia of F. solani are also broader
and more oval in shape with somewhat thicker walls; they are 8-16×2-4 µm. Macroconidia
develop after four to seven days from initially simple but later from short multibranched
conidiophores which soon merge to form effuse sporodochia. They are inequilaterally fusoid with
many of the spores having the widest diameter in the penultimate cell.
Specimen examined: Two varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun 19,
21 August 2018.
Lasiodiplodia theobromae (Pat.) Griff. & Maubl, Bull. Trimest. Soc. Mycol. Fr. 8:136 (1892)
(Fig. 3A)
Colonies are greyish brown, cottony, reverse brownish black. Hyphae septate, branched, dark
chocolate brown. Pycnidia globose, dark brown, ostiolate. Conidiophore short, hyaline. Conidia
dark brown, two-celled, ellipsoidal, 16−22 × 8−12 μm. Pycnidia formed with septate paraphyses
between the conidiogenous cells. The conidia measured 20-21.8 × 9.1-10.9 µm. They are initially
hyaline, thin-walled and aseptate, cylindrical to sub ovoid in shape.
Specimen examined: Three varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun 07,
11 August 2018.
Mucor Fresen (Fig. 3B)
Colonies are typically white to beige or grey and are fast-growing. Older colonies become grey
to brown in color due to the development of spores. Mucor spores or sporangiospores are simple
or branched and form apical, globular sporangia that are supported and elevated by a column-
shaped columella. Mucor can be differentiated from moulds of the genera Absidia, Rhizomucor
and Rhizopus by the shape and insertion of the columella, and the lack of rhizoids. Some Mucor
species produce chlamydospores. They produce mold with irregular, non-septate hyphae
branching at wide angles. The tip of the sporangiophore swells to form a globose sporangium that
contains uninucleate, haploid sporangiospores. An extension of the sporangiophore called the
columella which protrudes into the sporangium. The sporangium walls are easily ruptured to
release the spores, which germinate readily to form a new mycelium on appropriate substrates.
They may germinate to form hyphae or a sporangium.

306 KHATUN et al.
Specimen examined: Seven varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun
07, 11 May 2018.
Penicillium citrinum Thom in US Dept. Agr. Bur. Amin, Ind Bul. 118, pp. 61-63. Fig. 22.1910.
(Fig. 3C)
Penicillium citrinum produces septate, hyaline hyphae. Colonies are usually fast growing, in
shades of green, sometimes white, mostly consisting of a dense felt of conidiophores.
Microscopically, chains of single-celled conidia are produced in basipetal succession from a
specialised conidiogenous cell called a phialide. They are 6.5-12.0 µm in diameter and conidia
are 1.5-3.15 µm in diameter.
Specimen examined: Fourteen varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun
09, 10, 17 August 2017.
Penicillium aculeatum Raper & Fennell (1948). (Fig. 3D)
It is characterized by very restricted and comparatively deep colonies on Czapek agar,
variously buckeled and wrinkled, irregular in outline, medium sporing, often with a limited
overgrowth of red- pigmented hyphae, growing margins 2-3 mm wide, white to slightly pink, odor
almost lacking, reverse in vinaceous or purplish red in older areas. Conidiophores short,
commonly about 50 µ, rarely upto 100 µ, with walls appearing somewhat granular. Penicilli are
relatively shorter and broader than in the preceding species, usually appearing definitely inflated,
sterigmata 9-15 µm by 1.5-3.0 µm and conidia are strictly globose to subglobose, 2-3.5 µm in
diameter with walls comparatively heavy and strongly echinulate.
Specimen examined: Four varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun 14
May 2018.
Rhizopus oryzae Went & Prins. Geerl., (1895) (Fig. 3E
)
Rhizopus oryzae is a filamentous heterothallic microfungus that occurs as a saprotroph in soil,
dung, and rotting vegetation. It differs from R. oligosporus and R. microsporus by its larger
columellae and sporangiospores. It has variable sporangiosphoressuch as straight or curved,
swollen or branched, and the walls can be smooth or slightly rough. sporangiosphores are pale
brown to brown in colour. Sporangiosphores grow between 210-250 μm in length and 5-18 μm in
diameter. The sporangia in R. oryzae are globose or subglobose, wall spinous and black when
mature, 60-180 μm in diameter. The columellae are globose, subglobose or oval in shape. The
wall is generally smooth and pale brown in colour. The average diameter growth ranges from 30-
110 μm. It has abundant, root-shaped rhizoids. The stolons are smooth or slightly rough, almost
colorless or pale brown, 5-18 μm in diameter. The chlamydospores are abundant, globose ranging
from 10-24 μm in diameter, elliptical and cylindrical. Initially colonies are white becoming
brownish with age and can grow to about 1 cm thick.
Specimen examined: Three varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun 16,
21 May 2018.
Trichoderma viride Pers. (1794) (Fig. 3F)
Colony effuses, light green in colour. Conidiophores hyaline, much branched that cluster into
fascicles, bearing phialides single or in groups. Broad and straight/flexuous branches. They may
have conidial pigments that are either white or bright green in colour. Conidia are usually hyaline,

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF MICROMYCETES 307
powdery mass, 1-celled, ovoid shaped and borne in small terminal clusters. It is used in the
commercial production of enzyme cellulase.
Specimen examined: Ten varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun 12,
14, 17 September 2017.
Meyerozyma guilliermondii (Wick.) Kurtzman & M. Suzuki (Fig. 3G)
Meyerozyma guilliermondii (formerly known as Pichia guilliermondii) is a species of yeast of
the genus Meyerozyma whose asexual or anamorphic form is known as Candida guilliermondii.
Colonies are flat, moist, smooth and cream to yellow in colour on Sabouraud dextrose agar. It does
not grow on the surface when inoculated into Sabouraud broth. Pseudohyphae are short and few in
number. Cell reproduces by budding, ellipsoidal, ovoidal and clavate, occur singly and in pairs, or
short chains, pseudohyphae is formed. Colony flat, moist, smooth, cream to yellow in colour.
Specimen examined: Two varieties of cotton seeds (Gossypium hirsutum L.) A. Khatun 16
August 2018.
Molecular identification
Molecular characterization of the fungal species was performed according to Amer et al.
(2011) with some modifications. For further confirmation of these 29 fungi, ITS sequence based
molecular analysis was performed and 19 were confirmed up to species level (Fig. 5).
Genomic DNA was successfully isolated from the nineteen isolates. PCR was conducted using
ITS1 (Forward) and ITS4 (Reverse) primers and ~600 bp DNA band was amplified (Fig. 4).
Sequence analysis of the amplified DNA through BLAST search in GenBank was conducted and
found 92.60-99.81% similarity with partial sequence of 18S ribosomal RNA gene, complete
sequence of internal transcribed spacer 1, internal transcribed spacer 2, 5.8S ribosomal RNA gene
and partial sequence 28S ribosomal RNA gene of different isolates (Table 1).
Analysis of the nucleotide sequences of the amplified fragments allowed the identification of
the isolates at the species level (Table 1 and Fig. 4). ITS1 and ITS4 primers depicted isolate
species identities more than 90% sequence similarity. All fungal isolates were identified using the
sequences obtained through ITS1 and ITS4 primers. To confirm at the genomic sequence level,
PCR amplified bands (~ 600 bp) from nineteen samples were subjected to automated sequencing
followed by BLAST analysis (Fig. 4).
ITS sequences of nineteen samples were analyzed through NCBI-BLAST program database
search system. Results obtained from the BLAST database showed that 99.09% nucleotide
identities with Aspergillus aculeatus isolate KUASN10; 98.93% nucleotide identities with
Aspergillus fumigatus isolate HF11 and Aspergillus wentii strain CBS 131.49; 97.74% nucleotide
identities with Aspergillus flavus isolate En14; 96.70% nucleotide identities with Aspergillus
tamarii isolate MH3; 96.51% nucleotide identities with Aspergillus toxicarius strain CBS 129270;
99.11% nucleotide identities with Aspergillus subramanianii 18S rRNA gene (partial); 99.38%
nucleotide identities with Curvularia lunata strain AME-83; 98.47% nucleotide identities with
Lasiodiplodia theobromae strain E42F; 98.31% nucleotide identities with Rhizopus oryzae isolate
EV62; 97.24% nucleotide identities with Penicillium aculeatum strain LP65; 94.97% nucleotide
identities with Penicillium citrinum isolate 14R-2-F05; 99.74% nucleotide identities with
Fusarium moniliforme isolate CJBB12-18; 98.15% nucleotide identities with Fusarium solani
strain GuangX9 and Fusarium oxysporum isolate FLS 4; 92.60% nucleotide identities with
Fusarium fujikuroi isolate EFS3; 92.86% nucleotide identities with Mucor sp. isolate 580816;
99.81% nucleotide identities with Trichoderma viride strain TVJ-S-1 and 98.25% nucleotide
identities with Meyerozyma guilliermondii strain Q2 (Table 1).

308 KHATUN et al.
Table 1. Identification of fungal isolates using ITS sequence comparison with data from GenBank through BLAST
search.
Iso-
lates
No.
Length
(Base pair)
Acc. No. Description
Max
score Total
score Query
coverage
(%)
E-
value
Identity
(%)
1.
573 MN186997
Aspergillus aculeatus
isolate KUASN10 small
subunit ribosomal RNA gene, partial
sequence; internal transcribed spacer 1, 5.8S
ribosomal RNA gene, and internal transcribed
spacer 2, complete sequence; and large subunit
ribosomal RNA gene, partial sequence.
985
985
98%
0.0
99.09
2. 595 MN180857
Aspergillus flavus
isolate En14 small subunit
ribosomal RNA gene, partial sequence;
internal transcribed spacer 1, 5.8S ribosomal
RNA gene, and internal transcribed spacer 2,
complete sequence; and large subunit
ribosomal RNA gene, partial sequence
981 981 97% 0.0 97.74
3. 583 GU183175
Aspergillus fumigatus
isolate HF11 18S
ribosomal RNA gene, partial sequence;
internal transcribed spacer 1, 5.8S ribosomal
RNA gene and internal transcribed spacer 2,
complete sequence; and 28S ribosomal RNA
gene, partial sequence
1002 1002 98% 0.0 98.93
4. 608 FR733823
Aspergillus subramanianii
18S rRNA gene
(partial), ITS1, 5.8S rRNA gene, ITS2 and 28S
rRNA gene (partial), culture collection
CCF<CZE>:4008
1013 1013 95% 0.0 99.11
5. 603
MH562046
Aspergillus tamarii
isolate MH3 small subunit
ribosomal RNA gene, partial sequence;
internal transcribed spacer 1, 5.8S ribosomal
RNA gene, and internal transcribed spacer 2,
complete sequence; and large subunit
ribosomal RNA gene, partial sequence
952 952 97% 0.0 96.70
6. 611 MH865314
Aspergillus toxicarius
strain CBS 129270
small subunit ribosomal RNA gene, partial
sequence; internal transcribed spacer 1, 5.8S
ribosomal RNA gene, and internal transcribed
spacer 2, complete sequence; and large subunit
ribosomal RNA gene, partial sequence.
942 942 98% 0.0 96.51
7. 589 MH856464
Aspergillus wentii
strain CBS 131.49 small
subunit ribosomal RNA gene, partial
sequence; internal transcribed spacer 1, 5.8S
ribosomal RNA gene, and internal transcribed
spacer 2, complete sequence; and large subunit
ribosomal RNA gene, partial sequence
1002 1002 98% 0.0 98.93
8. 620 MG571435
Curvularia lunata
strain AME-83 small
subunit ribosomal RNA gene, partial
sequence; internal transcribed spacer 1 and
5.8S ribosomal RNA gene, complete
sequence; and internal transcribed spacer 2,
partial sequence
867 867 79% 0.0 99.38
9. 510 KY425745
Lasiodiplodia theobromae
strain E42F small
subunit ribosomal RNA gene, partial
sequence; internal transcribed spacer 1, 5.8S
ribosomal RNA gene, and internal transcribed
spacer 2, complete sequence; and large subunit
ribosomal RNA gene, partial sequence.
917
917
99% 0.0 98.47
10. 596 MG601177
Meyerozyma guilliermondii
strain Q2 small
subunit ribosomal RNA gene, partial
sequence; internal transcribed spacer 1, 5.8S
ribosomal RNA gene, and internal transcribed
spacer 2, complete sequence; and large subunit
ribosomal RNA gene, partial sequence
996 996 96% 0.0 98.25

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF MICROMYCETES 309
Table 1 (Contd.)
Iso-
lates
No.
Length
(Base pair)
Acc. No. Description
Max
score Total
score
Query
coverage
(%)
E-
value
Identity
(%)
11. 545 KX958025
Penicillium citrinum isolate 14R-2-F05 internal
transcribed spacer 1, partial sequence; 5.8S
ribosomal RNA gene and internal transcribed
spacer 2, complete sequence; and large subunit
ribosomal RNA gene, partial sequence.
873 873 98% 0.0 94.97
12. 590 HQ392496
Penicillium aculeatum strain LP65 18S ribosomal
RNA gene, internal transcribed spacer 1, 5.8S
ribosomal RNA gene, internal transcribed spacer
2, and 28S ribosomal RNA gene, partial sequence
454
454
86% 0.0 97.24
13. 568 KY785016
Fusarium solani strain GuangX9 small subunit
ribosomal RNA gene, partial sequence; internal
transcribed spacer 1, 5.8S ribosomal RNA gene,
and internal transcribed spacer 2, complete
sequence; and large subunit ribosomal RNA
gene, partial sequence
942 942 98% 0.0 98.15
14. 585 MK371768
Mucor sp. isolate 580816 internal transcribed
spacer 1, partial sequence; 5.8S ribosomal
RNA gene and internal transcribed spacer 2,
complete sequence; and large subunit ribosomal
RNA gene, partial sequence
845 845 100% 0.0 92.86
15. 560 KF439055 Trichoderma viride strain TVJ-S-1 28S ribosomal
RNA gene, partial sequence. 942 942 100% 0.0 99.81
16. 598 KU671029
Fusarium oxysporum isolate FLS 4 small subunit
ribosomal RNA gene, partial sequence; internal
transcribed spacer1, 5.8S ribosomal RNA gene,
and internal transcribed spacer 2, complete
sequence; and large subunit ribosomal RNA
gene, partial sequence.
942 942 98% 0.0
98.15
17. 586 MH084746
Fusarium fujikuroi isolate EFS3 small subunit
ribosomal RNA gene, partial sequence; internal
transcribed spacer 1, 5.8S ribosomal RNA gene,
and internal transcribed spacer 2, complete
sequence; and large subunit ribosomal RNA
gene, partial sequence
911 911 96% 0.0 92.60
18. 576 KC895528
Gibberella moniliforme isolate CJBB12-18 18S
ribosomal RNA gene, partial sequence; internal
transcribed spacer 1, 5.8S ribosomal RNA gene,
and internal transcribed spacer 2, complete
sequence; and 28S ribosomal RNA gene, partial
sequence.
993 993 96% 0.0 99.74
19. 621 MK108436
Rhizopus oryzae isolate EV62 small subunit
ribosomal RNA gene, partial sequence; internal
transcribed spacer 1, 5.8S ribosomal RNA gene,
and internal transcribed spacer 2, complete
sequence; and large subunit ribosomal RNA
gene, partial sequence.
1033 1033 95% 0.0 98.31
From the comparison between morphological and molecular identification, it was clear that out
of 19 fungal isolates morphological identification of one fungal isolate did not match with
molecular identification. It was Aspergillus ochraceous which was identified as Aspergillus
subramanianii by molecular identification (Table 2). Besides, there were four species of
Aspergillus, two species of Fusarium and two species of Penicillium which were difficult to
identify up to species label by morphological identifications. The species name of these fungi were
easily identified by this molecular technique. Furthermore, one unidentified fungus was detected
up to species level employing nucleotide sequences (Table 2).

310 KHATUN et al.
Table 2. Comparison between morphological and molecular identification of 19 fungal isolates.
lsolates
No. Morphological identification Molecular identification
1. A. flavus A. flavus isolate En14
2. A. fumigatus A. fumigatus isolate HF11
3. A. ochraceous Aspergillus subramanianii 18S rRNA gene (partial)
4. Aspergillus sp. 1 Aspergillus aculeatus isolate KUASN10
5. Aspergillus sp. 2 Aspergillus tamarii isolate MH3
6. Aspergillus sp. 3 Aspergillus wentii strain CBS 131.49
7. Aspergillus sp. 4 Aspergillus toxicarius strain CBS 129270
8. Curvularia lunata Curvularia lunata strain AME-83
9. Fusarium fujikuroi Fusarium fujikuroi isolate EFS3
10. Fusarium oxysporum Fusarium oxysporum isolate FLS 4
11. Fusarium sp. 1 Fusarium solani strain GuangX9
12. Fusarium sp. 2 Fusarium moniliforme isolate CJBB12-18
13. Lasiodiplodia theobromae Lasiodiplodia theobromae strain E42F
14. Mucor sp. Mucor sp. isolate 580816
15. Penicillium sp. 1 Penicillium citrinum isolate 14R-2-F05
16. Penicillium sp. 2 Penicillium aaculeatum strain LP65
17. Rhizopus oryzae Rhizopus oryzae isolate EV62
18. Trichoderma viride Trichoderma viride strain TVJ-S-1
19. Unidentified fungus Meyerozyma guilliermondii strain Q2
Fig. 4. Gel electrophoresis of the PCR products of 19 fungal isolates performed by ITS1 (F) and ITS4 (R)
primers and showing ~600 bp amplification.

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF MICROMYCETES 311
Fig. 5. DNA sequences of the PCR products of isolated fungi.
Among the isolated fungi, Aspergillus subramanianii, A. toxicarius, A. wentii, Penicillium
aculeatum, P. citrinum, Rhizomucor sp. and Meyerozyma guilliermondii have been reported as
new records for Bangladesh as these were not documented in relevant literature (Siddiqui et al.,
2007; Shamsi S, 2017; Nahar et al., 2019).
The present investigation suggests that molecular technique is more accurate and rapid means
of fungal identification. ITS-based molecular identification methods might be an important
complement to conventional mycological detection by culture.
Acknowledgements
The first author gratefully acknowledges to the “Ministry of Science and Technology”,
People’s Republic of Bangladesh for providing financial support in her research through NST
fellowship.
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(Manuscript received on 01 January, 2022; revised on 14 October, 2022)