Bangladesh J. Plant Taxon. 29(2): 361-371, 2022 (December) DOI: https://doi.org/10.3329/bjpt.v29i2.

63534
© 2022 Bangladesh Association of Plant Taxonomists

MORPHOLOGICAL AND MOLECULAR IDENTIFICATION OF

ENDOPHYTIC FUNGI ISOLATED FROM ZINGIBER OFFICINALE ROSC.
KAZI JANNATUL FERDOUS1, MD. HOSSAIN SOHRAB1, MST. NADIRA BEGUM2,
MD. RAKIBUL ISLAM AND MD. ABDUL MAZID*3
Department of Biochemistry and Molecular Biology, Faculty of Biological Sciences,
University of Dhaka, Dhaka-1000, Bangladesh

Keywords: Endophyte; Morphology; BLAST results; Phylogenetic identification.

Abstracts
This study was conducted to discover wide spectrum of endophyte diversity from
the Zingiber officinale Rosc. Endophytic fungi were obtained from different plant tissues.
All the isolated strains were identified up to genus level following described colony
morphology. The recognized five different morphotypes were subjected to sequence
analysis of internal transcribed spacer (ITS) gene. Fusarium proliferatum, Fusarium
solani and Cladosporium cladosporoides were identified based on comparison of the
BLAST results and phylogenetic identification. The presented study provides the
comprehensive explanation of the interrelation of morphological and molecular
homologies for the identification of the prospective fungi.

Introduction
Endophytic fungi are miscellaneous polyphyletic groups of microorganisms that can boom
asymptomatically in different healthy tissues (stems, leaves, and/or roots) of living plants. It is
estimated that over one million endophytic fungal species occur in the nature (Faeth and Fagan,
2002). The bioactive compounds produced by endophytic fungi can induce the production of a
plethora of known and novel biologically active secondary metabolites that can be utilized and
functional by human as important medicinal resources (Zhang et al., 2006; Firáková et al., 2007;
Rodriguez et al., 2009).
Zingiber officinale Rosc. (Ginger) is a herbaceous perennial plant of Zingiberaceae family.
The family Zingiberaceae has 52 genera and 1400 species. It is distributed throughout tropical
Africa, Asia, and the Americas. It has great traditional medicinal value being employed in many
indigenous medical systems since ancient time. Many members of Zingiberaceae are used in
Ayurvedic, Unani, and Homoeopathic systems of medicine. That is why this family is
ethnophamacologically important. Phytochemical investigation of the rhizomes of several
Zingiber sp. has disclosed the presence of bioactive compounds such as gingerols, shogaols,
diarylheptanoids, phenylbutenoids, flavanoids, diterpenoids and sesquiterpenoids (Sivasothy et al.,
2011). The gingerols are identified as the major active components in the fresh rhizome of the
plant. In addition, shogaols, dehydrated gingerol derivatives, are the predominant pungent
constituents in dried ginger (Jiang et al., 2006). This plant also reported to possess several
pharmacological activities such as antimicrobial activity, anti-diabetic activity, nephroprotective

*Corresponding author: E-mail: ma.mazid@du.ac.bd

1
Pharmaceutical Sciences Research Division, BCSIR Laboratories Dhaka, Bangladesh Council of Scientific and Industrial
Research (BCSIR), Dhaka-1205, Bangladesh.
2
Biological Research Division, BCSIR Laboratories Dhaka, Bangladesh Council of Scientific and Industrial Research
(BCSIR), Dhaka-1205, Bangladesh.
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh.
362 FERDOUS et al.

activity, hepatoprotective activity, larvicidal activity, anticancer activity, analgesic activity, anti-
inflammatory activity, immunomodulatory activity, antioxidant activity, anthelmintic property etc.
(Kumar et al., 2011). But searching of endophytic fungal miscellany from this plant remains
unacquainted.
Endophytic fungi show diversity that belongs to several taxonomic groups. Taxonomy uses
hierarchical classification as a way to facilitate scientists understands and organizes the diversity.
For the taxonomical identification, both the morphological and the molecular characteristics of the
organism are needed. Morphological identification is the conventional method of describing
physical features of the filamentous fungi that are given some possible clues of identification. Due
to the drawbacks of conventional methods, molecular techniques are used to investigate the
problems related to identification and classification of species. Identification of fungi to species
level is vital for both basic (ecology, taxonomy) and applied (genomics, bioprospecting)
applications in scientific research. As for genetic materials, phylogenetic analysis is another
alternative way of species identification. It is the study of evolutionary development of a gene to
understand the evolutionary relationships among species. Thus, in this report a detailed
characterization of endophytic fungi isolated from Zingiber officinale had been described with
respect to morphological and molecular approach for further preliminary screening of their
bioactive potentiality.

Materials and Methods

Plant collection and isolation of endophytes
Healthy and mature Z. officinale plants were collected from Dhamrai, Dhaka, Bangladesh and
the plant was recognized and validated by a taxonomist of Bangladesh National Herbarium
(BNH), Dhaka, Bangladesh. A receipt herbarium specimen under the accession number DACB
55762 of Z. officinale was assigned and deposited at the BNH.
Fungal endophytes were isolated from leaf, bark and petiole parts of the fresh and healthy
plant tissues following a revised surface sterilization method (Chowdhury et al., 2016; Khan et al.,
2016). For surface sterilization, the selected cleaned plant parts were undergone into a small
cutting (2–3 cm) over a sterile glass plate. The cutting edges were then subjected to subsequent
treatment with 70% ethanol, 1.3 M sodium hypochlorite and finally sterile distilled water
respectively. The treated plant samples were then soaked on sterile filter paper and placed in an
antibiotic (streptomycin 100 mg/l) containing water agar medium for incubation (at 28 ± 2°C).
The visible mycelium come out over 4-6 weeks was further transferred into Potato Dextrose Agar
(PDA) medium to isolate the endophytes by comparing the growth of the exophytes incubated in a
same manner collected from the unsterilized plant segments to be used for control study. The
isolated pure cultures were cultivated on PDA medium and for obtaining the crude fungal extracts
of each isolate, the cultured medium of all the fungal strains were extracted three times with ethyl
acetate.

Identification of the isolated endophytes

Morphological identification: Isolated endophytes were identified morphologically based on
macroscopic and microscopic features. Morphological characteristics such as growth pattern,
hyphae structure, the color of the colony and medium, aerial mycelium, surface texture,
sporulation and production of acervuli, the size and coloration of the conidia were examined in
3rd, 6th, 9th and 12th days of cultural growth on PDA medium until full growth of fungi and
compared with the standard taxonomic key (Devi and Prabakaran, 2014; Barnett and Hunter,
1972). Microscopic study of the isolated strains was done followed by staining with lactophenol
MORPHOLOGICAL AND MOLECULAR IDENTIFICATION OF ENDOPHYTIC FUNGI 363

cotton blue (LPCB) and examined under a bright-field and phase contrast microscope (Kruss,
Germany) with objective lens of 40 times magnification and 0.65 numerical apertures (Sadananda,
2014).
Molecular identification
To identify the species of the respective fungus, selected endophyte isolates were subjected to
molecular characterization by DNA amplification and sequencing of the internal transcribed
spacer (ITS) region. Here ITS4 (5ʹ-TCCGTAGGTGAACCTGCGG-3ʹ) (Invitrogen, USA) and
ITS5 (5ʹ- TCCTCCGCTTATTGATATGC-3ʹ) (Invitrogen, USA) were used as forward and
reverse primer, respectively (White et al., 1990). Fungal DNA isolation was carried out by using
DNeasy Minikit (QIAgen, USA) according to the manufacturer’s protocol. The target DNA
sequence was then amplified by polymerase chain reaction (PCR) using Hot StarT aq Master Mix
Kit (QIAgen, USA). ITS4 and ITS 5 primers, were mixed with Hot Star Taq Master Mix Kit and
DNA template in a total volume of 50μLwhere each PCR reaction mixture contained 5-10 ng of
genomic DNA, 1 µMeach of the primers ITS4 and ITS5 and 1 U of Hot Star Taq Polymerase. The
mixture was then applied to the thermal cycler (BioRad, USA) for 35 cycles using initial pre-heat
at 95°C for 2 minutes; denaturing for 1minute at 95°C, annealing for40 seconds at 56°C, extension
for 1 minute at 72°C, final extension for 10 minutes in 72°C. Approximately 550 bp PCR product
purification was carried out by using perfect Prep Gel Cleanup Kit (Eppendorf, USA) following
manufacturer’s protocol. The amplified pure fungal DNA (PCR product) was sequenced using
electrophoretic sequencing on an ABI370X1 DNA analyzer (Applied Biosystems, USA) using Big
dye Terminator v 3.1 cycle sequencing kit.

Phylogenetic analysis
The resulting sequences of the isolated endophytes were then subjected to nucleotide BLAST
in order to compare the regions of similarity of the query sequences against the deposited
biological sequences into the NCBI databases. To understand the evolutionary relationship,
phylogenetic tree of each isolated strain was build up using the selected database sequences
through the blast search along with the query sequence. Phylogenetic trees were constructed using
MEGA-X software following the statistical method of maximum likelihood including 1000
bootstrap replications.

Results and Discussion

Identification of the isolated fungi
A total 5 endophytes were isolated from the rhizome (bark) (ZOBE-1, ZOBE-2), petiole
(ZOPE-3) and leaf parts (ZOLE-1, ZOLE-2) of Zingiber officinale (Fig. 1). All the isolated
endophytes were morphologically identified up to the genus level and up to the species level
through molecular identification.

Morphological identification
According to the morphological characteristics, four endophytes belongs to Fusarium sp. (ZOBE-
1, ZOBE-2, ZOPE-3 and ZOLE-1); and another one belongs to Cladosporium sp. (ZOLE-2).
Identification was based on describing the colony characteristics of 12 days cultural growth
according to macroscopic and microscopic point of views explained in Tables 1,2 and 3 which
were also verified as confirmed by the previously described features.
364 FERDOUS et al.

Fig. 1. Isolated endophytic fungi from Zingiber officinale. (A) ZOBE-1 (Fusarium sp.), (B)
ZOBE-2 (Fusarium sp.), (C) ZOPE-3 (Fusarium sp.), (D) ZOLE-1 (Fusarium sp.) and
(E) ZOLE-2 (Cladosporium sp.)
MORPHOLOGICAL AND MOLECULAR IDENTIFICATION OF ENDOPHYTIC FUNGI 365

Table 1. Morphology of the fungal strains isolated from Rhizome (bark).

Strains Morphological characterization Identified genus

ZOBE-1 Macroscopic View Fusarium sp.
Upper view of the colony: Brown color observed in center with white side; Lower (Ignjatov et.al.,
view of the colony: light yellowish-brown; growth rate: moderate. Hyphae: soft and 2019)
aerial mycelium; Morphology of colony: villous, irregular and raised elevation.
Microscopic View
Mycelium: mass branched; Spores: single celled, small and large, Microconidia:
hyaline, delicate, slightly sickle-shaped or almost straight, Macroconidia: sickle-
shaped, one to three septa.
ZOBE-2 Macroscopic View Fusarium sp.
Upper view of the colony: White color; Lower view of the colony: Light purple; (Ignjatov et al.,
Growth rate: moderate; Hyphae: fertile, growing vertically; Morphology of colony: 2019)
villous, circular including entire margin.
Microscopic View
Mycelium: Branched and thread shaped; Spores: rod shaped, septed; Conidia:
slender sickle-shaped, one to three septa

Table 2. Morphology of the fungal strains isolated from petiole.

Strains Morphological characterization Identified genus

ZOPE-3 Macroscopic View Fusarium
Upper view of the colony: Purple and white; Lower view of the colony: deep purple sp.(Ignjatov et al.,
in center with white side; Growth rate: fast; Hyphae: septate; Morphology of colony: 2019; Zainudin
cottony, irregular. et al., 2017)

Microscopic View
Mycelium: Aerial, branched and unbranched; Spores: single and two celled,
cylindrical; Microconidia: small, oval, one or two celled; Macroconidia: typically
curved like a sickle, three to five septa expanded in the middle of their length.

Table 3. Morphology of the fungal strains isolated from leaf.

Strains Morphological characterization Identified genus

ZOLE-1 Macroscopic View Fusarium sp
Upper view of the colony: White; Lower view of the colony: Light yellow; growth .(Chehri, et.al.,
rate: rapid. Hyphae: soft and cottony mycelium; Morphology of colony: circular 2015)
including entire margin..
Microscopic View
Mycelium: Aerial; Spores: single and two celled, small and large, Microconidia: Oval
shaped, Macroconidia: curved.
ZOLE-2 Macroscopic View Cladosporium
Upper view of the colony: grey-olivaceous; Lower view of the colony: Light sp. (Torres et al.,
yellowish olive green; Growth rate: slow; Morphology of colony: velvety, irregular. 2017)
Microscopic View
Mycelium: aerial, sparse, diffuse, or sometimes abundantly formed; Spores: rod
shaped, septed; Conidia: subglobose, obovoid, ovoid to limoniform, aseptate;
Conidiophores: Straight, solitary, unbranched, terminal or lateral and without nodules.
366 FERDOUS et al.

Molecular Identification
The 5 fungal isolates had been identified at species level through the molecular identification
including ITS gene sequencing, BLASTN (NCBI) database queries and also interpretation of the
phylogenetic analysis respectively. Table 4 explained the list of those best matched organisms that
are obtained after BLASTN programs search of the respective strain sequences.

Table 4. Blast results outputs of the selected isolated strains.

SL Strains Query Percent Organisms with highest similarity including Accession No.
cover Identity
1. ZOBE-1 100% 100% Fusarium proliferatum (HF930594.1)
2. ZOBE-2 100% 100% Fusarium proliferatum (LS422790.1)
3. ZOPE-3 98% 100% Fusarium proliferatum (MT280199.1)
4. ZOLE-1 100% 99.64% Fusarium solani (KT184398.1)
5. ZOLE-2 100% 100% Cladosporium cladosporioides (MG572365.1)

Phylogenetic analysis
Phylogenetic analysis is a method to elucidate the evolutionary history and relationship
among a group of organisms. A phylogenetic tree is a diagram that represents evolutionary
relationships among organisms. In a phylogenetic tree, the relatedness of two species has a very
specific meaning. Two species are more related if they have a more recent common ancestor, and
less related if they have a less recent common ancestor. The species or groups of interest are found
at the tips of lines referred to as the tree's branches. The pattern of branching in a phylogenetic tree
reflects how species or other groups evolved from a series of common ancestors. Each branch
point (also called an internal node) represents a divergence event, or splitting apart of a single
group into two descendant groups known as a clade. The branch lengths estimate the genetic
distance, whereas the branch values represent the bootstrap confidence values.
Regarding the phylogram of Fig. 2 in which ZOBE-1 falls outside the polyphyletic group of
diverse species, is located just adjacent to Fusarium proliferatum (Acc. no. HF 930594.1) that are
strongly supported due to the highest bootstrap value of 82%. Morphological verifications and
also such distant relationship distinguishing ZOBE-1 as Fusarium proliferatum (Ignjatov, et.al.,
2019).
In Fig. 3, ZOBE-2 falls outside the polyphyletic group of diverse species, is located just
adjacent to Fusarium proliferatum (Accession no. LS 422790.1) that are weekly supported due to
low bootstrap value which may be caused by the poor alignment. However, according to the
exploration of BLASTN (NCBI) database queries where most of the search results obtained for the
same respective organism and also the previous morphological investigations, ZOBE-2 can be
confirmed as Fusarium proliferatum (Zainudin et al., 2017).
In Fig. 4, ZOPE-3 falls outside the polyphyletic group of diverse species, is located just
adjacent to Fusarium proliferatum (Accession no. MT 280199.1) that are weekly supported due to
low bootstrap value which may be caused by the poor alignment. However, according to the
exploration of BLASTN (NCBI) database queries where most of the search result obtained for the
same respective organism and also the previous morphological investigations, ZOBE-2 can be
confirmed as Fusarium proliferatum (Zainudin et al., 2017).
MORPHOLOGICAL AND MOLECULAR IDENTIFICATION OF ENDOPHYTIC FUNGI 367

Fig. 2. Phylogenetic relationship between ZOBE-1 and the other related species constructed using maximum
likelihood method (1000 bootstrap replication) including the bootstrap values supported each node.

Fig. 3. Phylogenetic relationship between ZOBE-2 and the other related species constructed using maximum
likelihood method (1000 bootstrap replication) including the bootstrap values supported each node.
368 FERDOUS et al.

Fig. 4. Phylogenetic relationship between ZOPE-3 and the other related species constructed using maximum
likelihood method (1000 bootstrap replication) including the bootstrap values supported each node.

Regarding the phylogram of Fig. 5 in which ZOLE-1 falls outside the polyphyletic group of
diverse species that are strongly supported due to bootstrap value of 36% (Accession no.
KT184398.1). Morphological verifications and also such distant relationship distinguishing
ZOLE-1 as Fusarium solani (Chehri et al., 2015).

Fig. 5. Phylogenetic relationship between ZOLE-1 and the other related species constructed using maximum
likelihood method (1000 bootstrap replication) including the bootstrap values supported each node.
MORPHOLOGICAL AND MOLECULAR IDENTIFICATION OF ENDOPHYTIC FUNGI 369

In case of Fig. 6, where ZOLE-2 serves as an outgroup positioning beyond the paraphyletic
clade of several Cladosprium sp., is more distantly related to Cladosporium cladosporioides
(Accession no. MG 572365.1) with a probably significant bootstrap value of 61%. Following that
arrangement, the ZOLE-2 strain can be registered as Cladosporium cladosporioides the
morphology of which can also viewed as same as the respective strain (Torres et al., 2017).

Fig. 6. Phylogenetic relationship between ZOLE-2 and the other related species constructed using maximum
likelihood method (1000 bootstrap replication) including the bootstrap values supported each node.

Discussion
This study was conducted to characterize both morphological and molecular examination
following phylogenetic analysis for targeting the proper identification of all the fungi isolated
from the plant Zingiber officinale. The isolated five strains belong to two genera like Fusarium
and Cladosporium. However, morphological analysis must be compared with the result of
molecular examination, as some characteristics are identical between species. For this reason the
recognized genera of the isolated fungi were further identified at the species level based on 5.8S-
rRNA-ITS sequences. In finding of evolutionary relationship, phylogenetic trees are commonly
constructed. A phylogenetic tree sorts organisms into clades or groups of organisms that
descended from a single ancestor using maximum parsimony. All the developed phylogenetic
trees of the respective strain were found to be reliable based on bootstrap values except for ZOBE-
2 and ZOPE-3 because of forming a weak monophyletic clade.
The genera Fusarium sp. contains over 300 species and widely distributed in various habitats,
aquatic, soil, and plant associated. From the literature it is suggested that they produce many
famous bioactive compounds such as equisetin (anti-HIV and anti-bacterial activities) (Jeong and
Moloney, 2015), coniosetin (anti-bacterial activity) (Segeth et al., 2003) and fusarisetin A
(inhibiting the tumor metastasis) (Jang et al., 2011), and so on. In light of this, the Fusarium genus
fungi have become a rich and hot source for discovering drug leads. The genus Cladosporium
includes more than 30 species, with C. cladosporioides as one of the most common species.
Cladosporium sp. was reported to produce several secondary metabolites, including cladosporin,
emodin, phytase, taxol, and other antibiotic and antifouling compounds (Quan et al., 2004; Zhang
et al., 2009; Xiong et al., 2009). Another study isolated and identified beneficial secondary
metabolites (brefeldin A) from an isolated active strain I(R)9-2, Cladosporium sp. (Wang et al.,
2007). Two naphthoquinones, namely anhydrofusarubin and methyl ether of fusarubin were
370 FERDOUS et al.

isolated from Cladosporium sp. by Md. Imdadul Huque Khana et al. The isolated compounds
showed potential cytotoxicity and prominent antibacterial properties (Khan et al., 2016).
Following such previous investigating report, it can be proposed that these identified endophytes
can be the great resources of novel antimicrobial or anticancer compounds. The present study
provides a basis for such further studies. Secondary metabolites of Fusarium sp. and
Cladosporium sp. can be the preference of our future research.

Conclusion
The current study defines the structural detection of all the fungal isolates that is confirmed
throughout the genetic analysis including their commencing from phylogenetic theory. Such
features of these potential fungi can be considered as the most suitable information in research
database.

Acknowledgement
Authors are grateful to Pharmaceutical Science Research Division, BCSIR Laboratories
Dhaka for providing with the necessary laboratory and instrumental facilities and also for all the
chemical and reagent supports.

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(Manuscript received on 15 May, 2021; revised on 17 November, 2022)

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