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volume 4, issue 6, 2025
224
MOLECULAR IDENTIFICATION OF ENDOPHYTIC FUNGI ISOLATED FROM
HYSSOPUS OFFICINALIS
Eshboev Farkhod
Senior Research Fellow, PhD,
Institute of the Chemistry of Plant Substances
Valieva Mufazzal
Master National University of Uzbekistan
Khayrullaeva Lobar
Assistant Lecturer,
Qarshi State University
Annotation:
In recent years, endophytic fungi of medicinal plants have gained considerable
attention as potential sources of novel bioactive compounds. These microorganisms, which
reside asymptomatically within plant tissues, are known to produce a wide range of secondary
metabolites with antimicrobial, antifungal, and antioxidant properties. In this study, a highly
active endophytic fungal isolate was obtained from the vegetative organs of
Hyssopus officinalis
and subjected to molecular identification. Using the ITS (Internal Transcribed Spacer) region
sequencing and BLAST analysis against the NCBI GenBank database, the isolate was identified
as
Colletotrichum elatum
. DNA extraction and sequencing were conducted using CTAB method,
PCR amplification with ITS5-ITS4 primers, and Sanger sequencing. The obtained sequence was
submitted to GenBank under the accession number OP476344.1. A phylogenetic tree was
constructed using MEGA 11 software to confirm taxonomic affiliation. This study reports for the
first time the isolation of
C. elatum
from
H. officinalis
, underlining its potential as a novel source
of bioactive compounds.
Keywords:
Endophytic fungi,
Hyssopus officinalis
,
Colletotrichum elatum
, secondary
metabolites, ITS sequencing, phylogenetic analysis, antimicrobial activity, BLAST, molecular
identification.
Introduction.
The biochemical and pharmaceutical industries rely on endophytic fungi as a
promising source of novel therapeutic biomolecules, which may include immunosuppressants,
anticancer drugs, plant growth promoters, antimicrobial agents, insecticides, antioxidants, and
antibiotics, thereby offering significant potential for medical applications [1]. Combating
pathogenic microorganisms is complicated by the high variability of pathogenic agents.
Morphological analysis methods are not sufficient for the objective assessment of fungal
biodiversity. In such cases, molecular genetic approaches, particularly polymerase chain reaction
(PCR) followed by sequencing of the amplicons, can be highly beneficial [3].
In our previous studies, among the isolates obtained from the vegetative parts of medicinal plants,
only one demonstrated strong antimicrobial activity. Therefore, this isolate was subjected to
molecular identification.
Results
. To study the diversity of phytopathogenic fungi, the nucleotide composition of the
nuclear ribosomal gene regions (ITS) was analyzed. Sequencing of the internal transcribed
spacer (ITS) and the large subunit (LSU) regions of rRNA, followed by comparative sequence
analysis, has become the "gold standard" for molecular identification of most fungi, particularly
those that are cultivable [2]. This strategy is rapid and accurate but depends on the quality of the
sequences available in existing databases.
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volume 4, issue 6, 2025
225
DNA was extracted using conventional methods, lysed in CTAB buffer, and purified with
chloroform. PCR amplification was performed using fungal-specific primers: ITS5–ITS4 [4,5].
The PCR-amplified ITS region was purified for subsequent sequencing. The amplified product
was cleaned using a commercial purification kit (QIAquick PCR Purification Kit) and then
subjected to gene sequencing. Sequencing was carried out using the Sanger method. The
resulting sequences were compared against database entries using the BLAST bioinformatics
tool to determine interspecies differences [3].
The molecular identification of the studied isolate was based on sequencing the ITS4 and ITS5
regions of the fungal genome. The obtained DNA sequence (578 bp) was submitted to GenBank
under the accession number OP476344.1. A BLAST search in the NCBI database confirmed the
isolate as
C. elatum
. This study reports
C. elatum
as being isolated from
H. officinalis
for the
first time. A phylogenetic tree of this strain was constructed using MEGA 11 software, based on
the obtained ITS sequence and the top nine related sequences from GenBank identified through a
BLAST search in the National Center for Biotechnology Information (NCBI) database (Figure 1).
Figure 1. Antimicrobial Activity Assay of the First Compound
Phylogenetic tree constructed using the ITS (Internal Transcribed Spacer) sequence of the isolate
obtained from the GenBank database after a BLAST search with MEGA 11 (Molecular
Evolutionary Genetics Analysis, version 11). The neighbor-joining tree was generated based on
1000 bootstrap replicates. All positions containing gaps and missing data were eliminated from
the dataset (complete deletion option).
References:
1. Conrad L Schoch, Keith A Seifert, Sabine Huhndorf, Vincent Robert, John L Spouge, C
André Levesque, Wen Chen; Fungal Barcoding Consortium; Fungal Barcoding Consortium
Author List; Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA
barcode marker for Fungi; 2012 April 7
2. White T.J, Bruns T, Lee S. & Taylor J. (1990) «Amplification and Direct Sequencing of
Fungal Ribosomal RNA Genes for Phylogenetics» page 13
3. Clement K.M. Tsui, James Woodhall, Wen Chen, K Andre Levesque, Anna Lau, Cor D Sean,
Christiana Bashien, Mohammad Dj. Nadjafzadeh, G Sibren de Hoog. Molecular methods for the
identification of pathogens and the detection of fungi in the environment. PMCID: PMC3359816
PMID: 22679603
4. Yurkov A.P., Kryukov A.A., Gorbunova A.O., Kojemyakov A.P., Stepanova G.V., Machs
E.M., Rodionov A.V., Shishova M.F.. Molecular genetic identification of fungus arbuscular
mycorrhiza. doi: 10.17816/ecogen16211-23\\ Genetic basis of ecosystem evolution.
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spotting of potatoes and tomatoes in modern mycology in Russia. // «Sovremennaya mikologiya
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