Characterization and Antagonistic Activity of New Causal Agent of Wilt Disease in Imperata cylindrica (Marasmius palmivorus)

Abstract

The genus Marasmius is distributed worldwide, along with a precise identification of this fungus as a pathogen on the Imperata cylindrical plant, although its bioactive role is not well understood. The current work was designed to examine the population of Marasmius palmivorus and its antagonistic patterns. Sequencing the ITS genetic region of this fungus occurs through verifying the cladistics nature, and taxonomic validity, in addition to studying the antagonistic potential of this fungus. M. palmivorus (MG717877.1) was recorded in this plant, which represents a new finding in Iraq mycobiota. Data from the sequence alignment were used to plot a phylogenetic tree, using the Neighbor-Joining method (NJ) in molecular evolutionary genetics analysis based on Mega 7. Phylogenetic trees show a close relationship of 99% between this strain of M. palmivorus (MG717877.1) and international strains. On the other hand, the maximum antagonistic effect of M. palmivorus has been found to act against Fusarium solani and F. thapsinum, and to a lesser degree against Penicillium sp., T. harzianum, and P. cyclopium after 6 to 8 days of incubation. Results confirm the precise nomination of this taxon is M. palmivorus, which may act as a bioherbicidal and could be used as bioagent against various phytopathogenic fungi.

Full text

*Correspondence: jka.uobsci.iq@gmail.com; +964783193359
(Received: 28 June 2019; accepted: 04 September 2019)
Hassan A. Tamur, Haider Jawad Al-Janabi, Jawad K. Abood Al-Janabi, Liqaa Y. Mohsin, Zahraa A.N. Al-Yassiry,
Characterizaon and Antagonisc Acvity of New Causal Agent of Wilt Disease in Imperata cylindrica (Marasmius palmivorus),
J Pure Appl Microbiol., 2019; 13(3): 1525-1536. hps://doi.org/10.22207/JPAM.13.3.24
© The Author(s) 2019. Open Access. This arcle is distributed under the terms of the Creave Commons Aribuon 4.0 Internaonal License which
permits unrestricted use, sharing, distribuon, and reproducon in any medium, provided you give appropriate credit to the original author(s) and
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Tamur et al. J Pure Appl Microbiol, 13(3), 1525-1536 | September 2019
Arcle 5684 | hps://doi.org/10.22207/JPAM.13.3.24
Print ISSN: 0973-7510; E-ISSN: 2581-690X
RESEARCH ARTICLE OPEN ACCESS
www.microbiologyjournal.org1525Journal of Pure and Applied Microbiology

Imperata cylindrica
(Marasmius palmivorus)
Hassan A. Tamur122,
Liqaa Y. Mohsin33
1Genec Engineering Department, Biotechniques College, Green University of Al Qasim, Iraq. 2Al-Mustaqbal
University College, Iraq. 3Department of Biology, College of Science, University of Babylon, Iraq.

      
Imperata cylindrical
Marasmius palmivorus 
ITS
M. palmivorus

      
            
M. palmivorus On the
M. palmivorusFusarium
solani and F. thapsinum, Penicillium sp., T. harzianum, and P. cyclopium
M. palmivorus,

 Marasmius palmivorus, Phylogenec structure, Antagonism.

www.microbiologyjournal.org1526
Tamur et al. J Pure Appl Microbiol, 13(3), 1525-1536 | September 2019 | hps://doi.org/10.22207/JPAM.13.3.24
Journal of Pure and Applied Microbiology

Marasmius is a genus of mushroom-
forming fungi in the family Marasmiaceae, order:
Agaricales, class: Agaricomycetes, division:
Basidiomycota1. In 2005, Kirk2 listed 955 species
belonging to Marasmius in the Index Fungorum.
There has been recent aenon in understanding
worldwide biodiversity and the evolution of
Marasmius3-7.
Marasmius palmivorus is most
phonecally like to a complex of the Marasmiellus
species, which is reportedly pathogenic in a range
of economically important monocots, including
bananas, oil palms, sugar cane, corn, maize, taro
(Colocasia esculenta), turf grass, cocoa, and reed
plants8-14. Marasmius is not closely related to the
species Marasmiellus, as it is best to use its present
taxon – where it was originally described15, and
unl more data from mulple genes and a large
Marasmiaceae dataset provide the informaon to
clarify its taxonomic posion16.
Members of the genus Marasmius are
saprotrophic fungi, oen in forests, that play a
key role in the breakdown of wood and leaf lier,
nutrient cycling, and soil genesis; this is where
they serve an important ecological role in the
biodegradaon of lignocellulosic material17. Fiy
cultures, invesgated for enzyme acvies and
anmicrobial and anoxidant properes, indicate
that the Marasmius species may have potenal
applicaons in biotechnology17.
Based on our knowledge, there is no
scienc report available to characterize Imperata
cylindrical wilt disease, caused by the Marasmiys
genus. Therefore, the current study was carried
out to determine the molecular sequence of the
causave agent of this disease by sequencing the
ITS-5.8s-ITS2 region of rDNA and invesgang the
antagonisc potenal of this agent against fungal
pathogens; however, this is poorly documented,
and may be used to develop new biocontrol
agents.


Fusarium solani, F. thapsinum, Penicillium
sp., T. harzianum and Penicillium cyclopium are
procured from the Advanced Mycology Unit,
Biology Department, College of Science, University
of Babylon.

Disease samples from stems and rhizomes
of I. cylindrical are grown close to the reed plant
or in agricultural elds, and were collected from
20 agricultural elds in Babylon Province, Iraq,
in November, 2017. Samples were transferred to
the laboratory, with inoculaon and incubaon
conducted as described14. Identification of
fungal isolates were based on morphological and
microscopic features, such as characteriscs of
colony and mycelium clamp connecons, using
40X Microscopic Objecve Lens18,19.

Due to the similarity among all isolates of
Imperata Cylindrical Wilt Disease Fungus (IcWDF),
only one isolate was chosen for the following
experiments. The microbial cultures include IcWDF
isolate, Fusarium solani, F. thapsinum, Penicillium
sp., T. harzianum, and Penicillium cyclopium, which
were separately inoculated in petri dishes with
potato dextrose agar (PDA) (pH 7.0), followed
by incubaon for 5 days at 26 ± 2oC. To make the
slants, 20 ml of PDA was poured into glass tubes
and left until solidified. Fungal isolates were
maintained at 50C in a refrigerator, followed by
subculturing at regular intervals (every 30 days)20.

The inoculum of IcWDF was prepared
with 250 ml of millet seeds (Panicum miliaceum
L.) in conical asks, according to the procedure
described by Dewan and Sivasithamparam (1989)21
and others14,22.

Twenty uniform I. cylindrical plants at
similar age were selected from vegetable elds
and from the channels of irrigaon and planted
as described by Tamur et al.14. I. cylindrical plants
were grown for 2 months under glasshouse
condions (25±5 oC). The pots were then inoculated
individually with a causal agent, by mixing the
previously prepared inoculum in the pot soil at
a rate of 5% each. Ten pots without inoculaon
served as controls. The symptoms of disease were
reported at 15 days aer inoculaon14.

The fungal isolate from IcWDF was
recultured on PDA, as previously menoned, and
incubated at 28 ± 2oC for 3 days. The fungus cells
were collected and then transported to a new
sterilized tube for DNA extraction, which was

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Tamur et al. J Pure Appl Microbiol, 13(3), 1525-1536 | September 2019 | hps://doi.org/10.22207/JPAM.13.3.24
achieved with a special puricaon kit (Genomic
DNA purification kit, Zymogene, Orlando, FL,
USA), and used according to the manufacturer’s
protocol.

Detecon of ITS gene was conducted by
using primers for amplicaon. A fragment 565 bp
of ITS was amplied using a forward primer (ITS1
F: 5' - TCCGTAGGTGAACCTGCGG -3') and a reverse
primer (ITS4 R:5' TCCTCCGCTTATTGATATGC-3')
(Primer set supplied by IDT (Integrated DNA
Technologies, Coralville, IA, USA). The PCR
amplicaon was performed in a volume of 25µl,
containing 1.5µl DNA, 5 µl Taq PCR Pre Mix (Intron
Biotechnology, Seoul, S. Korea), 1 µl of each primer
(10 pmol), at which point dislled water was added
into a tube for a total volume of 25 µl. The thermal
cycling condions were as follows: denaturaon at
94°C for 3 min, followed by 35 cycles of 94°C for
45 s, 52°C for 1 min, and 72°C for 1 min, with nal
incubaon at 72°C for 7 min, using a thermal cycler
(Gene Amp, PCR system 9700; Applied Biosystem,
Waltham, MA, USA). The PCR products were
separated by 1.5% agarose gel electrophoresis and
visualized by exposure to ultraviolet light (302 nm)
aer red staining (Intron, Seoul, S. Korea).
Gene sequencing was done at the
Naonal Instrumentaon Center for Environmental
Management (nicem) online (hp://nicem.snu.
ac.kr/main/?en_skin=index.html), biotechnology
lab, with a DNA sequencer 3730XL, Applied
Biosystems, Waltham, MA, USA). A homology
search was conducted using a Basic Local Alignment
Search Tool (BLAST) program, available at the
National Center of Biotechnology Information
(NCBI) online at (hp:// www.ncbi.nlm.nih.gov)
and the BioEdit program. Aer morphological and
molecular idencaon, these experiments were
carried out:
Marasmius palmivorus Temperature
The growth of M. palmivorus was
measured at various temperatures (15, 20, 25,
and 30oC). 20 ml PDA were added to sterile petri
dishs, each plate, was inoculated by taking 0.5 cm
from the edge of colonies. Plates were separated
into 5 groups before incubation for 3 days (4
replicates each). Radial growth of this fungus (from
two intersecng lines in the center of the dish.)
was daily esmated on an Petri plate unl full
expansion of growth, with dierent temperature
treatments clearly determined.
pH
Dierent ranges of pH (4.5, 5.5, 6.5, 7.5,
and 8.5) were established for the growth of M.
palmivorus, using a sterilized petri-dishes (20 ml
of PDA each). The inoculation, the incubation
process, and the fungal growth measurement were
achieved as described in the previous paragraph
(for temperature).
  Marasmius palmivoru  
nutrient sources
Dierent nutrient sources were prepared
by collecng leaves of wheat, I. cylindrical, and
reed (youngest expanded). Leaf samples were
dried in the shade, as well as in an oven (60÷C) for
48 h, and grinded into ne powder by an electronic
blender14. All leaf powders were stored in sterilized
containers unl usage23.
Preparaon of media was achieved by
dissolving 2 g of each nutrient source, in 100
ml of dislled water with or without dextrose
using a magnec srrer. Supplementaon with
dextrose and agar growth medium were carried
out at the same rate as for PDA, then mixed gently
and autoclaved at 15 psi for 15 min, with PDA
as a control treatment. Autoclaved media were
poured into petri plates for each type, with the
plates inoculated aer solidicaon with fungal
ssue (0.5 cm) taken from the edge of an M.
palmivorus colony (4 days old). Three replicates of
each source were taken and incubated for 6 days
at 28 ± 20C24. Radial growth was regularly recorded
as menoned before.
  Marasmius palmivorus:

The antagonistic activity of M .
palmivorus isolate was screened for the growth
of pathogenic and nonpathogenic fungi, including
Fusarium solani, F. thapsinum, Penicillium sp., T.
harzianum, and Penicillium cyclopium by using the
dual culture technique25. Five mm discs of tested
fungus were placed individually in the center of
each PDA plate half, whereas the other half was
inoculated with IcWDF followed by incubaon
at 26 ± 2°C, in a controlled experiment, with
each fungus grown on PDA. Growth inhibitory
antagonisc acvity of the tested fungus against
phytopathogenic fungi was assessed in terms of

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percent inhibion in cm by using this formula26:
Inhibion % = (r1-r2/r1) ×100,
where, r1 is the diameter of
phytopathogenic fungal growth without IcWDF,
while r2 is represents the radial growth of the
pathogenic fungus with IcWDF. Furthermore, all
treatments were carried out in three replicates.

The current work were arranged in a
randomized complete block design. Data were
assessed by analysis of variance. The level of
significance was determined by Fisher’s least
signicant dierence (LSD) comparisons at the 5%
probability level.

Marasmius palmivorus

Infecon of I. cylindrical by IcWDF fungus
was appeared as wilt symptoms on stems and
rhizomes, which produced blight symptoms on the
leaves. Symptoms primarily iniated on the base
of the stem’s lower secon as brown to black rot
lesions. Mycelium which was grown rapidly show
u as white coony growth around the plant base
(Fig. 1A).
IcWDF isolate was tentavely idened
as M. palmivorus, according to colony features.
Microscopic observaon revealed that this fungus
produced wilt colonies on petri dishes and covered
the plate within 3 to 4 days at 28 ± 2oC, with
hairy-branched and hyaline mycelium (Fig. 1B).
Colonies were creamy aer 10 days of inoculaon.
Septaon is recognized in the fungal mycelium (Fig.
1C), with clamp connecons clearly disnguished
(Fig. 1D). Spores, and reproductive aspects
were not observed under the light microscope
examinaon, with a temperature range of 28 ± 2oC.
The macroscopic fruing body of M. palmivorus
was not detected under lab condions.

The signs and symptoms of M. palmivorus
were conrmed according to Koch’s postulates.
Dierent symptoms of I. cylindrical wilt disease
were recorded aer 2 weeks and found to be
similar to symptoms observed during natural
 Disease symptoms of wilt disease caused by M. palmivorus on I. cylindrical plants at 15 days aer inoculaon
(A), morphological feature of M. palmivorus growing on PDA at 28 ± 2oC aer 4 days of incubaon (B), septal wall
of fungal hyphae (C), and clamp connecons (D), under a light compound microscope, 25 µm (40X-1 at 10 days
aer incubaon)

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 Phylogenec tree, depicng the relaonship of M. palmivorus
infection. In controlled experimentation, no
disease symptoms appeared. Death of I. cylindrical
plants tended to occur aer 3 weeks, while the
infecon process increased in moist circumstances.

In this study, the conrmaon process
of one isolate, M. palmivorus, was conducted by
convenonal PCR technique to detect the presence
of a specic gene, with sequence analysis of the
ITS1-5.8s-ITS2 region, and BLAST analysis from
the NCBI database. Aer sequencing, this isolate
was idened. The extracted genomic DNA of
these isolates was used as a template spacer
(ITS). The PCR amplicaon products show that
M. palmivorus yielded about 565 pb. The product
is shown in Fig. 2.
PCR product, the band size of 565 bp. The product was electrophoresis on 1.5% agarose at 5 volt/cm2. Also,
1x TBE buer for 1.5 h, DNA ladder (100), lane (1 to 2) PCR product of band size 565 bp, visualized under UV light
Phylogenec analysis by rDNA sequencing
found a genec variaon among isolates from M.
palmivorus in other countries. BLAST results were
explored by searching with partial nucleotide
sequences at the gene bank database (Table 1).
A phylogenec isolate tree in Iraq showed
a close relaonship with M. palmivorus in India,
idened at 99% (4 KC771224.1), in the Philippines
at 98% (4 KR05 6289.1), and in Kenya at 98% (9
KT27 3356.1). M. palmivorus in Malaysia was
idened at 98% (7 JQ65 3440.1), with a distant
relaonship to M. palmivorus (MG717877) in Iraq
(Fig. 3 and Table 1).
 Marasmius palmivorus
Temperature
The results of this study revealed a
substanal eect of temperature (Fig. 4) for the
growth of M. palmivorus. The opmum growth
occurred at 30°C after a 3-day incubation,
followed by 25°C, then 20°C, while the lowest
growth occurred at 15oC. The rising or lessening
temperatures below 15°C, or higher than 30°C,
caused obstrucon of M. palmivorus growth.

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 Eect of dierent temperatures: 15oC (A), 20oC (B), 25oC (C), and 30oC (D) in the growth diameter of M.
palmivorus on PDA aer 3 days of incubaon (LSD(0.05)=0.263)
pH
As a constant, pH (4.5, 5.5, 6.5, 7.5, and
8.5) has a signicant eect on the growth of M.
palmivorus with PDA, at temperatures 28 ± 2˚C.
Maximum growth of M. palmivorus was obtained
at pH 7.5 (8 cm), followed by pH 6.5 (6.3 cm), with
the radial growth of this fungus increasing at pH
5.5 (6 cm), which was less than pH 7.5 and 6.5, and
higher than pH 8.5 (5 cm). Growth of this fungus
decreased to 4.5 cm at pH 4.5 at the end of the
incubaon (Figs. 5A and 5B).
  Marasmius palmivorus  
nutrient sources
Growth of M. palmivorus varied
considerably, according to the composition of
growth media, which was most profound with
wheat and reed leaves, supplemented with
powdered material in the medium (Fig. 6).
Therefore, the growth diameter of M. palmivorus
was dramacally aected by the source of the
added nutrient. Aer 4 days of incubaon, the
growth of the tested fungus signicantly diered in
Eect of dierent pH levels (4.5, 5.5, 6.5, 7.5, and 8.5 on colony growth of M. palmivorus aer 4 days of
incubaon)
Homology sequence idened for local Marasmius palmivorus isolate
Accession Gene country Source Compability
ID: MG251431.1 18S ribosomal RNA India Marasmius palmivorus 99%
ID: MF100969.1 18S ribosomal RNA USA Marasmius palmivorus 98%
ID: KR056289.1 18S ribosomal RNA Philippines Marasmius palmivorus 98%
ID: KJ865843.1 18S ribosomal RNA India Marasmius palmivorus 98%
ID: JQ653445.1 18S ribosomal RNA Malaysia Marasmius palmivorus 98%
ID: JQ653435.1 18S ribosomal RNA Malaysia Marasmius palmivorus 98%
ID: MF100965.1 18S ribosomal RNA USA\ California Marasmius palmivorus 98%
ID: KT273356.1 18S ribosomal RNA Kenya Marasmius palmivorus 98%
ID: KR056290.1 18S ribosomal RNA Philippines Marasmius palmivorus 98%
ID: JQ653446.1 18S ribosomal RNA Malaysia Marasmius palmivorus 98%

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Eect of dierent pH levels (4.5, 5.5, 6.5, 7.5, and 8.5) on growth diameter of M. palmivorus aer 4 days
of incubaon (LSD(0.05)=0.377)
  Colony growth of M. palmivorus on culture
media from powdered leaves of the following plants
with dextrose (1) and without dextrose (2): A (wheat),
B (reed), C (I. cylindrica), and D (PDA) aer 4 days of
incubaon at 30oC
wheat (6.8), reed (9), caladium (8.5) supplemented
with dextrose, compared to PDA. In contrast, the
average growth reached 5.5, 8.3, and 7.1 in wheat,
reed, and caladium, respecvely, which were not
supplemented with dextrose (compared to PDA).
Thus, proporonal fungal growth was increased in
all formulated media with and without dextrose,
except in the wheat treatment, which was not
supplemented with dextrose (Fig. 7).

Substantial antagonistic effect of M.
palmivorus was appeared against F. solani and
F. thapsinum, in the range of 70-100% at 6 to 8
days aer incubaon, respecvely. The growth
inhibitory percentages of RBDF isolate were 33%
(Penicillium sp.), 14-62% (T. harzianum), and 20-
Growth diameter of M. palmivorus on culture media of powdered leaves of the following plants with dextrose
(1) and without dextrose (2): A (Wheat), B (Reed), C (I. cylindrica), and D (PDA) aer 4 days of incubaon at 30oC.
(LSD(0.05)=0.631)

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50% (P. cyclopium) within the 6th – 8th days aer
incubaon, respecvely. The most remarkable
antagonism was observed with T. harzianum, in
which Marasmiellus sp. started to grow over the
already-grown ssues of T. harzianum. Another
excing results about antagonism were occurred
between Penicillium sp. and M. palmivorus, where
in spite of the fast growth of the Penicillium sp.
covering nearly 70% of the petri area, the fungal
mycelia of M. palmivorus rapidly approached
Penicillium sp.; this then directly inhibited its
growth upon contact (Figs. 8 & 9).
Antagonisc eciency of M. palmivorus towaed: F. solani (A1), F. solani + M. palmivorus- front face (A2), F.
solani + M. palmivorus- reverse face (A3), F. thapsinum- alone (B1), F. thapsinum + M. palmivorus- upper face (B2),
F. thapsinum + M. palmivorus- lower face (B3), Penicillium sp. alone (C1), Penicillium sp. + M. palmivorus- front face
(C2), Penicillium sp + M. palmivorus - reverse face (C3), T. harzianum (D1), T. harzianum. + M. palmivorus-4 d.a.i.
(D2), T. harzianum + M. palmivorus- 8 d.a.i. (D3), Penicillium cyclopium (E1), Penicillium cyclopium. + M. palmivorus-
fron face (E2), Penicillium cyclopium + M. palmivorus- reverse face (E3), Control, M. palmivorus (F) at 28 ± 2oC

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
Imperata cylindrical plants are known
to invade agriculture elds and irrigaon channel
systems in the majority of agriculture lands in
Iraq. In the present study, the observaons of
disease symptoms and the distribuon of disease
demonstrated that the rate of wilt disease on I.
cylindrical by M. palmivorus varied according to
agricultural region, weed intensity, and type of soil.
Based on colony characteristics, like
the appearance of clamp connecons in fungal
tissues (mycelium), this fungus was identified
as Marasmius palmivorus. This nding is closely
harmonious with the observaons presented by
Singer (1973)8.
The generic situaon of M. palmivorus
is quesonable, since it’s morphology is quite
similar to that in Marasmiellus than to Marasmius
sensu stricto19. The original was published as M.
palmivorus by Corner (1996)27. In 2005, Wilson and
Desjardin7 formally proposed including this taxon
as Marasmiellus palmivorus, but the proposal
was not accepted, and there is no taxon available
as Marasmiellus palmivorus. Some workers read
the formal proposal of Wilson and Desjardin, but
wrongly named their collected taxon Marasmiellus
palmivorus; they published some of this work as
well16.

The transformave history was deduced
by ulizing the Neighbor-Joining method (NJ)28.
The ideal tree with the enrety branch length
= 0.01559401 is appeared. The tree is drawn to
scale, with branch lengths in the similar units
as the evoluonary distances used to conclude
the phylogenec tree. These separaons were
processed with the most extreme Composite
Likelihood Method29, and are in units of the
number of base substitutions per site. The
invesgaon included 21 nucleode sequences.
Codon positions included were
1st+2nd+3rd+Noncoding. All posions containing
gaps and missing data were eliminated. There
were a enre of 325 locaons in the nal dataset.
Evoluonary analyses were conducted in MEGA730.
The mulple sequence alignment analysis
of paral 18S ribosomal RNA gene sequence for
local M. palmivorus isolate and the NCBI gene
bank, using Mega 7, was a mulple alignment
analysis tool. Sequence comparisons of the ITS
region are broadly utilized in taxonomy and
molecular phylogeny, as it is direct to enhance
from lile amounts of DNA (due to the high copy
number of rRNA genes), including that there is
a high level of variaons between rmly related
species
It has been demonstrated that paral
18S ITS1 5.8S ITS2, and paral 28S ribosomal RNA
gene sequence data on an individual strain with
the nearest neighbor, and exhibing a similarity
score of < 97%, represents a new species; however,
the meaning of similarity scores > 97% is not
as clear. The rst step of alignment analysis for
all sequences in this study with other selected
references used the (Clustal W) program step in
MEGA 6.0. This program demonstrated an accurate
degree of identity with all world sequences,
including those in this study. The results with
Inhibion percentage in A (F. solani), B (F. thapsinum), C (Penicillium sp.), D (T. harzianum) and E (Penicillium
cyclopium) grown in PDA aer 6 and 8 days of incubaon during the antagonism with Marasmius palmivorus

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(Clustal W) are important, because they were
directly used in the phylogenec tree construcon,
and agreed with previous studies using specic
primers, The rmly related taxa possess similar
fragment distribuons, while the remotely related
taxa are increasingly dissimilar, thus providing
impressive phylogenec data 31.
The mulple alignment analysis showed
similarity and dierences in 18S ribosomal RNA
gene nucleotide sequences. This corresponds
to the study by Tom ovsk et al.32, in which it
was revealed that all Trametes species form a
single clade. The ITS region showed the highest
fungi ecology33 and was recommended to be the
universal fungal barcode sequence34.
A nucleode was used with a maximum
likelihood of detecng the relaonship of world
and local sequences, which was the preferred
method of nucleode sequences in MEGA 6.0: it
uses the NJ method, a simplied version of the
Minimum Evoluon (ME) method. The NJ method
produces an unrooted tree, as it does not require
a constant rate of evolution. Finding the root
requires an out-group taxon28. Further studies
with dierent gene sequences will resolve this in
the phylogenec analysis of fungi35.
Dua and Acharya (2018) reported that
nrITS and nrLSU sequences of M. palmivorus
were 760 and 593 bp, respecvely, which were
deposited in the GenBank database with accession
numbers MG251431 (for nrITS) and MG251441
(for nrLSU). Based on BLAST searches in the NCBI
database, they had the most similarity to the taxon
from Malaysia [GenBank JQ653433; identities
= 717/726(99%), gaps = 3/726(0%)], Hawaii
[GenBank AY639434; idenes = 592/593(99%),
and gaps = 0/593(0%)]16.
Marasmius palmivorus
Culture media is viewed as one of the
fundamental necessies for the development and
improvement of fungi. M. palmivorus was found
to grow a lot quicker in supplemented growth
medium of powdered leaves, comprising of wheat,
reed, and caladium, compared with PDA; this
suggests that there are preferable growth sources
with the possibility to substute the ulizaon of
PDA for culturing M. palmivorus.
The alteraon in growth paerns was
studied by Thiruchchelvan et al.36
, which observed
that at 4 days’ incubaon of fungus on PDA, King
yam and Elephant foot yam media were more
preferable for fungal growth than that in lter
paper, sago nutrient agar, and water agar. Another
study acheived by Ravimannan et al.37 showed
the ulity of legume seeds (cowpea, green gram,
black gram, and soya meat) for the growth of
Trichoderma, Scleroum, Fusarium, Aspergillus,
and Penicillium sp.
The antagonisc eect of Basidiomycetes
mushroom on the growth of plant pathogenic
fungi can be used as a promising biocontrol
system. The results of this study show the
broad spectrum of antagonistic activity of M.
palmivorus to pathogenic fungi, such as F. solany,
F. thapsinum, P. cyclopium, and T. harzianum. M.
palmivorus surprisingly colonizes the anbioc
eect of T. harzianum, which indicates overgrowth
of M. palmivorus or production of antifungal
substance(s) against the tested fungi.
However, the growth of M. palmivorus
was found to be suppressed by Penicillium sp., as
they reached the vicinity of the other. This might
be due to the producon of toxic substance(s)
by Penicillium sp., which undermine the growth
of M palmivorus. In the present scenario, one
interesng aspect to be invesgated is the possible
mechanisc insight behind the biocontrol acvies
of such a fungus. One potenal mechanism of
antagonism behavior of M. palmivorus can be
associated with nutrients and niche compeon.
In addion, the anbiosis eect of M. palmivorus
against soil-borne fungi, as well as weed plants,
may also be associated with the producon of
volale and nonvolale compounds. Previously,
it was documented that the antagonisc eect
of a living cell might be due to the release of
extracellular bioacve molecules.
The present results disagree with
those of Thiruchchelvan et al.36, in which they
report that Trichoderma sp. causes growth
inhibion of Marasmiellus sp. up to 92% under
in vitro conditions. The antifungal activity of
T. harzianum to fungal pathogens have been
reported: Aspergillus niger, A. avus, Phytophthora
sp., Fusarium oxysporum, Rhizoctonia solani,
Penicillium notatum, and Alternaria solani38.

To the best of our informaon, this is
the rst record of the presence of M. palmivorus

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Journal of Pure and Applied Microbiology
in Iraq, and likely around the world for weeds.
Using M. palmivorus as a biocontrol agent against
I. cylindrical plants is a new approach. The earliest
experimental studies also show the selective
acon of M. palmivorus against weeds, without
causing any deterioraons to neighboring plant
seedlings. However, further studies are required
to explore its selecve herbicidal acvity in the
near future.

Authors would like to thank the Ministry of Science
and Technology - Babylon Secon - for providing
formal facilies to Mr. Tamur, and the Department
of Biology, University of Babylon, for technical
assistance. Authors are also thankful to Prof. Ali
H. K. El-Bahadili for scienc advice.
CONFLICT OF INTEREST
The authors declares that there is no
conict of interest.

All authors listed have made a substanal,
direct and intellectual contribuon to the work,
and approved it for publicaon.

This work was nancially supported by
the Department of Biology, College of Science,
University of Babylon, Iraq.

All datasets generated or analyzed during
this study are included in the manuscript.
ETHICS STATEMENT
This arcle does not contain any studies
with human parcipants or animals performed by
any of the authors.
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