Available via license: CC BY
Content may be subject to copyright.
R E S E A R C H Open Access
Potential of phytopathogenic fungal
isolates as a biocontrol agent against some
weeds
Youssef M. M. Mohammed
1*
and Mohamed E. I. Badawy
2
Abstract
Bioherbicides afford satisfactory approach to overcome weed problem. Selection of new bioherbicides from
phytopathogenic microbiota is essential for modern agricultural management, especially mycoherbicides. Thus, in
the present investigation, 4 phytopathogenic fungi including Alternaria alternata YMM1, A.tenuissima YMM3,
Chaetomium globosum YMM2, and Nigrospora oryzae YMM4 were isolated from infected leaves with chlorotic rings
of Rumex dentatus and Sonchus oleraceus as common weeds in Egypt. These fungi were identified based on
morphology and sequence analysis of ITS1-5.8SrDNA-ITS2 of rRNA genes. Mycoherbicidal metabolites were
produced using submerged cultures in potato dextrose broth. Their bioherbicidal activities were evaluated using
seed germination and leaf disk puncture assays. It was found that the most promising fungal strain against major
tested weeds was N.oryzae YMM4. Thus, phytopathogenic microbiota are considered a great resource for the
construction of a new bioherbicide for managing weeds.
Keywords: Phytopathogenic fungi, Molecular identification, Herbicidal action, Weed control
Background
Weeds are the main problem in intensive agriculture
and compete for the same nutrients of crop plants
(Sahin, 2019). Therefore, the quality of crops and yields
has decreased significantly and caused enormous eco-
nomic losses (Bajwa et al. 2018). Weed control is vital to
increase crops’yields. Management approaches mainly
rely on synthetic herbicide systems for increasing crop
productivity (Cordeau et al. 2016). However, synthetic
chemicals are inadequate due to their resistance to deg-
radation, environmental risks, and the development of
resistant varieties of weeds (Schütte et al. 2017). Resist-
ant individuals among weed populations are developed
through the intensive uses of the same synthetic
herbicides for long times (Matthews 2018). Hence, these
synthetic herbicides need to be replaced with environ-
mentally friendly biological herbicides (bioherbicides).
Bioherbicides are ecofriendly materials and usually
have shorter half-life than synthetic herbicides (Javed
et al. 2016). Fungi are well investigated to bioherbicidal
production for weedy management (Guo et al. 2020).
Phytopathogenic molds generate metabolites that play a
great role in the improvement of weed control. Produc-
tion of bioherbicides from plant pathogens has been
confirmed to be an effective approach for weed control
(Júnior et al. 2019). Broth cultures of several fungi
showed considerable herbicidal activity in both crude
and purified forms against various weeds (Sica et al.
2016). Bioherbicidal production for management of
weeds involves a sequence of steps, including isolation
and selection of phytopathogenic fungi from infected
weeds and broth medium were used for bioherbicides
production (de Souza et al. 2015).
The phytopathogenic fungal biodiversity in diseased
weeds of cultivated areas in Egyptian agriculture has
© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
* Correspondence: youssefmoustafa@ymail.com
1
Department of Botany and Microbiology, Faculty of Science, Damanhour
University, Damanhour 22516, Egypt
Full list of author information is available at the end of the article
Egyptian Journal o
f
Biolo
g
ical Pest Contro
l
Mohammed and Badawy Egyptian Journal of Biological Pest Control (2020) 30:92
https://doi.org/10.1186/s41938-020-00295-0
been seldom studied, especially for production of bio-
active metabolites. Therefore, the main objective of the
present study was to isolate phytopathogenic fungi from
infected leaves with chlorotic rings of Rumex dentatus
and Sonchus oleraceus as common weeds in Egypt. In
addition, in vitro cultivation of the isolated phytopatho-
genic fungi in the laboratory was done to obtain filtrates
containing biologically active metabolites with herbicidal
action and evaluate their herbicidal effects against some
weeds.
Materials and methods
Culture media
Acetonitrile, methanol, and water of HPLC grade were
purchased from Sigma-Aldrich Co. (Spruce Street, St.
Louis, MO, USA). Glucose, magnesium sulphate, yeast
extract, peptone, beef extract, dimethyl sulfoxide
(DMSO), and agar were supplied from El-Gomhoria for
pharmaceutical and chemicals Co., Alexandria, Egypt.
Potato dextrose agar (PDA) was purchased from Oxoid
(Basingstoke, Hampshire, RG24 8PW, UK). Other che-
micals and solvents were purchased from El-Nasr
Pharmaceutical Chemicals Co., Qalyub, Egypt, and used
without further purification. All media were prepared
immediately and autoclaved at 121 °C for 20 min.
Sampling area and collection of weed specimens
Plant weeds (Rumex dentatus and Sonchus oleraceus)
with visible diseased symptoms of samples were col-
lected from wheat field and placed in sterilized glass bot-
tles. The symptoms of all diseased weeds were
characterized by necrotic (an irregular brownish) spots
with chlorotic halo around them. The seeds and leaves
used in the present study were collected from weeds
growing in fields of cultivated crops. Sampling locality
was a cultivated area at Jarrar, Abu Hommos, Beheira
Governorate, Egypt. The weed species were identified by
the help of available literature (Täckholm 1974; Boulos
1999,2002,2005 and 2009).
Isolation and selection of phytopathogenic fungi
Phytopathogenic fungi were isolated from the infected
leaves of R.dentatus and S.oleraceus weeds. The leaves
were surface sterilized by 95% ethyl alcohol for 1 min,
then dipped in 10% sodium hypochlorite for one minute,
followed by washing in sterile water. The infected spots
of leaves were directly transferred to a Petri plate con-
taining PDA medium. Inoculated plates were incubated
at 28 °C and observed after 7 days (Babu et al. 2003 and
Aybeke 2017). Fungal colonies were selected for purifica-
tion by repeated streaking on agar plates of the PDA.
The pure colonies obtained were transferred to a fresh
PDA slant, subcultured, and stored at 4 °C.
Identification of phytopathogenic fungi
Phytopathogenic fungi were identified morphologically
on the basis of macroscopic (naked eye) and microscopic
characteristics (Moubasher 1993) after culturing on the
PDA medium at 28 °C for 7 days. For molecular charac-
terizations of fungal isolates, total genomic DNA prepa-
rations of fungal cultures in potato dextrose broth (PDB)
were made according to the protocol of Quick-DNA™
Fungal Microprep Kit (Zymo research #D6007). The ex-
tracted DNA preparations were examined using 1%
agarose gel electrophoresis. The internal transcribed spa-
cer (ITS) region (ITS1-5.8SrDNA-ITS2) of rRNA genes
was amplified, using the primer pairs of ITS1 (5′-TCC
GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC
TCC GCT TAT TGA TAT GC-3′) (White et al. 1990).
The 5.8S rRNA gene and the two flanking internal tran-
scribed spacer regions (ITS1-5.8S rDNA-ITS2) of rRNA
genes were PCR amplified according to the protocol of
Maxima Hot Start PCR Master Mix (Thermo K1051).
The amplifications were performed in a ThermoHybaid
PCR Sprint Thermal Cycler (Thermo Electron, USA).
PCR products were purified according to the protocol of
GeneJET™PCR Purification Kit (Thermo K0701). Ampli-
cons, along with the marker DNA (DNA Ladder), were
visualized by 1 % agarose gel electrophoresis after stain-
ing with ethidium bromide to confirm the size and pur-
ity of the amplified PCR products. The amplified PCR
products of ITS1-5.8SrDNA-ITS2 were sequenced on
both strands using ITS1 and ITS4 primers at GATC
Company by use ABI 3730xl DNA sequencer. The
nucleotide sequences determined were deposited in
GenBank. For the identification of the isolates, the nu-
cleotide sequences obtained were compared with those
sequences already deposited in the data bank of the Na-
tional Centre for Biotechnology and Information (NCBI)
using the nucleotide basic local alignment search tool
(BLASTn) to find the most closely related sequences.
The identification of the species was determined based
on the best sequence alignment score. The obtained
DNA fragments were also subjected to a phylogenetic
study by means of comparative sequence analysis of the
ITS regions including the 5.8S rDNA sequences. This
was achieved by generating a neighbor-joining distance-
based tree using the software MEGA 6.0.
Preparation of fungal inocula
For inoculum preparations, PDA Petri plates were
inoculated by phytopathogenic fungi from slants and
incubated at 28 °C for 7 days. Then, the culture surfaces
on the agar plates were scraped with 10 ml of sterilized
water, using isolation needle. The spores were obtained
and the suspensions were counted using a
hemocytometer to obtain the desired concentrations of
2.5 × 10
5
conidia/ml and used as inoculum sources.
Mohammed and Badawy Egyptian Journal of Biological Pest Control (2020) 30:92 Page 2 of 9
Preparation of fungal bioactive metabolites with
herbicidal action
For the production of fungal filtrates with herbicidal ac-
tivity, the fungal culture broths were prepared using
250-ml Erlenmeyer conical flasks containing 100 ml of
PDB medium inoculated by 2 ml of spore suspension
(2.5 × 10
5
conidia/ml) at pH 6 and in static conditions at
28 °C (Babu et al. 2003 and Guo et al. 2020). After 7 days
of incubation, fungal mycelia were separated by filtration
on a pre-weighed Whatman filter paper (No. 4), washing
twice with distilled water and dried to a constant weight
at 60 °C and reweighed. Fungal dry biomass was mea-
sured gravimetrically as the difference in weight. The
culture filtrate for each fungus (≈500 ml) was centri-
fuged at 10,000 rpm for 10 min and the supernatant was
lyophilized.
Bioherbicidal activity of crude fungal filtrates
The lyophilized product (0.1 g) that was obtained from
the fungal filtrate was dissolved in 50 ml distilled water
and sterilized, using 0.45 μm Minisart membrane filter.
In vitro bioherbicidal activity
The in vitro bioherbicidal activities of the filtrates of
phytopathogenic fungi were assayed by seed germination
bioassay and seedling morphology. All seeds were
treated by 0.5% sodium hypochlorite for 10 min and
washed numerous times with sterile distilled water be-
fore germination assay immediately.
Evaluation of bioherbicidal potential of crude filtrate,
using an in vitro seed germination bioassay was carried
out on filter paper (Whatman No. 4) in Petri dishes (10
cm in diameter) against R.dentatus L., S.oleraceus L.,
Avena fatua L., Polypogon monspeliensis L., Setaria viri-
dis L., Echinochloa crus-galli L. Beauv, E.colona L., and
Plantago major L. seeds. Crude filtrate of each phyto-
pathogenic fungus (2 ml daily) was poured on the filter
paper in Petri plate containing 100 seeds and incubated
for 10 days under alternation between light (12 h using
2000 Lux) and dark (12 h) at 25 °C. Germination (%),
plumule heights, and radical lengths of seedlings were
examined. Control treatments were carried out, using
sterile PDB medium instead of crude filtrates. Each trial
was examined with three replications.
In vivo bioherbicidal activity
The in vivo bioherbicidal activities of crude filtrates were
tested by leaf disk puncture assay against leaves of the
abovementioned weeds. Crude filtrate of each phyto-
pathogenic fungus (20 μL) was applied on leaf disks of
the selected plants. The disks (13-mm diameter) were
cut from weed leaves, then located on wetted filter paper
(Whatman No. 4) within Petri plates, and punctured by
sterile needle in the center before addition of fungal
filtrates. Leaf disks were kept under constant light condi-
tions and 25 °C. After 5 days of incubation, symptoms
were examined visually. The degree of phytotoxicity (%)
was scored according to severity of the symptoms on leaf
disks. Control treatments were carried out, using sterile
PDB medium instead of crude filtrates (Guo et al. 2020).
Each trial was examined with three replications.
Isolation of fungal extracts and GC/MS analysis
For preparation of fungal extracts, culture filtrates of
phytopathogenic fungi (500 ml) were lyophilized. A
weight of 0.1 g was dissolved in 50 ml distilled water and
stored at 4 °C in the dark. The compositions of the ex-
tracts were analyzed (Ekman & Holmbom 1989) by gas
chromatography/mass spectrometry (GC/MS) with the
following specifications: A Trace GC Ultra/Mass Spec-
trophotometer ISQ (Thermo Scientific) instrument was
equipped with flame ionization detector (FID) and a DB-
5 narrow bore column. Helium (average velocity 39 cm
s
−1
) was used as the carrier gas (flow rate of 1 ml/min),
and the temperature program was 120 °C/min, raised at
6 °C/min to 320 °C, injector temperature was 260 °C, and
detector temperature was 320 °C with post run (off) at
320 °C. The GC/MS was equipped with a ZB-5MS
Zebron capillary column (length 30 m × 0.25 mm ID,
0.25-μm film thickness; Agilent). A sample (1 μl) was
injected at 250 °C, with split/split-less injector (50:1 split
ratio) in the split-less mode flow with 10 ml/min. All
mass spectra were recorded in the electron impact
ionization (EI) at 70 electron volts. The mass spectrom-
eter was scanned from 50 to 500 m/z at five scans per
second. Scan time is 1.5 s and mass range is 40 to 300
amu. Peak area percent was used for obtaining quantita-
tive data with the Xcalibur software (Thermo Scientific)
without the use of response factor correction. Constitu-
ents were identified by comparing their mass spectra
with MS library (NIST, Mainlib, Reblib and Wiley) data
(Adams 1995). Quantification of constituents was ob-
tained by integrating the peak area of the
chromatogram.
Statistical analysis
Experimental data are presented as mean ± standard
error and the analysis of variance (ANOVA) of data was
conducted and mean property values were compared (p
≤0.05) to Fisher’s least significant difference (LSD)
method by Minitab 16.1.0 program (Minitab Inc., PA,
USA).
Results and discussion
Fungal isolation and characterizations
Four phytopathogenic fungal strains were isolated from
infected lesions of weed leaves. YMM1 and YMM2 were
isolated from R.dentatus and YMM3 and YMM4 from
Mohammed and Badawy Egyptian Journal of Biological Pest Control (2020) 30:92 Page 3 of 9
S.oleraceus. These fungal isolates were identified based
on morphology and phylogenetic analysis. According to
morphological features, the isolates belonged to 3
different genera, Alternaria (YMM1 and YMM3), Chae-
tomium (YMM2), and Nigrospora (YMM4).
Purified genomic DNA was prepared for PCR to amp-
lify the ITS regions, using ITS1 and ITS4 primers, which
resulted in fragments, ranging in length between 531
and 610 bp (Fig. 1). The same primers were then separ-
ately used for nucleotide sequencing of the purified
DNA fragments. After editing, the obtained sequences
were submitted to the GenBank and the received acces-
sion numbers were shown in Table 1. ITS regions of
rDNA are widely used to study the variability in fungi at
the species levels (Bonito et al. 2010 and Mohankumar
et al. 2010).
The database matching results of the sequences ob-
tained in the present study confirmed that the 4 exam-
ined isolates belonged to 3 different genera (Table 1).
The observed high identity results (99 %) to database se-
quences allowed the identification of the isolates at the
species level. These included 2 Alternaria spp. (A.alter-
nata YMM1 and A.tenuissima YMM3), one
Chaetomium sp. (C.globosum YMM2), and one Nigros-
pora sp. (N.oryzae YMM4).
In a similar study, A.alternata was isolated from dis-
eased leaves of Lantana camara (Saxena & Pandey
2002). In addition, a phytopathogenic fungus Septoria
polygonorum was isolated from sporulating lesions on
Polygonum pensylvanicum weed (Mitchell 2003). Five
isolates of Phoma spp. were isolated from Setaria viridis
(Zhang et al. 2003). Fusarium oxysporum was isolated
from Striga hermonthica stems (Marley & Shebayan
2005). Colletotrichum graminicola and Gloeocercospora
sorghi were collected from sporulating lesions on
Johnson grass and were used for the control of shatter-
cane weed (Mitchell et al. 2008). Phyllosticta cirsii,a
phytopathogen was isolated from diseased leaves of Cir-
sium arvense (Tuzi et al. 2010).
Based on the ITS of rDNA sequences obtained from
the experimental isolates and their close relatives, a
phylogenetic neighbor-joining tree that reflected evolu-
tionary relationships was constructed for each of the
three genera using MEGA 6.0 (Fig. 2).
Effect of fungal filtrates on germination and growth
parameters of weeds
In the present study, weed species competing with 2
major crops (wheat and rice) were selected. The ability
of fungal cultures filtrates to inhibit seed germination
and seedling growth of different weeds was examined
and compared by controls. Data in Table 2demon-
strated that all crude filtrates from the 4 phytopatho-
genic fungal strains significantly reduced radical lengths
and plumule heights of R.dentatus,A.fatua,P.monspe-
liensis,S.viridis,S.oleraceus, and P.major than the con-
trols. In addition, crude filtrate of N.oryzae YMM4
showed the strongest reduction effect against seedling
elevation and seed germination (%) of major tested
plants. Furthermore, seedlings of E.colona and E.crus-
galli were sensitive and significantly inhibited by crude
filtrate of N.oryzae YMM4. In addition, culture filtrate
of N.oryzae YMM4 was the most active agent against
weeds seeds germination. No seed germination occurred
in case of E.crus-galli treated by crude filtrate of N.ory-
zae YMM4. However, culture filtrates of A.tenuissima
Fig. 1 Agarose gel electrophoresis of the ITS regions amplified using
ITS1 and ITS4 PCR primers. The gel shows (respectively from left to
right) GeneRuler 100 bp DNA Ladder and the 530 to 600 bp
fragments of the fungal strains YMM1, YMM3, YMM2, and YMM4
amplification products
Table 1 ITS-based identification of fungal isolates and their accession numbers
Experimental genotypes Reference genotypes Identity
Isolates Accession number Strains Accession number
YMM1 MG711600 Alternaria alternata isolate ZB11060991 KX783398.1 99%
YMM3 MG711602 Alternaria tenuissima strain WGS11789 JX406513.1 99%
YMM2 MG711603 Chaetomium globosum strain CCTCC AF 206003 DQ854987.1 99%
YMM4 MG711604 Nigrospora oryzae strain UC3 KU554580.1 99%
Mohammed and Badawy Egyptian Journal of Biological Pest Control (2020) 30:92 Page 4 of 9
Fig. 2 Neighbor-joining phylogenetic trees constructed based on the alignment of the ITS sequences of phytopathogenic fungal genotypes
using MEGA 6 software
Mohammed and Badawy Egyptian Journal of Biological Pest Control (2020) 30:92 Page 5 of 9
Table 2 Phytotoxicity and effects of culture filtrates of Alternaria alternata YMM1, Chaetomium globosum YMM2, Alternaria tenuissima
YMM3, and Nigrospora oryzae YMM4 on weeds
Weed Fungal filtrate Radical length (cm) ± SE Plumule height (cm) ± SE Germination (%) ± SE Phytotoxicity (%) ± SE
Rumex dentatus Control 1.30 ± 0.03
a
2.50 ± 0.06
a
100.0 ± 1.10
a
0.0 ± 0.0
d
YMM1 0.90 ± 0.02
b
1.60 ± 0.03
b
90.00 ± 1.15
b
80 ± 1.63
a
YMM2 0.60 ± 0.02
cd
1.20 ± 0.05
c
90.00 ± 1.73
b
79 ± 1.41
a
YMM3 0.70 ± 0.05
c
1.30 ± 0.03
c
99.63 ± 0.32
a
38.7 ± 1.89
b
YMM4 0.50 ± 0.03
d
0.60 ± 0.04
d
06.00 ± 0.29
c
19 ± 1.41
c
Avena fatua Control 9.00 ± 0.12
a
15.00 ± 0.58
a
100.0 ± 1.20
a
0.0 ± 0.0
c
YMM1 1.50 ± 0.06
c
4.00 ± 0.12
c
95.00 ± 0.58
b
2 ± 0.41
a
YMM2 2.00 ± 0.06
b
7.00 ± 0.52
b
99.33 ± 0.33
a
0.97 ± 0.05
b
YMM3 1.50 ± 0.05
c
7.00 ± 0.46
b
95.17 ± 0.44
b
0.93 ± 0.09
b
YMM4 2.03 ± 0.03
b
3.00 ± 0.06
c
70.00 ± 0.58
c
0.97 ± 0.05
b
Polypogon monspeliensis Control 0.20 ± 0.01
a
2.60 ± 0.06
a
100.0 ± 2.10
a
0.0 ± 0.0
d
YMM1 0.20 ± 0.01
a
1.80 ± 0.12
b
90.00 ± 1.15
c
99.3 ± 0.94
a
YMM2 0.10 ± 0.02
b
1.50 ± 0.09
c
90.00 ± 1.73
c
99.3 ± 0.94
a
YMM3 0.10 ± 0.01
b
2.00 ± 0.12
b
95.00 ± 0.58
b
89.7 ± 0.47
b
YMM4 0.10 ± 0.01
b
1.80 ± 0.05
b
95.00 ± 0.29
b
18.7 ± 1.89
c
Setaria viridis Control 2.00 ± 0.12
a
7.00 ± 0.23
a
99.33 ± 0.33
a
0.0 ± 0.0
c
YMM1 0.80 ± 0.06
b
3.40 ± 0.06
c
60.00 ± 1.73
c
19.3 ± 0.94
a
YMM2 0.60 ± 0.03
bc
4.50 ± 0.06
b
99.00 ± 0.58
a
4.87 ± 0.19
b
YMM3 0.50 ± 0.02
c
3.40 ± 0.05
c
95.00 ± 1.73
b
4.8 ± 0.28
b
YMM4 0.80 ± 0.05
b
2.60 ± 0.12
d
20.00 ± 0.58
d
4.93 ± 0.09
b
Sonchus oleraceus Control 2.00 ± 0.06
a
3.00 ± 0.12
a
99.67 ± 0.33
a
0.0 ± 0.0
d
YMM1 1.40 ± 0.06
b
2.50 ± 0.05
b
99.27 ± 0.63
a
29.3 ± 0.94
a
YMM2 1.00 ± 0.03
c
2.00 ± 0.05
c
90.00 ± 1.73
b
9.4 ± 0.85
b
YMM3 0.80 ± 0.01
d
1.00 ± 0.02
d
08.00 ± 0.29
c
3.77 ± 0.33
c
YMM4 0.50 ± 0.03
e
0.90 ± 0.01
d
10.33 ± 0.88
c
4.73 ± 0.38
c
Plantago major Control 1.10 ± 0.06
a
1.50 ± 0.06
a
99.00 ± 0.58
a
0.0 ± 0.0
c
YMM1 0.40 ± 0.03
b
0.70 ± 0.06
c
90.00 ± 1.73
c
89.3 ± 0.94
a
YMM2 0.50 ± 0.01
b
1.30 ± 0.05
b
95.00 ± 1.73
b
89 ± 1.41
a
YMM3 0.40 ± 0.01
b
0.80 ± 0.05
c
98.67 ± 0.88
ab
29 ± 1.41
b
YMM4 0.50 ± 0.05
b
0.70 ± 0.03
c
10.00 ± 0.58
d
29.2 ± 1.18
b
Echinochloa colona Control 0.50 ± 0.03
b
4.00 ± 0.11
b
99.33 ± 0.67
a
0.0 ± 0.0
d
YMM1 0.20 ± 0.01
e
3.50 ± 0.10
c
98.33 ± 1.20
a
3.93 ± 0.09
c
YMM2 1.50 ± 0.02
a
5.00 ± 0.17
a
89.00 ± 2.08
b
8.83 ± 0.24
a
YMM3 0.30 ± 0.02
d
2.80 ± 0.03
d
81.00 ± 3.79
c
8.83 ± 0.24
a
YMM4 0.40 ± 0.02
c
1.70 ± 0.04
e
81.33 ± 2.96
c
7.77 ± 0.33
b
Echinochloa crus-galli Control 0.50 ± 0.02
c
3.51 ± 0.06
b
97.67 ± 0.33
a
0.0 ± 0.0
e
YMM1 0.50 ± 0.01
c
3.00 ± 0.17
c
59.67 ± 1.45
b
19 ± 1.41
c
YMM2 0.60 ± 0.03
b
2.50 ± 0.04
d
50.00 ± 1.15
c
39 ± 1.41
b
YMM3 0.90 ± 0.04
a
5.00 ± 0.12
a
98.67 ± 0.88
a
9.67 ± 0.47
d
YMM4 0.00 ± 0.00
d
0.00 ± 0.00
e
0.00 ± 0.00
d
79 ± 1.41
a
Values are means of three replicates and given as mean ± standard error. Different letters in the same column indicate significant differences according to Fisher’s
least significant difference (LSD) method (P≤0.05). Rating of injury symptoms due to phytotoxicity: 0% = no injury, 10–25% = feeble, 25–50% = mild, 50–75% =
moderate, and 75–100% = severe injury, plants killed
Mohammed and Badawy Egyptian Journal of Biological Pest Control (2020) 30:92 Page 6 of 9
YMM3 and C.globosum YMM2 improved seedling
growth of E.crus-galli and E.colona, respectively.
Previous studies confirmed the bioherbicidal activities
of culture filtrates of diverse phytopathogenic genera in-
cluding: Drechslera,Diaporthe,Chaetomium,Alternaria,
and Nigrospora against different weed species (Saxena &
Pandey 2002; Babu et al. 2003; Shabana 2005; Lee et al.
2008; Mitchell et al. 2008; Varejão et al. 2013; Akbar &
Javaid 2015; de Souza et al. 2015; de Oliveira Bastos
et al. 2017). Zhang et al. (2013) found that the secondary
metabolites of the Pythium aphanidermatum including
4-hydroxy-3-methoxycinnamic acid and indole deriva-
tives showed an herbicidal activity on the radical and
coleoptile elongation of Digtaria sanguinalis. Akbar and
Javaid (2015) concluded that cultural filtrates of Drech-
slera australiensis and D.hawaiiensis contain herbicidal
metabolites that resulted in about 58% reduction in bio-
mass of R.dentatus.Coniolariella sp. produced mevalo-
cidin, a secondary metabolite phytotoxin that revealed a
wide range of herbicidal actions (Sica et al. 2016).
Bioherbicidal activity of fungal filtrates on weed leaves
According to leaf disk puncture bioassay; the herbicidal
effects of fungal crude filtrates varied according to plant
species and filtrate type, and a variety of the symptoms
appeared including chlorosis and necrosis. Culture fil-
trates of A.alternata YMM1 and C.globosum YMM2
caused severe injury symptoms against leaf disks of P.
major,P.monspeliensis, and R.dentatus. Also, they
cause mild injury symptoms against S.oleraceus and E.
crus-galli, respectively. Similarly, culture filtrates of A.
tenuissima YMM3 showed sever injury symptoms
against leaf disks of P.monspeliensis and mild injury
symptoms against R.dentatus and P.major. Likewise,
culture filtrate of N.oryzae YMM4 exhibited severe in-
jury symptoms against leaf disks of E.crus-galli and mild
injury symptoms against P.major (Table 2). Similar
symptoms were observed in several previous studies by
bioherbicides (Varejão et al. 2013; de Souza et al. 2015
and Pes et al. 2016). In the present study, culture fil-
trates of fungal species had a non-selective toxicity
against a wide range of tested weeds. These activities
may be due to the toxins produced by phytopathogens
and cause disease symptoms.
Characterizations of fungal metabolites in crude extracts
GC/MS analyses of the crude extracts from A.alternata
YMM1, C.globosum YMM2, A.tenuissima YMM3, and
N.oryzae YMM4 (Fig. 3) led to the identification of 15,
22, 23, and 15 different components, representing 70.98,
Fig. 3 GC-MS chromatograms of the cultures filtrates of Alternaria alternata YMM1 (a), Chaetomium globosum YMM2 (b), Alternaria tenuissima
YMM3 (c), and Nigrospora oryzae YMM4 (d) indicating various metabolites peaks
Mohammed and Badawy Egyptian Journal of Biological Pest Control (2020) 30:92 Page 7 of 9
91.27, 66.46, and 82.87% of the total extracts for the
tested fungi, respectively. The identified compounds
were tested according to their elution order on a ZB-
5MS Zebron capillary column. The results showed some
constituents’similarities among extracts of the 4 tested
fungi.
Conclusion
Four fungal phytopathogens A.alternata YMM1, C.glo-
bosum YMM2, A.tenuissima YMM3, and N.oryzae
YMM4 were isolated from R.dentatus and S.oleraceus
weeds and their bioherbicidal activities were character-
ized. Their broth cultures showed a strong phytotoxic
activity on the tested weeds. Within these 4 phytopatho-
genic fungi, N.oryzae YMM4 showed the greatest bio-
herbicidal activity. This genus has previously been
recorded as a producer of bioherbicidal metabolites.
Thus, N.oryzae YMM4 metabolites might be used as a
bioherbicide to control several weeds in Egypt. In the fu-
ture, it would be of interest to further investigate the po-
tential of this isolate under controlled conditions and/or
field, such as those underway in Egypt, as a biological
control against some weeds to confirm the current
results.
Abbreviations
ANOVA: Analysis of variance; BLASTn: Basic local alignment search tool;
DMSO: Dimethyl sulfoxide; EI: Electron impact ionization; FID: Flame
ionization detector; GC/MS: Gas chromatography/mass spectrometry;
ITS: Internal transcribed spacer region; LSD: Fisher's least significant
difference; NCBI: National Centre for Biotechnology and Information;
PDA: Potato dextrose agar; PDB: Potato dextrose broth
Acknowledgements
The authors would like to extend their sincere appreciation to the Misr El
Kheir Foundation: Science, Technology and Innovation (Cordeau et al.)
Program to support this work by Lyophilizer instrument under the project
code LGA05130114.
Authors’contributions
All authors contributed equally to the work presented in this paper. The
authors read and approved the final manuscript.
Funding
Not applicable
Availability of data and materials
The data that support the findings of this study are available from the
corresponding author, Youssef M. M. Mohammed, upon reasonable request.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Botany and Microbiology, Faculty of Science, Damanhour
University, Damanhour 22516, Egypt.
2
Department of Pesticide Chemistry
and Technology, Faculty of Agriculture, Alexandria University, 21545
El-Shatby, Alexandria, Egypt.
Received: 27 April 2020 Accepted: 30 June 2020
References
Adams RP (1995) 10. Identification of essential oil components by gas
chromatography. Mass Spectrom:237–243
Akbar M, Javaid A (2015) Management of Rumex dentatus (toothed dock) by
fungal metabolites under field conditions. Int J Agric Biol 17:187–192
Aybeke M (2017) Fusarium infection causes genotoxic disorders and antioxidant-
based damages in Orobanche spp. Microbiol Res 201:46–51
Babu RM, Sajeena A, Seetharaman K (2003) Bioassay of the potentiality of
Alternaria alternata (Fr.) Keissler as a bioherbicide to control waterhyacinth
and other aquatic weeds. Crop Prot 22:1005–1013
Bajwa AA, Shabbir A, Adkins SW (2018) Interference and impact of parthenium
weed on agriculture. Parthenium weed: Biol Ecol Manage 7:57
Bonito GM, Gryganskyi AP, Trappe JM, Vilgalys R (2010) A global meta-analysis of
Tuber ITS rDNA sequences: species diversity, host associations and long-
distance dispersal. Mol Ecol 19:4994–5008
Boulos L (1999) Flora of Egypt, vol 1. Al-Hadara Publishing, Cairo, Egypt, p 419
Boulos L (2002) Flora of Egypt, vol 3. Al-Hadara Publishing, Cairo, Egypt, p 373
Boulos L (2005) Flora of Egypt, vol 4. Al-Hadara Publishing, Cairo, Egypt, p 617
Boulos L (2009) Flora of Egypt Checklist. Revised Annotated Edition. Al-Hadara
Publishing, Cairo, p 410
Cordeau S, Triolet M, Wayman S, Steinberg C, Guillemin JP (2016) Bioherbicides:
dead in the water? A review of the existing products for integrated weed
management. Crop Prot 87:44–49
de Souza ARC, Baldoni DB, Lima J, Porto V, Marcuz C, Ferraz RC, Kuhn RC,
Jacques RJS, Guedes JVC, Mazutti MA (2015) Bioherbicide production by
Diaporthe sp. isolated from the Brazilian Pampa biome. Biocatal Agric
Biotechnol 4:575–578
Ekman R, Holmbom B (1989) Analysis by gas chromatography of the wood
extractives in pulp and water samples from mechanical pulping of spruce.
Nordic Pulp and Paper Res J (Sweden).
Guo QY, Cheng L, Zhu HX, Li W, Wei YH, Chen HY, Guo LZ, Weng H, Wang J
(2020) Herbicidal activity of Aureobasidium pullulans PA-2 on weeds and
optimization of its solid-state fermentation conditions. J Integr Agric 19:
173–182
Javed S, Yousaf Z, Rashid M, Saleh N, Zahoor M, Ramzan H, Yasin H, Qamar NR,
Aftab A (2016) Pericarp of Trapa natans var.bispinosa (Roxb.) Makino as an
organic herbicide.
Júnior FWR, Scariot MA, Forte CT, Pandolfi L, Dil JM, Weirich S, Carezia C, Mulinari
J, Mazutti MA, Fongaro G (2019) New perspectives for weeds control using
autochthonous fungi with selective bioherbicide potential. Heliyon 5:e01676
Lee HB, Kim JC, Hong KS, Kim CJ (2008) Evaluation of a fungal strain,
Myrothecium roridum F0252, as a bioherbicide agent. Plant Pathol J
24:453–460
Marley P, Shebayan J (2005) Field assessment of Fusarium oxysporum based
mycoherbicide for control of Striga hermonthica in Nigeria. BioControl 50:
389–399
Matthews J (2018) Management of herbicide resistant weed populations.
Herbicide resistance in plants. CRC Press, In, pp 317–336
Mitchell JK (2003) Development of a submerged-liquid sporulation medium for
the potential smartweed bioherbicide Septoria polygonorum. Biol Control 27:
293–299
Mitchell JK, Yerkes CN, Racine SR, Lewis EH (2008) The interaction of two
potential fungal bioherbicides and a sub-lethal rate of glyphosate for the
control of shattercane. Biol Control 46:391–399
Mohankumar M, Vijayasamundeeswari A, Karthikeyan M, Mathiyazhagan S,
Paranidharan V, Velazhahan R (2010) Analysis of molecular variability among
isolates of Aspergillus flavus by PCR-RFLP of the ITS regions of rDNA. J Plant
Prot Res 50:446–451
Moubasher AH (1993) Soil fungi in Qatar and other Arab countries. University of
Qatar, The centre for scientific and applied research
Pes MP, Mazutti MA, Almeida TC, Curioletti LE, Melo AA, Guedes JV, Kuhn RC
(2016) Bioherbicide based on Diaporthe sp. secondary metabolites in the
control of three tough weeds. Afr J Agric Res 11:4242–4249
Sahin H (2019) A Review on parameters affecting the choice of alternative (non-
chemical) weed control methods. Eur J Eng Res Sci 4:16–19
Saxena S, Pandey AK (2002) Evaluation of an indigenous isolate of Alternaria
alternata (LC# 508) for use as a mycoherbicide for Lantana camara L. Crop
Prot 21:71–73
Mohammed and Badawy Egyptian Journal of Biological Pest Control (2020) 30:92 Page 8 of 9
Schütte G, Eckerstorfer M, Rastelli V, Reichenbecher W, Restrepo-Vassalli S,
Ruohonen-Lehto M, Saucy AGW, Mertens M (2017) Herbicide resistance and
biodiversity: agronomic and environmental aspects of genetically modified
herbicide-resistant plants. Environ Sci Eur 29:5
Shabana YM (2005) The use of oil emulsions for improving the efficacy of
Alternaria eichhorniae as a mycoherbicide for waterhyacinth (Eichhornia
crassipes). Biol Control 32:78–89
Sica VP, Figueroa M, Raja HA, El-Elimat T, Darveaux BA, Pearce CJ, Oberlies NH
(2016) Optimizing production and evaluating biosynthesis in situ of a
herbicidal compound, mevalocidin, from Coniolariella sp. J Ind Microbiol
Biotechnol 43:1149–1157
Täckholm V (1974) Students’Flora of Egypt, 2nd edn. Cairo University, Giza,
Egypt, p 888
Tuzi A, Andolfi A, Cimmino A, Evidente A (2010) X-ray crystal structure of
phyllostin, a metabolite produced by Phyllosticta cirsii, a potential
mycoherbicide of Cirsium arvense. J Chem Crystallogr 40:15
Varejão EVV, Demuner AJ, Barbosa LCDA, Barreto RW (2013) Phytotoxic effects of
metabolites from Alternaria euphorbiicola against its host plant Euphorbia
heterophylla. Quimica Nova 36:1004–1007
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of
fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to
methods and applications 18:315–322
Zhang W, Wolf TM, Bailey KL, Mortensen K, Boyetchko SM (2003) Screening of
adjuvants for bioherbicide formulations with Colletotrichum spp. and Phoma
spp. Biol Control 26:95–108
Zhang LH, Zhang JL, Liu YC, Cao ZY, Han JM, Juan Y, Dong JG (2013) Isolation
and structural speculation of herbicide-active compounds from the
metabolites of Pythium aphanidermatum. J Integr Agric 12: 1026-1032
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Mohammed and Badawy Egyptian Journal of Biological Pest Control (2020) 30:92 Page 9 of 9
