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Biocontrol of okra-rot-causing Cochliobolus spicifer-CSN-20 using
secondary metabolites of endophytic fungi associated with
Solenostemma arghel
Fatma F. Abdel-Motaal
a,b,
⁎,Noha M. Kamel
a,b
, Magdi A. El-Sayed
a,c
, Mohamed Abou-Ellail
d
a
Botany Department, Faculty of Science, Aswan University, Aswan 81528, Egypt
b
Unit of Environmental Studies and Development, Aswan University, Aswan 81528, Egypt
c
Molecular Biotechnology Program, Biological Sciences Department, Faculty of Science, Galala University, New Galala City, Suez, Egypt
d
Department of Genetics, Faculty of Agriculture and Natural Resources, Aswan University, Aswan 81528, Egypt
abstractarticle info
Article history:
Received 3 August 2021
Received in revised form 8 April 2022
Accepted 9 April 2022
Available online xxxx
Rot disease is responsible for serious economic losses relatedto okra (Abelmoschusesculentus) crops cultivated in
Upper Egypt. Colonies with a consistent morphology were isolated from the infected okra stems and leaves and
subjected to morphological and molecular examinations. The causal pathogen was identified as Cochliobolus
spicifer based on morphological fungus descriptions, as well as on the amplified 28S rDNA and internal tran-
scribed spacer region (ITS) sequences, which showed 99%–100% similarity to the sequences of C. spicifer-CSN-
20 strains. The volatile and non-volatile organic compounds (VOCs and n-VOCs, respectively) produced by the
endophytic fungi that are associated with the medicinal plant Solenostemma arghel,namelyFusarium solani-F4-
1007, Penicillium verrucosum-F2-1006, and Aspergillus terreus-F5-1008, inhibited the growth of the C.spicifer
pathogen by 34.2%,31.4%, and 30.5%, respectively. In total,27 VOCs were identified by GC/MS, among which
eight were specifictoA. terreus-F5-1008, eight to P. verrucosum-F2-1006,and three to F. solani-F4-1007, whereas
nine VOCs were commonlyproduced by the three endophyticfungi. Moreover, F. solani-F4-1007-produced VOCs
and n-VOCs exhibited the highest antifungal activity, with 37.27% and 37.1% inhibition against C.spicifer colony
growth, respectively. The potent antifungal VOCs produced by F. solani-F4-1007were identified as 3,4-dihydro-2
h-1,5-(3″-t-butyl) benzodioxepine, 4-(2-hydroxyethyl) phenol,and phenylethyl alcohol usingGC/MS. Therefore,
F. solani-F4-1007 was tested as a potential biocontrol agent against C. spicifer-CSN-20 using an in-planta assay.
Okra plants treated with endophytic F. solani-F4-1007did not show any disease symptoms, whereas those that
were not treated with F. solani-F4-1007 exhibited severe disease symptoms when challenged with inoculation
of the C. spicifer pathogen. Our results demonstrated the contribution of the endophytic fungus F. solani-F4-
1007 as a potential biocontrol agent against the C. spicifer pathogen, to improve okra growth.
© 2021 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords:
Abelmoschus esculentus
Cochliobolus spicifer
Endophyte
Rot disease
Solenostemma arghel
Volatiles
Non-volatiles
1. Introduction
Okra (A. esculentus L.) is an economically important vegetable crop
worldwide and an affordable source of protein, carbohydrates, vitamins,
and minerals that exhibits potential for cultivation as an essential oil-
seed crop (Ofori et al., 2020). However, okra issusceptible to manyfun-
gal pathogens, such as Pythium spp., Rhizoctonia spp., Phytopthora spp.,
Fusarium oxysporum, and Erysiphe cichoracearum, which cause severe
disease symptoms, including damping-off of seedlings, root rot, leaf
wilting, and powdery mildew (Jukte et al., 2016). Cercospora spp. is
also a major fungal pathogen that causes early leaf spots in okra in Au-
gust, resulting in complete defoliation of susceptible cultivars. The path-
ogen appears on the surface of lower leaves as olivaceous- to sooty-
colored growth, and severely infected leaves wither, wilt, and die from
coalescing lesions. In addition, Alternaria and Phyllosticta leaf spots
have also been reported in okra, with brown/reddish borders to black
circular spots (Awasthi, 2015).
The growingworld population combined with serious infectious dis-
eases in crop plants ledto the extensive use of fungicides to increase ag-
riculture production, resulting in a high accumulation of persistent and
non-biodegradable fungicides in various components of water, air, and
soil ecosystems (Ons et al., 2020). For example, Strobilurin is a natural
fungicide produced by mushrooms that is widely used in combating ag-
ricultural pathogens, such as white mold, rot, early and late leaf spots,
rusts, and rice blast (Bartlett et al., 2002;Feng et al., 2020). This agent
Annals of Agricultural Sciences 67 (2022) xxx
⁎Corresponding author at: Botany Department, Faculty of Science, Aswan University,
Aswan 81528, Egypt.
E-mail address: fatma.fakhry@aswu.edu.eg (F.F. Abdel-Motaal).
AOAS-00329; No of Pages 10
https://doi.org/10.1016/j.aoas.2022.04.001
0570-1783/© 2021 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Contents lists available at ScienceDirect
Annals of Agricultural Sciences
journal homepage: www.elsevier.com/locate/aoas
Please cite this article as: F.F. Abdel-Motaal, N.M. Kamel, M.A. El-Sayed, et al., Biocontrol of okra-rot-causing Cochliobolus spicifer-CSN-20 using
secondary metabolites of endophyti..., Annals of Agricultural Sciences, https://doi.org/10.1016/j.aoas.2022.04.001
exhibited non-target effects during long-term use and raised serious
public health concerns. In addition, the excessive use of fungicides ren-
ders the phytopathogens insensitive to them and leads to the develop-
ment of resistant fungal pathogens. Therefore, an effective, affordable,
and eco-friendly alternative approach is needed to maintain agriculture
production with minimum negative effects on the environment.
Medicinal-plant-associated endophytes demonstrate antagonistic ac-
tivity against disease-causing phytopathogens and are capable of pro-
ducing antimicrobial, insecticidal, and antioxidant secondary
metabolites (Kamel et al., 2020;Kusari et al., 2012;Tariq et al., 2009;
Puri et al., 2005). Therefore, the endophytes associated with medicinal
plants are considered as an alternative biocontrol agent and a bioactive
metabolite reservoir for crop protection. For example, non-volatile or-
ganic compounds (n-VOCs), alkaloids, and some terpenes were de-
tected in the extracts of the medicinal plant Euphorbia geniculata and
its associated fungi (Kamel et al., 2020). Similarly, volatile organic com-
pounds (VOCs) derived from endophytes showed potent antifungal ac-
tivity and were recognized as an important biocontrol agent that
inhibited pathogen growth and promoted plant growth (Farag et al.,
2006). An analysis of the VOCs produced by two endophytic fungi,
Sarocladium kiliense and Penicillium oxalicum, isolated from the medici-
nal plant Aloe dhufarensis revealed the presence of fatty acids, fatty acid
methyl esters, furfuryl alcohol, 1,2-diols, and amide, and exhibited high
antifungal activity against Fusarium sp. and Cladosporium sp. pathogens
(Al-Rashdi et al., 2020).
In this study, first, we isolated and identified the causal pathogen of
okra rot disease usin g morphological and molecular techniques. Second,
we screened for and identified the various n-VOCs and VOCs produced
by three S. arghel strains associated with the endophytic fungi
F. solani-F4-1007, P. verrucosum-F2-1006, and A. terreus-F5-1008.
Third, the antimicrobial activity of endophyte-produced n-VOCs and
VOCs was examined against different phytopathogens. Finally, the can-
didate endophytic fungus Fusarium solani-F4-1007 was tested against
okra rot disease using an in-planta assay. Our study showed that
F. solani-F4-1007 seems to be suited for use as a biocontrol agent to
manage the existing and future invasive pathogens of okra.
2. Material and methods
2.1. Sample collection
In this study, okra plants showing black-spot symptoms on the
stems and leaves, in addition to yellowing of the leaves, for the first
time in Upper Egypt at the Aswan University farm (Aswan city, Egypt)
were used. The diseased organs were collected, placed in sterilized
bags, and brought to the laboratory for pathogen isolation.
2.2. Isolation of pathogens
The symptomatic parts of infected okra stems and leaves were sur-
face sterilized with ethanol (70%) for 1 min and NaClO (1%) for 1 min
and then washed four times with sterilized distilled water. Small parts
were aseptically cut and plated on potato dextrose agar (PDA) plates
for 4 days at 28 °C. Within 2 days, fungal mycelia visibly grew from
the cultured organ pieces. Using the hyphal tip method of purification,
a single hypha was transferred and inoculated on fresh PDA plates and
then examined using a microscope (Ghuffar et al., 2018).
2.3. Morphological and molecular identification and assessment of Koch's
postulates
The isolated fungal species were identified morphologically based
on their colony and hyphal characteristics (Christensen and Raber,
1978). The molecular identification of pathogens was performed by
rDNA gene sequencing. The CTAB method (Gontia-Mishra et al., 2014)
was used to extract DNA from 5-day-old fungal cultures. The partial
fragment of the rDNA gene was amplified using two fungal primers,
ITS1 and ITS4 (Suarez et al., 2005). The PCR products were analyzed
by electrophoresis on 1% agarose gels. The bands were eluted and se-
quenced at the Korea Solgent Company. The NCBI website (BLAST)
was used to analyze the sequences. The MEGA 6 software was used
for the construction of a phylogenetic tree (Tamura et al., 2013).
2.4. Pathogenicity test
The pathogenicity of the candidate fungi was confirmed by a re-
inoculation test according to Berner et al. (2007), in which the pathogen
was cultured on PDA medium and incubated at 28 °C for 11 days. The
harvested conidia were suspended in sterilized water at 1× 10
6
co-
nidia/mL; the Okra plants were sprayed with a conidialsuspension, cov-
ered with transparentplastic bags, and incubated at 28 °C for 2 weeks. In
the same conditions, the control plants were sprayed with sterile dis-
tilled water. Five replicates were performed per group.
In another experiment, healthy leaves were plated in sterilized Petri
dishes containing wet sterilized filter paper. Next, the mycelial disks of
the pathogen were inoculated on the leaf surface, and the control (path-
ogen-free condition) was inoculated in plates containing agar disks
alone. All plates were incubated at 28 °C for 4 days.
2.5. Solenostemma arghel endophytic fungi
The three endophytic fungi isolated from the medicinal plant
S. arghel (i.e., Aspergillus terreus-F5-1008, Fusarium solani-F4-1007, and
Penicillium verrucosum-F2-1006) were obtained from the deposited
lab collection of the Faculty of Science, Aswan University. Each isolate
was sub-cultured on PDA medium and allowed to flourish at 28 °C.
2.6. Extraction of n-VOCs from endophytic fungi
The endophytic fungi isolated from S.arghel were inoculated as
6.0 mm disks in 1000-mL flasks containing 400 mL of Potato Dextrose
Broth medium with shaking (200 rpm) for 10 days. Cultures were fil-
tered, and the filtrate was partitioned with the ethyl acetate (EtOAc)
solvent. The EtOAc extract was separated using a separating funnel
and vacuum dried (Abdel-Motaal et al., 2010).
2.7. Detection of n-VOCs and metabolic profiling based on thin-layer chro-
matography (TLC)
The ethyl acetate extracts of the studied fungi were spotted on TLC
Silica gel 60 F254 plates (Merck ® Darmstadt, Germany). For the sepa-
ration of the bands of the compounds, the plates were developed in a
mobile phase of diethyl ether:n-hexane (2:1 v/v). The TLC-developed
plates were observed under UV light at 254 and 365 nm (Abdel-
Motaal et al., 2009). To detect the full presence of n-VOCs, the plates
were sprayed with 0.1%sulphuric vanillin (w/v) and heated, to develop
colored spots.
2.8. GC–MS analysis of the VOCs released from endophytes
The VOCs extracted from the studied endophytes were analyzed and
identified by GC/MS. The GC–MS analyses were carried out as follows:
Instrument: TRACE GC Ultra Gas Chromatograph (THERMO Scientific
Corp., USA), connected with a thermal mass spectrometer detector
(ISQ Single Quadrupole Mass Spectrometer). The GC–MS system was
equipped with a TR-5 MS column (30 × 0.32 mm i.d., 0.25 μmfilm thick-
ness). Analyses were performed using helium as a carrier gas at a flow
rate of 1.0 mL/min and a split ratio of 1:10 using the following temper-
ature program: 60 °C for 1 min; temperature increase to 240 °C at 4.0 °C/
min, followed by a 1-min hold. The injector and detector were held at
210 °C. Diluted samples (1:10 hexane, v/v) of 1 μLofthemixtures
were always injected. Mass spectra were obtained by electron
F.F. Abdel-Motaal, N.M. Kamel, M.A. El-Sayed et al. Annals of Agricultural Sciences 67 (2022) xxx
2
Fig. 2. TLC chromatogaram showing the differences of secondary metabolites between P. verrucosum-F2-1006, F. solani-F4-1007 and A. terreus-F5-1008. Plates were visualized under
254 nm (a) and blue under 365 nm (b) and derivatized with Sulphuric vanillin 0.1% w/v (c). (For interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
Fig. 1. Rot and yellowing symptoms on Okra plant infected by C.spicifer-CSN-20 in the field (a, b, c).Pathogenicity test(d). C.spicifer-CSN-20 under microscope (e) andcolony shape (f) .
Phylogenetic relationships of isolated pathogen, Cochliobolus spicifer-CSN (Synonymy, Curvularia spicifer) and selected fungi derived from NCBI Genbank based on nuclear ribosomal in-
ternal spacer sequence (ITS) (g).
F.F. Abdel-Motaal, N.M. Kamel, M.A. El-Sayed et al. Annals of Agricultural Sciences 67 (2022) xxx
3
ionization at 70 eV, using a spectral range of m/z40–450. The identifica-
tion of the chemical constituents of the VOCs was de-convoluted using
the AMDIS software (www.amdis.net)andidentified by its retention in-
dices (relative to C
8
–C22 of n-alkanes), amass spectrum matching au-
thentic standards, or the Wiley spectral library collection and NSIT
library database.
2.9. Antifungal activity of the n-VOCs extracted from endophytic fungi
against C. spicifer-CSN-20
Fungal ethyl acetate extracts were dissolved in DMSO, added to PDA
medium atvarious concentrations (0.5, 1.0, and 2.0 mg/mL), and shaken
well to homogenize. A 0.5-cm mycelial disk was transferred into the
center of the plate (6.0 cm in diameter) according to the “poisoned
food method,”which was used to check the antimicrobial effect against
the pathogen (Balouiri et al., 2016). According to Singh and Tripathi
(1999), the diameters of the fungal growth in the treated and control
plates were measured after 3 days, and the inhibition percentage was
calculated.
2.10. Antifungal activity of the VOCs released from endophytic fungi against
C. spicifer-CSN-20
Pathogen cultures were exposed to the VOCsproduced by the endo-
phytes F5-1008, F4-1007, and F2-1006. Briefly, in three 15-cm Petri
dishes (5.0 cm), each endophyte culture (3-days old) was plated oppo-
site to a cultureof the pathogen, or, as the control, the pathogen was cul-
tured alone in a plate with PDA medium. The plates were wrapped with
Parafilm and incubated at 28°C until the pathogen culture in the control
reached the maximum (Strobel et al., 2001). The diameter of the patho-
gen colony was measured and compared with that of the control.
The inhibition percentage in the above two experiments was calcu-
lated as follows:
%Inhibition ¼Dc−Ds
Dc 100
where Dc is the average colony diameter in the control, and Ds is the av-
erage colony diameter in the experimental treatments (Singh and
Tripathi, 1999).
2.11. In vitro screening of the antagonistic potential of Solenostemma
arghel endophytic fungi against C. Spicifer-CSN-20
The antagonistic effects of the endophytic fungi were tested in vitro
against C. Spicifer-CSN-20 by dual culture assay. The control plates were
prepared by culturing the pathogenic fungus against an agar plug. Tests
were carried out in three replicates, plates were incubated at 28 ± 2 °C,
and the growth diam eter of C.spicifer-CSN-20 was measured. The inhibi-
tion percentage wascalculated after 7 days of culture based on the path-
ogen growth inhibition percentage, as described above.
2.12. In vivo assay of the endophytic fungal treatments on the disease resis-
tance of okra plants against the C. spicifer-CSN-20 pathogen
According to the results of the dual culture assay and the antifungal
activity of endophytic fungi against the okra pathogen, the seeds of okra
were soaked for 2 h in a spore suspension of the most effective endo-
phytic fungi, whereas the control was soaked in sterilized distilled
water. Subsequently,5-week-old endophyte-treated and endophyte-
untreated plants were sprayed with the fungal pathogen suspension
culture, whereas the control plants were sprayed with sterilized dis-
tilled water. All plants were covered with plastic bags for 48 h, to keep
humidity at 75%–80%, and the pots with and without pathogen treat-
ment were kept in a growth chamber (Awan et al., 2018). All experi-
ments were performed in triplicate.
2.13. Statistical analysis
One-way analysis of variance was used to analyze the results ob-
tained with the help of the Minitab 18 software (www.minitab.com).
Tukey's testwas run to verify the significance of the differences detected
between the control and the treatments (P≤0.05). The values shown in
the Figures are the means ± standard errors. Corrplot in the R program
(R- 3.4.3, https://www.r-project.org/)was used for correlation analysis.
Control
Endophyte
Treated
b
a
c
Fig. 3. Antifungal activity of S.arghel endophytic fungal extracts against C. spicifer-CSN-20
(the Okra pathogen) (a). Microscopic examination of the pathogen, C. spicifer-CSN-20 in
control which showed formation of hyphae and conidia while only hypha were formed
in treated plates with endophytes extract (b). The inhibition % of the pathogen when
treated with S.arghel endophytic fungal extracts with the concentrations of (0.5, 1.0,
and 2.0 mg/mL) (c).
F.F. Abdel-Motaal, N.M. Kamel, M.A. El-Sayed et al. Annals of Agricultural Sciences 67 (2022) xxx
4
3. Results and discussion
3.1. Disease symptoms and pathogen colonization
Rot disease causes severe symptoms and the loss of many crops. It
appears as spots of various sizes occurring on the stems and leaves
(Awasthi, 2015). These spots may vary in color, from gray to brown
and black, and as the disease progresses, yellowing, dieback, and poor
vigor can be observed as common symptoms, resulting in plant death
(Amaradasa and Amundsen, 2016). Our results showed that C. spicifer-
CSN-20is the causal agent of stem rot and leaf-yellowing disease in
okra (Fig. 1a–c); moreover, this is the first report of C. spicifer-CSN-20
as an okra pathogen. Esuruoso et al. (1975) recorded Alternaria
alternata,Curvularia lunata (Cochliobolus lunatus), and Cladosporium
cladosporioides as okraseed-borne fungal diseases in Nigeria.
3.2. Morphological characterization of the pathogen
Based on the morphological and cultural characteristics viz.,colonies
were olive-green to dark brown colored, with septate, well-branched,
and brown mycelia. Solitary and flexuous conidiophores were mid to
dark brown. Conidia were cylindrical, straight, or oblong with rounded
ends and 2–3 septate (Fig. 1e,f). The pathogen was identified as
Cochliobolus spicifer (Synonymy, Curvularia spicifer) based on fungal
morphology and microscopic features (Ellis, 1976).
According to the NCBI-BLAST search, the ITS sequence showed 99%–
100% similarity with all C.spicifer strains. The ITS sequences of C.spicifer
were deposited in the GenBank database with accession number
LC520251.1. The nucleotide sequence alignments of C. spicifer-CSN-20
and other Cochliobolus species derived from NCBI GenBank were used
to construct a neighbor-joining phylogenetic tree using the Mega 4 soft-
ware (Fig. 1g).
3.3. Pathogenicity test
Healthy okra plants sprayed with C.spicifer-CSN-
20exhibitedsymptoms of the disease after 1 week of incubation, which
included yellowing of the leaves and spots on stems (Fig. 1d). In con-
trast, the control plant did not show any morphological symptoms.
Cochliobolus spicifer-CSN-20was re-isolated from the diseased parts,
whereas the control was fungus free.
3.4. Comparative analysis and antifungal activity of the VOC and n-VOC
compounds produced by S. arghel endophytes
A TLC analysis clarified the variation of the n-VOC content in the
studied endophytes (Fig. 2). The culture extracts of the fungus
F. solani-F4-1007 contained additional compounds that could be
observed under short and long UV (quenched under 254 nm and blue
under 365 nm). These compounds were terpenoid and phenolic com-
pounds. It is known that the medicinal plant S. arghel contains a variety
of compounds and exerts diverse bioactivities with no toxicity (Abu-
Odeh and Talib, 2021). Particular differences were observed in the met-
abolic profile between the fungus Fusarium solani-F4-1007 and the
other two fungi, P.verrucosum-F2-1006 and A. terreus-F5-1008 (Fig. 2).
The crude extract of the endophytic fungus F. solani-JK10 isolated
from the root of themedicinal plant Chlorophora regia demonstrated po-
tential antibacterial activity (Kyekyeku et al., 2017).
The antifungal activities of the non-volatile or diffusible compounds
produced by S. arghel plant endophytes demonstrated a significant ef-
fect against the okra pathogen (Fig. 3a). All concentrations of the se-
lected fungal endophytes were active against the okra pathogen, with
variable inhibitory effects. Fusarium solani-F4-1007 produced the most
active substances in the EtOAc fraction, which showed the best antifungal
activity, with an inhibition rate of 37.1% at a concentration of 2.0 mg/mL,
followed by the endophyte P. verrucosum-F2-1006 and A. terreus-F5-1008,
which exhibited inhibition rates of 34.9% and 19.8%, respectively, at the
same concentration. The biological activity of F. solani-F4-1007 may be
attributed to the presence of terpenoid and phenolic compounds, which
are not produced by the other examined fungi. In the microscopic exam-
ination, sporulation was also affected, as no conidia were produced in all
colonies grown in the presence of endophyte extracts in contrast with the
control (Fig. 3bandTable 4).
Solenostemma arghel endophytic fungi showed variability in VOCs, as
assessed using GC–MS. Aspergillus terreus-F5-1008 emitted the highest
number of VOCs (15 compounds), among which the most abundant
constituent was di-isooctylphthalate, with a relative value of 67.82%
(Table 1 and Fig. 4a). The major VOCs from the fungus F5-1008 isolated
from soil were methyl 12,15-octadecadienoate (31.989%) and n-
hexadecanoic acid (15.31%), which exhibited significant mosquitocidal
activity (Ragavendran and Natarajan, 2015).Most of the VOCs identified
from the F5-1008 endophyte in this study (Table 1) were reported pre-
viously as antimicrobial agents, such as2H-pyran-3-ol, tetrahydro-
2,2,6-trimethyl-6-(4-methyl-3-cyclohexen-1-yl (Fahmy, 2020).
Ten active compounds were identified from the fungus F. solani-F4-
1007, with the most abundant constituents being di-isooctyl phthalate
and phenylethyl alcohol, with a peak areaof 67.93% and 10.41%,respec-
tively. The relative values of the remaining compounds were almost
similar and ranged between 1.47% and 3.66% (Table 2 and Fig. 4b).
The main components of the endophyte F. solani isolated from Taxus
baccata were 1-tetradecene, 8-octadecanone, 8-pentadecanone,
octylcyclohexane, and 10-nonadecanone (Zheng et al., 2021). Many
previous reports confirmed the biological activity of VOCs such as di-
isooctyl phthalate, which exhibits a strong antimicrobial activity com-
pared with other known antibiotics used for sepsis treatment (Amer
et al., 2019), i.e., 1-docosanol (Balachandar et al., 2018), 1-eicosanol
Table 1
Identification of VOCs emitted from the endophyte A. terreus-F5-1008 (compounds name, structure, Formula, Retention time (RT), Molecular weight (M.W), and Peak area%).
Chemical compounds Formula RT M.W Peak area %
p-Benzoquinone, 2-methyl-(2,5-Cyclohexadiene-1,4-dione, 2-methyl C
7
H
6
O
2
4.78 122 2.88
2-Coumaranone C
8
H
6
O
2
8.57 134 1.74
1,4-Benzenediol, 2-methyl C
7
H
8
O
2
11.20 124 3.84
15-Methyltricyclo [6.5.2 (13,1,4). 0(7,15)]pentadeca-1,3,5,7,9,1,1,13-heptene C
16
H
14
14.37 206 1.40
1-Hexadecanol C
16
H
34
O 15.81 242 0.85
2H-Pyran-3-ol, tetrahydro-2,2,6-trimethyl-6-(4-methyl-3-cyclohexen-1-yl)- C
15
H
26
O
2
18.80 238 0.78
1-Nonadecene C
19
H
38
19.55 266 1.76
7,9-Di-tert-butyl-1-oxaspiro (4,5)dec,a-6,9-diene-2,8-dione C
17
H
24
O
3
21.78 276 3.44
9-octadecenoic acid (Z)- C
18
H
34
O
2
22.54 282 2.84
1-Eicosanol C
20
H
42
O 22.94 298 2.64
1-Docosanol C
22
H
46
O 26.06 326 1.05
1-Chlorooctadecane C
18
H
37
Cl 26.14 288 0.89
Methoxyacetic acid, 3-tetradecyl ester C
17
H
34
O
3
27.59 286 1.63
Heptacosane C
27
H
56
28.98 380 2.81
Diisooctyl phthalate C
24
H
38
O
4
30.99 390 67.82
F.F. Abdel-Motaal, N.M. Kamel, M.A. El-Sayed et al. Annals of Agricultural Sciences 67 (2022) xxx
5
(Chatterjee et al., 2018), 1-hexadecanol (Jaddoa et al., 2016;Susanti
et al., 2013),and other compounds, such as the antioxidantagents1-
nonadecene and 1-docosanol (Amudha et al., 2018) and 3,4-dihydro-
2H-1,5-(3″-T-butyl) benzodioxepine (Akshatha et al., 2016;Mohamed
et al., 2020), and, finally, hexadecanoic acid, as an anticancer and anti-
inflammatory agent (Aparna et al., 2012;Ravi and Krishnan, 2017).
All of these active valuable compounds were produced by the endo-
phyte Fusarium solani-F4-1007 in this study. Among the 15active com-
pounds produced by the endophyte P. verrucosum-F2-1006, 7,9-Di-
tert-butyl-1-oxaspiro (4,5) dec,a-6,9-diene-2,8-dione and 1-eicosanol
demonstrated the highest peak areas (18.98% and 15.16%, respectively)
(Table 3 and Fig. 4c). The volatile compounds9, 12-octadecadienoyl
chloride, hexadecanoic acid, and 2,2-dideutero octadecanal possess
anticancer activities (Lalitha et al., 2019). The remaining VOCs emitted
from the endophyte F2-1006 exhibit antimicrobial characteristics, as
described above.
The Venn diagram reported in Fig. 4d shows that five VOCS (1-
eicosanol, 7,9-Di-tert-butyl-1-oxaspiro(4,5) dec,a-6,9-diene-2,8-dione,
di-isooctylphthalate, 1-hexadecanol, and 1-nonadecene) were emitted
from all endophytic fungi. Interestingly, all of these VOCs that were
common among the studied endophytes demonstrate antimicrobial ac-
tivity, as described above. 1-Docosanol was recorded in two endophytes
(F5-1008 and F4-1007); 9-octadecenoic acid (Z) was shared between
two endophytes, F5-1008 and F2-1006; whereas, hexadecanoic acid
was specific to the endophytes F4-1007 and F2-1006. Out ofthe27
VOCs emitted from the three studied endophytes, eight VOCs were
Aspergillus terreus
Fusarium solani
Penicillium verrucosum
Fig. 4. GC-mass analysis of volatile organic compound profiles of A. terreus-F5-1008(a),F.solani-F4-1007(b), P. verrucosum-F2-1006(c),Venn diagram showed similarity and differences
between VOCs emitted from the three endophytes (d).
F.F. Abdel-Motaal, N.M. Kamel, M.A. El-Sayed et al. Annals of Agricultural Sciences 67 (2022) xxx
6
specific to the fungus F5-1008, and an additional eight compounds were
specified to the endophyte F2-1006; whereas only three VOCs were par-
ticularly released from the endophyte F4-1007 (Fig. 4dandTable 4).
3.5. In vitro antagonistic activity of endophytic fungi against C.spicifer-CSN-20
Exposure to the VOCs emitted from endophytic fungi (F. solani-F4-
1007, P. verrucosum-F2-1006, and A. terreus-F5-1008) in sealed plates
slowed down the growth of the okra pathogenic fungus C.spicifer-
CSN-20 (Fig. 5a). Specifically, the VOCs emitted from the endophytic
fungus F. solani-F4-1007 yielded the strongest reduction in the growth of
C.spicifer-CSN-20(37.27%) (Fig. 5b). Conversely, the endophytes F2-1006
and F5-1008 showed similar (25.4% and 24.38%, respectively) inhibition
against C.spicifer-CSN-20 (Fig. 5b). The high activity of the VOCs emitted
from F4-1007 would classify them as specific compounds in this fungus.
These VOCs were identified as 3,4-dihydro-2 h-1,5-(3″-t-butyl)
benzodioxepine, 4-(2-hydroxyethyl) phenol, and phenylethylalcohol
(Table 4), or the synergy between these specific VOCs of endophyte F4-
1007 and other VOCs shared with the remaining two endophytes.
Phenylethyl alcohol demonstrates antimicrobial activity (Lilley and
Brewer, 1953). Recently, this VOC was used as a potent preservative
agent in emulsions, cleansing solutions, and conditioners (Sirilun et al.,
2017). The second VOC, 3,4-dihydro-2 h-1,5-(3″-t-butyl) benzodioxepine,
demonstrates a potent antioxidant activity (Akshatha et al., 2016;
Mohamed et al., 2020).
The dual culture assay demonstrated that the tested endophytic
fungi obtained from S.arghel (A. terreus-F5-1008, F. solani-F4-1007,
and P. verrucosum-F2-1006) inhibited the growth of C.spicifer-CSN-20
(i.e., the okra pathogen) through VOCs and the other effect through
spreading in media by n-VOCs and eliminate the pathogen growth.
Fig. 6(a) shows that the antagonistic isolates changed the shape of the
phytopathogen colonies from a circle (as in the control) to an elongated
ellipse. In comparison with the control, the above-mentioned isolates
demonstrated a significant inhibitory effect on the growth of pathogen
colonies. The antagonistic activity of the selected endophytic fungi
yielded varying degrees of inhibition against the phytopathogenic C.
spicifer-CSN-20, with the highest inhibition percentage demonstrated
by the endophyte F4-1007, followed by F2-1006 and F5-1008, with in-
hibition rates of 34.2%, 31.4%, and 30.5%, respectively (Fig. 6b). Our re-
sults clearly showed that the S.arghel endophytes affected the growth
of the phytopathogenic fungus C. spicifer-CSN-20. This inhibition, espe-
cially in the case of theendophyte F4-1007, may be attributed to the se-
cretion of inhibitory substances, such as phenolic and terpenoid
compounds (Fig. 2), in co-effect with the VOCs produced by the antag-
onists. This inhibition differs according to thenature, quantity, and qual-
ity of antibiotics/inhibitory substances (Alwathnani and Perveen, 2012).
Purification or concentration of active molecules may enhance the bio-
control process through the increase of the inhibition percentage com-
pared with the crude extract.
3.6. Effect of F. solani-F4-1007-seed treatments on the resistance of okra
plants against the disease caused by C. spicifer-CSN-20
Accordingto the positive results described above, which clarified the
potency of S. arghel endophytic fungi in limiting the growth of the okra
Table 2
Identification of VOCs emitted from the endophyte F. solani-F4-1007 (compounds name,
structure, Formula,Retention time (RT), Molecular weight (M.W), and Peak area%).
Chemical compounds Formula RT M.W Peak
area %
Phenylethyl Alcohol C
8
H
10
O 6.17 122 10.41
4-(2-Hydroxyethyl) phenol C
8
H
10
O
2
12.76 138 3.57
3,4-Dihydro-2 h-1,5-(3″-t-butyl)
benzodioxepine
C
13
H
18
O
2
14.36 206 3.55
1-Hexadecanol C
16
H
34
O 15.81 242 2.34
1-Nonadecene C
19
H
38
19.54 266 3.56
7,9-Di-tert-butyl-1-oxaspiro (4,5) dec,
a-6,9-diene-2,8-dione
C
17
H
24
O
3
21.78 276 1.62
Hexadecanoic acid C
16
H
32
O 22.54 256 1.88
1-Eicosanol C
20
H
42
O 22.94 298 3.66
1-Docosanol C
22
H
46
O 26.05 326 1.47
Diisooctyl phthalate C
24
H
38
O
4
31.03 390 67.93
Table 3
Identification of VOCs emitted from the endophyte P. verrucosum-F2-1006 (compounds
name, structure,Formula, Retentiontime (RT), Molecular weight(M.W), and Peak area%).
Chemical compounds Formula RT M.W Peak
area %
1,4-Benzenediol, 2-(1,1dimethylethyl)
-5-(2-propenyl)
C
13
H
18
O
2
14.36 206 5.46
1-Hexadecanol C16H34O 15.81 242 3.23
1-Docosene C
22
H
44
19.54 308 8.51
Nonadecane C
19
H
40
15.95 268 1.54
1-Nonadecene C
19
H
38
19.54 266 8.51
7,9-Di-tert-butyl-1-oxaspiro (4,5)
dec,a-6,9-diene-2,8-dione
C
17
H
24
O
3
21.78 276 18.98
Hexadecanoic acid C
16
H
32
O
2
22.54 256 9.59
9-Octadecenoic acid (Z)- C
18
H
34
O
2
22.54 282 9.59
1-Eicosanol C
20
H
42
O 22.94 298 15.16
1,3,5-Triazine-2,4-diamine, 6-chloro-n-ethyl C
5
H
8
ClN
5
23.04 173 1.56
6-Heptadecyne, 1-chloro- C
17
H
31
Cl 23.20 270 2.05
9,12-Octadecadienoyl chloride, C
18
H
31
ClO 25.67 298 3.75
2,2-Dideutero octadecanal C
18
H
34
D
2
O 28.91 270 5.02
Hexanoic acid, 2-ethyl-,oxybis
(2,1-ethanediyloxy-2,1-ethanediyl) ester
C
24
H
46
O
7
30.03 446 2.58
Diisooctyl phthalate C
24
H
38
O
4
30.97 390 4.21
Table 4
Volatile Organic Compounds specific for each endophyte and the common volatile compound between each other.
Fungal species No., of
compounds
Volatile organic compounds
A. terreus-F5-1008;
F. solani-F4-1007;
P. verrucosum-F2-1006
5 1-Eicosanol; 7,9-Di-tert-butyl-1-oxaspiro(4,5)dec,a-6,9-diene-2,8-dione; Diisooctyl phthalate; 1-Hexadecanol; 1-Nonadecane
A. terreus-F5-1008
F. solani-F4-1007
1 1-Docosanol
A. terreus-F5-1008;
P. verrucosum-F2-1006
1 9-Octadecenoic acid (Z)-
F. solani-F4-1007;
P. verrucosum-F2-1006
1 Hexadecanoic acid
A. terreus-F5-1008 8 2-Coumaranone; Methoxyacetic acid, 3-tetradecyl ester; 15-Methyltricyclo [6.5.2 (13,1,4). 0(7,15)]pentadeca-1,3,5,7,9,1,1,13-heptene;
p-Benzoquinone, 2-methyl-(2,5-Cyclohexadiene-1,4-dione, 2-methyl; 1,4-Benzenediol, 2-methyl; Heptacosane; 1-Chlorooctadecane;
2H-Pyran-3-ol, tetrahydro-2,2,6-trimethyl-6-(4-methyl-3-cyclohexen-1-yl)
F. solani-F4-1007 3 3,4-Dihydro-2 h-1,5-(3″-t-butyl) benzodioxepine; 4-(2-Hydroxyethyl) phenol; Phenylethyl Alcohol
P. verrucosum-F2-1006 8 9,12-Octadecadienoyl chloride; Nonadecane; 2,2-Dideutero octadecanal; 6-Heptadecyne, 1-chloro; 1-Docosene; 1,4-Benzenediol,
2-(1,1dimethylethyl) -5-(2-propenyl); 1,3,5-Triazine-2,4-diamine, 6-chloro-n-ethyl; Hexanoic acid, 2-ethyl-,oxybis
(2,1-ethanediyloxy-2,1-ethanediyl) ester
F.F. Abdel-Motaal, N.M. Kamel, M.A. El-Sayed et al. Annals of Agricultural Sciences 67 (2022) xxx
7
0
5
10
15
20
25
30
35
40
A. terreus F. solani P. verrucosum
Pathogen Inhibion %
*
*
*
Control A. terreus F. solani- P. verrucosum
a
b
Fig. 5. Inhibition of the okra pathogen, C. spicifer-CSN-20 by volatiles organic compounds emitted from A. terreus-F5-1008, F. solani-F4-1007, P. verrucosum-F2-1006 growing in sealed
plates (a) with C. spicifer-CSN-20 to the left; the inhibition percentage of the pathogen by endophytes (b).
0
5
10
15
20
25
30
35
40
A. terreus F. solani P. verrucosum
Pathogen Inhibition %
*
a
b
Fig. 6. Synergy between volatile and non-volatile organic compounds through antagonistic activity of S. arghel fungi against the pathogen C. spicifer-CSN-20 (a). Histogram showing the
antagonism activityof S. arghel endophyticfungi against C. spicifer-CSN-20 (b), valuesare mean ± stander errors(SEs) of three independent replicates (n = 3). LetterA indicate significant
differences p< 0.05 (ANOVA after Tukeys test analysis).
F.F. Abdel-Motaal, N.M. Kamel, M.A. El-Sayed et al. Annals of Agricultural Sciences 67 (2022) xxx
8
pathogen, the F4-1007 fungus was most effective. Therefore, this endo-
phytic fungus was selected for the examination of its ability to protect
okra plants from C.spicifer-CSN-20 attack. Symptoms were observed
in endophyte-free plants at 25 days after inoculation with C.spicifer-
CSN-20, whereas the endophytic fungus Fusarium solani-F4-1007
completely protected the okra leaves and stems from rot disease
(Fig. 7).
Esuruoso et al. (1975) recorded eight seed-borne fungal diseases in
okra from Nigeria, none of which had previously been reported as a
seed-borne disease in okra. These fungi were A. alternata,Curvularia
lunata (Cochliobolus lunatus), and Cladosporium cladosporioides.
For disease management, the application of plant/herbal extracts,
which is an alternative to fungicides, and the use of trace elements
have to be utilized. Biocontrol agents blended with fungicides are prom-
ising for seed- and soil-borne disease management. Crop rotation and
many other cultural practices seem to demonstrate little effect on the
management of foliar disease.
Several studies showed that the interaction between plants and var-
ious endophytic fungi was associated with beneficial effects, such as
plant growth promotion and biocontrol potential against plant patho-
gens (Abdel-Motaal et al., 2020). In the present study, the above-
mentioned endophytic fungus, F4-1007, considerably inhibited the
growth of the phytopathogen C. spicifer-CSN-20 in vitro. It was also ef-
fective as a biological control agent against this okra pathogen. Previous
studies reported that several antagonistic specieshave been confirmed to
be effective as biocontrol agents in controlled laboratory conditions (Zhao
et al., 2011), such as Penicillium species (Sabuquillo et al., 2006), Aspergil-
lus species (Abdel-Motaal et al., 2020), and F. solani, which were isolated
from cotton plants as endophytic fungi (Wei et al., 2019). F. verticillioides
was also reported to reduce the aggressiveness of the Ustilago maydis
pathogen in maize (Rodriguez Estrada et al., 2012).
4. Conclusion
In this study, the causal pathogen of okra rot disease was identified
as C. spicifer-CSN-20 based on morphological and molecular character-
izations. This is the first report of C. spicifer as a phytopathogenic fungus
in okra plants. To identify a suitable biological control for this serious
disease, the antifungal activities of the VOCs and n-VOCs produced by
the beneficial endophytic fungi A. terreus-F5-1008, F. solani-F4-1007,
and P. verrucosum-F2-1006 isolated from the medicinal plant S. arghel
were examined against C. spicifer. Our results clearly showed a strong
inhibitory effect of VOCs and n-VOCs against C. spicifer.Specifically,
VOCs and n-VOCs from F. solani-F4-1007 exhibited the highest inhibi-
tion percentage against C. spicifer rot disease compared with other en-
dophytic fungi. In addition, F. solani-F4-1007-treated okra plants
showed no disease symptoms compared with F. solani-F4-1007-
non-treated okra plants when inoculated with the C. spicifer patho-
gen. Our results indicated that the F. solani-F4-1007 endophyte is a
potential and effective biocontrol agent that can manage okra rot
diseasethroughthesecretionofvarious bioactive VOCs and n-
VOCs.TreatmentsofokraseedswithF. solani-F4-1007 endophyte
spores or VOC or n-VOC extracts can be an alternative cost-
effective approach for the control of okra rot disease. Further studies
will be conducted to examine the interaction mechanisms between
arghel endophytes and the okra pathogen C. spicifer and whether
these endophytes produce mycotoxins as a defensive strategy
against the C. spicifer pathogen. In addition, we will examine the ef-
fects of endophyte treatments on the nutritional values of okra
plants, as an important issue for the consumer.
Funding sources
There is no fund for this research.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
We are grateful to Prof. Abouelhamd Hassan Mohamed, Chemistry
department, Faculty of Science, Aswan University for his valuable advice
in drawing the compound structure to accomplish this work.
Fig. 7. Effectof the fungal endophyte F.solani on Okra plant protection by treated the plant with theendophyte and then spraying withpathogen comparing with plant sprayedonly with
the pathogen C.spicifer-CSN-20 causing rot disease and that plant sprayed with sterilized distilled water and not treated with endophyte (control).
F.F. Abdel-Motaal, N.M. Kamel, M.A. El-Sayed et al. Annals of Agricultural Sciences 67 (2022) xxx
9
References
Abdel-Motaal, F.F., Nassar, M.S., El-Zayat, S.A., El-Sayed, M.A., Ito, S., 2009. Responses of
fungi to tropane alkaloids produced by a medicinal plant Hyoscyamus muticus
(Egyptian henbane). Folia Microbiol. 54, 207–212.
Abdel-Motaal, F.F.,El-Sayed, M., El-Zayat, S., Nassar, M.M.,Ito, S., 2010. Choanephora rot of
floral tops of Hyoscyamus muticus caused by Choanephora cucurb itarum. J. Gen.
Plant Pathol. 76, 358–361.
Abdel-Motaal, F., Kamel, N., El-Zayat, S., Abou-Ellail, M., 2020. Early blight suppression
and plant growth promotion potential of the endophyte aspergillus flavus in tomato
plant. Ann. Agric. Sci. 65, 117–123.
Abu-Odeh, A.M., Talib, W.H., 2021. Middle East medicinal plants in the treatment of dia-
betes: a review. Molecules 26, 742. https://doi.org/10.3390/molecules26030742.
Akshatha, J.V., Prakash, H.S., Nalini, M.S., 2016. Actinomycete endophytes from the ethno
medicinal plants of southern India: antioxidant activity and characterization studies.
J. Biol. Act. Prod. Nat. 6, 166–172.
Al-Rashdi, K.H.F., Al-Sadi, M.A., Al-Riyamy, Z.B., Maharachchikumbura, S.N.S., Al-Sabahi,
N.J., Velazhahan, R., 2020. Endophytic fungi from the medicinal plant Aloe
dhufarensis lavranos exhibit antagonistic potential against phytopathogenic fungi.
S. Afr. J. Bot. https://doi.org/10.1016/j.sajb.2020.05.022 In press.
Alwathnani, A.H., Perveen, K., 2012. Biological control of fusarium wilt of tomato by an-
tagonist fungi and cyanobacteria. Afr. J. Biotechnol. 11, 1100–1105.
Amaradasa, B.S., Amundsen, K., 2016. Transcriptome profiling of buffalograss challenged
with the leaf spot pathogen Curvularia inaequalis. Front. Plant Sci. 7, 715. https://
doi.org/10.3389/fpls.2016.00715.
Amer, M.S.,Barakat, K.M., Hassanein, A.E.A., 2019. Phthalate derivativesfrom marine Pen-
icillium decumbens and its synergetic effect against sepsis bacteria. Biointerface Res.
Appl. Chem. 9, 4070–4076.
Amudha, P.,Jayalakshmi, M., Pushpabharathi, N.,Vanitha, V., 2018. Identification of bioac-
tive components in Enhalus acoroides seagrass extract by gas chromatography–mass
spectrometry. Asian J. Pharm. Clin. Res. 11, 131–137.
Aparna, V., Dileep, K.V., Mandal, P.K., Karthe, P., Sadasivan, C., Haridas, M., 2012. Anti-
inflammatory property of n-hexadecanoic acid: structural evidence and kinetic as-
sessment. Chem. Biol. Drug Des. 80, 434–439.
Awan, A.Z., Shoaib, A., Khan, A.K., 2018. Variations in total phenolics and antioxidant en-
zymes cause phenotypic variability and differential resistant response in tomato ge-
notypes against early blight disease. Sci. Hort. 239, 216–223.
Awasthi, L.P., 2015. Recent Advances in theDiagnosis and Managementof Plant Diseases.
Springer, New Delhi.
Balachandar, R., Karmegam, N., Saravanan, M., Subbaiya, R., Gurumoorthy, P., 2018. Syn-
thesis of bioactive compounds from vermicast isolated actinomycetes species and
its antimicrobial activity against human pathogenic bacteria. Microb. Pathog. 121,
155–165.
Balouiri, M., Sadiki,M., Ibnsouda, K.S., 2016. Methodsfor in vitro evaluating antimicrobial
activity: a review. J. Pharm. Anal. 6, 71–79.
Bartlett, D.W., Clough, J.M., Godwin, J.R., Hall, A.A., Hamer, M., Parr-Dobrzanski, B., 2002.
The strobilurin fungicides. Pest Manag. Sci. 58, 649–662.
Berner, D.K., Smallwood, E.L., McMahon, M.B., Luster, D.G., 2007. First report of leaf spot
caused by Cladosporium herbarum on Centaurea solstitialis in Greece. Plant Dis. 91,
463. https://doi.org/10.1094/PDIS-91-4-0463A.
Chatterjee, S., Karmakar, A., Azmi, S.A., Barik, A., 2018. Antibacterial activity of long-chain
primary alcohols from Solena amplexicaulis leaves. Proc. Zool. Soc. 71, 313–319.
Christensen, M., Raber, B.K., 1978. Synoptickey to Aspergillus nidulans group species and
related Emericella species. Mycol. Res. 71, 177–191.
Ellis, M.B., 1976. More Dematiaceous Hyphomycetes. Commonwealth Agricultural Bu-
reau, Farnham, Surrey, UK.
Esuruoso, O.F., Ogundiran, S.A., Chheda, H.R., Fatokun, D.O., 1975. Seed-borne fungi and
some fungal diseases of okra in Nigeria. P J. Dis. Reptr. 59, 660–663.
Fahmy, N.M.,2020. Isolation andcharacterizationof Streptomyces sp.NMF76 with poten-
tial antimicrobial activity from mangrove sediment, Red Sea, Egypt. Egypt. J. Aquat.
Biol. Fish. 24, 479–495.
Farag, M.A., Ryu, C.M., Sumner, L.W., Paré, P.W., 2006. GC–MS SPME profiling of
rhizobacterial volatiles reveals prospective inducers of growth promotion and in-
duced systemic resistance in plants. Phytochemistry 7, 2262–2268.
Feng, Y., Huang, Y., Zhan, H., Bhatt, P., Chen, S., 2020. An overview of strobilurin fungicide
degradation: current status and future perspective. Front. Microbiol. 11, 389. https://
doi.org/10.3389/fmicb.2020.00389.
Ghuffar,S., Irshad, G., Naz, F.,Rosli, H.B., Hyder,S., Mehmood, N., Zeshan,M.A., Mayer, C.G.,
Gleason, M.L., 2018. First report of two Penicillium spp. causing post-harvest fruit rot
of grapes in Pakistan. Plant Dis. 104,1037. https://doi.org/10.1094/PDIS-10-17-1616-
PDN.
Gontia-Mishra, I.,Tripathi, N., Tiwari, S.A., 2014. Simpleand rapid DNA extractionprotocol
for filamentous fungi efficient for molecular studies. Indian J. Biotechnol. 13,
536–539.
Jaddoa, H.H., Imad, H.H., Ghaidaa, J.M., 2016. Analysis of volatile metabolites released by
Staphylococcus aureus using gas chromatography-mass spectrometry and determi-
nation of its antifungal activity. Orient. J. Chem. 4, 8–24.
Jukte, S.R.,Badgujar, S.L., Suryawanshi, A.P., Dey, U., Kuldhar, D.P., 2016. Symptomatology,
isolation, identification and pathogenicity test of damping off disease in okra. Int.
J. Plant Prot. 9, 358–361.
Kamel, N.M.,Abdel-Motaal, F.F.,El-Zayat, S.A., 2020. Endophytic fungi from the medicinal
herb Euphorbia geniculata as a potential source for bioactive metabolites. Arch.
Microbiol. 202, 247–255.
Kusari, S., Verma, V.C., Lamshoeft, M., Spiteller, M., 2012. An endophytic fungus from
Azadirachta indica a. Juss. that produces azadirachtin. World J. Microbiol. Biotechnol.
28, 1287–1294.
Kyekyeku, O.J., Kusari, S., Adosraku, K.R., Bullach, A., Golz, C., Strohmann, C., Spiteller, M.,
2017. Antibacterial secondary metabolites from an endophytic fungus, Fusarium
solani JK10. Fitoterapia 119, 108–114.
Lalitha, G., Nazeema, T.H., Anitha, P., 2019. GC-MS analysis of bioactive components on
the leaves extract of Elaeagnus conferta roxb. Int. Res. J. Pharm. 10, 83–89.
Lilley, B.D., Brewer, J.H., 1953. The selective antibacterial action of phenyl ethyl alcohol.
J. Am. Pharm. Assoc. 42, 6–8.
Mohamed, E., Kasem, A.M.M., El-khatib, A., 2020. Allelopathic potential of Egyptian halo-
phytes Arthrocnemum macrostachyum and Halocnemum strobilaceum from two
coastal areas. Allelopathy J. 50, 225–241.
Ofori, J., Tortoe, C., Agbenorhevi, J.K., 2020. Physicochemical and functional properties of
dried okra (Abelmoschus esculentus L.) seed flour. Food Sci. Nutr. 8, 4291–4296.
Ons, L., Bylemans, D., Thevissen, K., Cammue, B.P.A., 2020. Combining biocontrol agents
with chemical fungicides for integrated plant fungal disease control. Microorganisms
8, 1930. https://doi.org/10.3390/microorganisms8121930.
Puri, S.C., Verma, V., Amna, T., Qazi, G.N., Spiteller, M., 2005. An endophytic fungus from
nothapodytes foetida that produces camptothecin. J. Nat. Prod. 68, 1717–1719.
Ragavendran, C., Natarajan, D., 2015. Insecticidal potency of aspergillus terreus against
larvae and pupae of three mosquito sp ecies Anopheles stephensi, Culex
quinquefasciatus, and Aedes aegypti. Environ. Sci. Pollut. Res. 22, 17224–17237.
Ravi, L., Krishnan, K., 2017. Cytotoxic potential of N-hexadecanoic acid extracted from
Kigelia pinnata leaves. Asian J. Cell Biol. 12, 20–27.
Rodriguez Estrada, E.A., Jonkers, W., Kistler, H.C., May, G., 2012. Interactions between Fu-
sarium verticillioides,Ustilago maydis,andZea mays: an endophyte, a pathogen, and
their shared plant host. Fungal Genet. Biol. 49, 578–587.
Sabuquillo, P., De Cal, A., Melgarejo, P., 2006. Biocontrol of tomato wilt by Penicillium
oxalicum formulations in different crop conditions. Biol. Control 37, 256–265.
Singh, J., Tripathi, N.N., 1999. Inhibition of storage fungi of blackgram Vigna Mungo by
some essential oils. Flavour Fragr. J. 14, 1–4.
Sirilun, S., Chaiyasut, C., Sivama ruthi, S.B., Peerajan, S., Kumar, N., Kesika, P., 2017.
Phenethyl alcohol i s an effective non-traditional preservative agent for cosmetic
preparations. Asian J. Pharm. Clin. Res. 10, 129–133.
Strobel, G.A., Dirkse, E., Sears, J., Markworth, C., 2001. Volat ile antimicrobia ls from
muscodoralbus a novel endophytic fungus. Microbiol. 147, 2943–2950.
Suarez, M.B., Walsh, K., Boonham, N., 2005. Development of realtime PCR (Taq-Man) as-
says for the detection and quantification of Botrytis cinerea in planta. Plant Physiol.
Biochem. 43, 890–899.
Susanti, D., Awang, N.A., Qaralleh, H., Mohamed, H.S.I., Attoumani, N., 2013. Antimicrobial
activityand chemical composition of essential oil of Malaysian Etlingera elatior (Jack)
RM smith flowers. J. Essent. Oil-Bear. Plants 16, 294–299.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evo-
lutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.
Tariq, S., Khan, R., Sultana, V., Ara, J., Ehteshamul-Haque, S., 2009. Utilization of endo-root
fluorescent pseudomonas of chili for the management of root diseases of chili. Pak.
J. Bot. 41, 3191–3198.
Wei, F., Zhang, Y., Shi, Y., Feng, H., Zhao, L., Feng, Z., Zhu, H., 2019. Evaluation of the bio-
control potential of endophytic fungus Fusarium solani CEF559 against Verticillium
dahliae in cotton plant. Biome d Res. Int. 2019, 3187943. https://doi.org/10.1155/
2019/3187943.
Zhao, Q., Dong, C., Yang, X., Mei, X., Ran, W., Shen, Q., Xu, Y., 2011. Biocontrol of Fusarium
wilt disease for Cucumis Melo melon using bio-organic fertilizer. Appl. Soil Ecol. 47,
67–75.
Zheng, R., Li, S., Zhang, X., Zhao, C., 2021. Biological activities of some new secondary me-
tabolites isolated fr om endophytic fungi: a review study. Int. J. Mol. Sci. 22, 959.
https://doi.org/10.3390/ijms22020959.
F.F. Abdel-Motaal, N.M. Kamel, M.A. El-Sayed et al. Annals of Agricultural Sciences 67 (2022) xxx
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