Novel Research in Microbiology Journal (2023), 7(5): 2138-2151 (Print) (ISSN 2537-0286)

Research Article (Online) (ISSN 2537-0294)

https://nrmj.journals.ekb.eg/
DOI: 10.21608/nrmj.2023.318807

Antifungal activity of essential oils emulsions, their bioactive compounds, and biological
control of Fusarium wilt of Majorana hortensis

Ali, A.A.M.1*; Hassanin, M.M.H.2; Abdel-Rahman, T.F.M.2; Mergawy, M.M.3

1
Plant Pathology Department, Faculty of Agriculture and Natural Resources, Aswan University, Aswan, Egypt;
2
Plant Pathology Research Institute, Agricultural Research Center (ARC), Giza, Egypt; 3Central Laboratory of
Organic Agriculture (CLOA), Agricultural Research Center (ARC), Giza, Egypt
*
Corresponding author E-mail: abdallahali@agr.aswu.edu.eg

Received: 29 August, 2023; Accepted: 24 September, 2023; Published online: 27 September, 2023

Abstract

This investigation was conducted to evaluate the activity of some essential oils emulsions
and a biocide (Bio-Cure F) for controlling Fusarium wilt disease of marjoram (Majorana
hortensis) caused by Fusarium oxysporum. The fungal filtrate of F. oxysporum isolated from
the infected marjoram plants significantly decreased the marjoram seed germination and
caused wilting of its seedlings. The essential oil emulsions of thyme; basil, and marjoram,
Copyright policy inhibited the in vitro fungal growth of F. oxysporum, and significantly suppressed its
sporulation and spore germination. However, thyme oil was the most effective one, which
NRMJ allows the
author(s) to hold the
completely inhibited the fungal growth at a concentration of 2000 µg/ ml, and completely
copyright, and to retain prevented its sporulation and spore germination. The tested essential oils were analysed for
publishing rights without their bioactive components using Gas Chromatography (GC), which were recorded as follows:
any restrictions. This
work is licensed under the
linalool and eugenol were detected in basil; terpinen-4-ol, β-phyllandrene, and sabinene in
terms and conditions of marjoram oil, and thymol, 1.8-cineole, ρ-cymene, and α-terpinene in thyme oil. In the
the Creative Commons greenhouse experiments, Actamyl 70 % wp (wettable powder) and Bio Cure F were the most
Attribution (CC BY)
license effective treatments, which significantly decreased the disease incidence and increased the
(https://creativecommons. plant growth parameters, compared with the untreated control. On the other hand, all
org/licenses/by/4.0/)
treatments increased the peroxidase and polyphenol oxidase antioxidant enzyme activities,
compared with the control; however, Actamyl 70 % (wp) and Bio Cure F had the superiority;
recording 1.27 and 1.23 (units/ ml enzyme) for peroxidase, and 0.57 and 0.51 (units/ ml
enzyme) for polyphenol oxidase, respectively. The results of the present study indicated the
strong antifungal potential of the thyme essential oil. Meanwhile, Bio-Cur F biocide can be
effectively used as a potent biofungicide to controlling the marjoram wilt incited by F.
oxysporum.

Keywords: Marjoram, Bio-Cur F, Thyme essential oil, Fusarium oxysporum

2138
Novel Research in Microbiology Journal, 2023
 Ali et al., 2023

1. Introduction

Marjoram (Majorana hortensis L.), which belongs recorded in the plant essential oils (Singh et al., 2007;
to the family Labiatae, has many different uses, and is Wang et al., 2009). These plant-derived compounds
considered as one of the most important medicinal and have antibacterial and antifungal potentials (Koga et
aromatic crops in Egypt. It is used in the food and al., 1999; Ahmad et al., 2010). Thyme essential oil
pharmaceutical industries, such as tooth pastes and and the water extract of licorice have high inhibitory
cough medicine, and as a carminative in the case of efficacy against F. oxysporum; Colletotrichum capsici,
digestive disorders (Bellakhdar et al., 1988). F. and Pythium aphanidermatum, which infect Capsicum
oxysporum caused wilt and root-rot in the Egyptian annuum L. (Arora et al., 2023). Moreover, thyme
marjoram plantations; affecting the plant stand and essential oil has inhibited the germination of spores
growth (Hilal et al., 1990; El-Gebaly, 1998). The most and mycelial growth of Alternaria linariae; the causal
dangerous fungus; F. oxysporum, affect the marjoram agent of the early blight disease of tomato (Saltos-
seedlings and cause pre- and post-emergence Rezabala et al., 2022).
damping-off, in addition to wilt and root rot disease in
several plants (Hassanin, 2007). A previous study Biological control of the fungal plant diseases is
conducted by El-Garhy, (1994) reported that the considered as an effective alternative strategy to the
fungal filtrate of F. oxysporum made the veins of lentil chemical control (El-Rafai et al., 2003). The efficacy
leaves brown; thus causing necrotic spots. of the biocontrol agents against the plant pathogens
has been reported by many researchers (Lebeda and
In the open fields, the fungal pathogens have Cohen, 2011). Several studies attributed the protection
developed resistance to the chemical fungicides; as a effects of the biocontrol agents to antibiosis in the
result of the long-term application of these fungicides infection courts (Matei and Matei, 2008). Among the
(Brent and Hollomon, 1998). There are several adverse most common mechanisms used by the biocontrol
environmental effects associated with the intensive agents to controlling the plant pathogenic fungi are
application of fungicides, including teratogens and mycoparasitism; competition for nutrients and space,
carcinogens; high toxicity, negative effects on the stimulating the plant's defense, and the release of
humans, and pollution of the environment (Pathak et bioactive compounds (De Curtis et al., 2010). In a
al., 2022). Thus, there is an urgent need for the recent study conducted by Rahman et al., (2023), the
development of ecofriendly antifungal agents. tested isolates of Trichoderma harzianum inhibited the
bacterial wilt disease; reduced the disease incidence,
Among the benefits of the essential oils are that and enhanced the tomato plant growth.
they are less toxic to the humans and the environment,
as well as that they contain a variety of antimicrobial The objective of the present work was to evaluate
compounds (Hammer et al., 2003). ρ-cymene; 1,8- the effect of some essential oils emulsions and a
cineole, and other thymol constituents are primarily biocide (Bio-Cure F) on controlling the marjoram wilt
responsible for the anti-fungal activity of thyme disease incited by F. oxysporum.
essential oil against some fungal species, including
Aspergillus; Penicillium, Alternaria, Cladosporium, 2. Materials and methods
and Rhizopus (Šegvić Klarić et al., 2007). The bio-
active components such as pinene; limonene, cineole, 2.1. The pathogenic F. oxysporum isolate
eugenol, terpineol, geraniol, thymol, menthol, and
The F. oxysporum isolate used in this study, which
terpinene are among the bioactive compounds
was isolated from wilted marjoram seedlings (sweet

2139
Novel Research in Microbiology Journal, 2023
 Ali et al., 2023

marjoram); was kindly provided by the fungal 40, 60, 80, and 100 %) and the control (un-inoculated
collection of the Ornamental, Medicinal and Aromatic medium). Thirty-day-old healthy marjoram seedlings
Plant Diseases, Plant Pathology Research Institute, (10 cm in height) were planted in each vial; five
ARC, Giza, Egypt. seedlings/ vial and three vials were used for each
treatment and for the control. All vials were kept at 28-
2.2. Effect of F. oxysporum filtrate on marjoram 30 °C for 7 d in the growth chamber. The seedlings
seed germination were watered whenever needed. The percentages (%)
As reported by Hassanin, (2007), a five-mm disc of wilted seedlings were recorded 72 h after
was cut from a ten-day-old active growing culture of inoculation.
F. oxysporum ing a erile cor orer, and inoc la ed 2.4. Preparation of essential oils emulsions
a ep ically in o a 5 -ml rlenmeyer fla con aining
ml of ape ro h medi m. he n-inoculated Basil; marjoram, and thyme essential oils were
flasks served a he con rol . he inoc la ed and obtained from the Medicinal Plants Research
con rol fla ere inc a ed a 7 for 5 d. f er Department, Horticultural Research Institute, ARC,
inc a ion, a ha man o. fil er paper a ed o Giza, Egypt. The oil emulsions were prepared as
filtrate the growing culture; followed by sterilization reported by Hassanin et al., (2017). About 5 ml of the
of the obtained filtrate using a Millipore filter (0.45 non-ionic surfactant Tween 80 was added slowly to 10
µm). Using sterile dist. water, the culture filtrate was ml of each essential oil; followed by gentle stirring
prepared at five concentrations; mainly 20; 40, 60, 80, until a homogeneous mixture was obtained. Sterilized
and 100 %. The marjoram seeds (i.e., sweet marjoram dist. water was added to reach a final volume of 100
seeds obtained from the Medicinal and Aromatic ml, and then stirred for 30 min. using a magnetic
Plants Department, Horticulture Research Institute, stirrer (Hotplate Magnetic Stirrer, model: JSHS-18D,
ARC, Giza, Egypt) were surface sterilized for 2 min. JS Research company, Gongju-City, Korea).
using 1% sodium hypochlorite, and then washed twice
with sterile dist. water. The sterilized seeds were then 2.5. Evaluation of the in vitro antifungal potential of
placed aseptically on a sterilized filter paper in a petri the essential oils emulsions against F. oxysporum
plate (10 seeds/ plate), and then 5 ml of each fungal
2.5.1. Effect of oil emulsions on the mycelial growth
filtrate concentration/ plate was added aseptically to
of F. oxysporum
the seeds. The control plates involved the addition of 5
ml of sterile dist. water. Three replicate plates were The method described by Zambonelli et al.,
used for each treatment. The plates were incubated at (1996); Hassanin et al., (2017) was applied with a
27 °C for 10 d, and 3 ml/ plate of sterile dist. water slight modification. The oil emulsions of basil;
was added aseptically to each plate after 5 d of marjoram, and thyme were added individually to
incubation, to maintain a proper level of humidity potato dextrose agar (PDA) medium before
(Hassanin, 2007). In all treatments, the level of solidification at five concentrations; mainly 1000;
marjoram seed germination (%) was recorded. 2000, 3000, 4000, and 5000 µg/ ml (v/v).
Chloramphenicol antibiotic (0.1 mg/ l) was also added
2.3. Effect of the fungal filtrate on the percentages to prevent bacterial contamination. Three plates were
of wilted seedlings used for each treatment and for the untreated control
This experiment was carried out as reported by (without oil emulsion). A single disc (5-mm) of F.
Hassanin, (2007). Glass vials (i.e., 30 ml small bottles) oxysporum was added aseptically at the center of each
were used, which contained the previously prepared plate and incubated at 27 °C. After incubation, the
individual concentrations of the fungal filtrate (i.e., 20; inhibition (%) of the fungal growth was calculated
when the control plates were completely covered with

2140
Novel Research in Microbiology Journal, 2023
 Ali et al., 2023

the fungal growth; according to the formula defined by Thyme essential oil emulsion at a rate of 2 ml/ l; a
Ahmed, (2013) as follows: fungicide Actamyl 70 % wp at the rate of 1 g/ l (i.e.,
Common name: Thiophanate methyl, Arysta Life
Inhibition (%) = (G1-G2)/ G1×100 Science SAS, France), and a biocide Bio-Cure F 1.15
Where; G1= Linear growth of the fungus in control % wp (T. viride 1 × 106 cfu/ g) (M/S.T. Stanes
plate, G2 = Linear growth of the fungus in the treated plate Company, Limit-India) at the rate of 6 g/ l, were
evaluated for their abilities to controlling Fusarium
2.5.2. Effect of the oil emulsions on the fungal wilt of marjoram in the greenhouse.
sporulation
For preparation of the F. oxysporum inoculum, the
Using a sterile needle, spores of F. oxysporum fungus was grown on an autoclaved sorghum grain
were harvested from the colonies' edge after 7 days of medium (50 g washed sand + 100 g corn + 100 ml
incubation at 27 °C in PDA plates, which treated dist. water) in 500 ml glass bottles; followed by
individually with an oil emulsion at a concentration of incubation for 15 d at 27 °C. For cultivation of the
3000 µg/ ml, and from the untreated control plates. marjoram seedlings, 25-cm-diameter plastic pots that
These spores were inoculated into test tubes containing were filled with formalin sterilized soil (equal amounts
10 ml of sterile dist. water. The tubes were centrifuged of sand; peat moss, and clay) were used, and these
at 1000 rpm for 3 min. to disperse the spores, and soils were infested individually with F. oxysporum
finally a drop of the resulting spore suspension was inocula at the rate of 1 % (w/w). Throughout the
counted using a hemocytometer, in reference to experiment, 60 d old seedling roots (5 seedlings/ pot)
Hassanin, (2013). were dipped for 20 min. in the three previously
prepared treatments or in water only (as a control) and
2.6. Biochemical analysis of the tested essential oils
then planted in the infested soil; with 3 replicate pots
using Gas Chromatography
for each treatment. After 90 d from planting, the
The biochemical constituents of the tested oil percentages of disease incidence (%); survival of
emulsions were determined at the Laboratories of the plants, plant height (cm), root length (cm), and plant
National Research Centre (NRC), Giza, Egypt; using fresh and dry weight (g) were recorded. The disease
Gas Chromatography (GC) technique, as described by incidence percentages (%) were recorded according to
Elhalawany et al., (2019). In this method, GC was the formula reported by Ahmed, (2013) as follows:
performed using a Ds Chrome 6200 Gas Number of infected plants
Chromatograph. The machine was composed of a Disease incidence (%) = -------------------------------- × 100
flame ionization detector; a 5 % phenyl Total plant numbers
polysillphenylene-siloxane (30 m × 0.25 mm ID ×
. 5 μm film hic ne ), and ol mn: BPX-5. The
The percentages (%) of plant survival were determined
temperature program ranged from 70 to 200 °C at an
by subtracting the number of living plants by the initial
average of 10 °C/ min., while the used temperature for
number of the cultivated plants. The dry weight of
the detector was 280 °C. Nitrogen flowed at the rate of
marjoram plants was determined by drying them in an
30 ml/ min.; hydrogen at 30 ml/ min., and air at 300
oven (40 °C) overnight. The plants were then left to
ml/ min.
cool and weighed using a scale.
2.7. Evaluation of the in vivo inhibitory efficacy of
2.8. Determination of activities of the defense-
the essential oils emulsions and the biocide against
related enzymes in the treated marjoram plants
Fusarium wilt of marjoram under greenhouse
conditions

2141
Novel Research in Microbiology Journal, 2023
 Ali et al., 2023

To measure the activity of the peroxidase (POD) 3. Results

and polyphenoloxidase (PPO) antioxidant enzymes in
the treated marjoram plants; approximately 72 h after 3.1. F. oxysporum filtrate effect's on the marjoram
each treatment, fresh samples of marjoram plants plant
(approximately 10 g/ treatment) were collected and
3.1.1 Effect of F. oxysporum filtrate on the
ground in 10 ml of 0.1 M sodium phosphate buffer
marjoram seed germination
(pH 6.8) using a mortar and pestle. Through four
layers of cheesecloth; the samples were filtered, the Data demonstrated in Fig. (1) show the effect of
filtrates were centrifuged for 20 min. at 6 ºC at 3000 five concentrations of F. oxysporum filtrate on
rpm (Aina et al., 2012), and the resulting filtrates were marjoram seed germination. The inhibition
used for enzyme analyses. percentages for seed germination ranged from 53.3 % -
100 %, which were significantly increased by
2.8.1. Peroxidase (POD) activity
increasing the filtrate's concentration. On the other
Pyrogallol's oxidation to purpurogallin in the hand, complete inhibition of seed germination was
presence of H2O2 was measured obtained upon using a 60 % concentration of the
spectrophotometrically at 425 nm to determine the fungal filtrate.
peroxidase enzyme's activity. The reaction mixture
3.1.2. Effect of F. oxysporum filtrate on the
was composed of 0.3 ml of the sample filtrate; 0.1 ml
percentages of wilted seedlings
of 1 % H2O2, 0.5 ml of 0.1 M sodium phosphate buffer
solution (pH 7.0), and 0.3 ml of 0.05 M pyrogallol. The results presented in Fig. (1) demonstrate that
The remaining 3 ml were made up with dist. water. the F. oxysporum fungal filtrate induced wilt
The absorbance change per min. (Abs/ min.) was used symptoms in the marjoram seedlings after 72 h of
to measure the POD enzyme activity (Aina et al., treatment; as it raised the percentage of wilted
2012). seedlings compared with the control. The percentages
of wilted seedlings were shown to be positively
2.8.2. Polyphenol oxidase (PPO) activity
correlated with the fungal filtrate concentration,
The activity of the PPO enzyme was determined whereas a concentration of 100 % resulted in 100 % of
colorimetrically using the previous method described wilted seedlings.
by Quiles et al., (2005). The reaction mixture
3.2. Effect of essential oils emulsions on the in vitro
consisted of 1 ml of the sample filtrate; 1 ml of 103 N
growth of F. oxysporum
Catechol, and 1 ml of 0.2 M sodium phosphate buffer
(pH 7.0). The remaining 6 ml were made up with dist. The data presented in Table (1) proved that
water. The change in absorbance of the reaction increasing the concentration of the essential oils was
mixture per min. at 495 nm was used to measure the correlated with a decrease in the linear fungal growth.
PPO enzyme activity. Compared with the control, the most effective oil
emulsion against F. oxysporum was thyme at a
2.9. Statistical analysis
concentration of 2000 µg/ ml, which completely
Using a total randomized design with three inhibited the growth; followed by basil at a
replicates per treatment, this study was designed as a concentration of 4000 µg/ ml, while the marjoram oil
factorial experiment (Snedecor and Cochran, 1989). completely inhibited the fungal growth at 5000 µg/ ml.
Based on the Least Significant Difference (L.S.D) test Finally, the oil emulsions of basil; marjoram, and
at 0.05, this statistical analysis was conducted using thyme completely inhibited the growth of F.
MSTAT-C version (4) statistical package. oxysporum at a concentration of 5000 µg/ ml.

2142
Novel Research in Microbiology Journal, 2023
 Ali et al., 2023

120
100 100 100 100
100
Effect on marjorum plant

86.7
80
80
60
60 53.3
% inhibition of
46.7
seed
40 germination
% of Wilted
20 seedlings
20
0 0
0
0 20 40 60 80 100
% Fungal filtrate concentrations

Fig. 1. Effect of F. oxysporum filtrate's on seed germination and wilted seedlings of marjoram, compared with the control; after
72 h of inoculation. The Error bars represent the standard deviation (SD)

Table 1. Effect of different concentrations of the essential oils emulsions on the in vitro mycelial growth of F.
oxysporum

Essential oils Different concentrations (µg/ ml) of the oils emulsions

emulsions
Control 1000 2000 3000 4000 5000
a b a b a b a b a b a b
Basil 9.0 0.0 5.8 35.55 3.2 64.44 1.7 81.11 0.0 100 0.0 100

Marjoram 9.0 0.0 9.0 0.0 7.6 15.55 5.0 44.44 2.8 68.88 0.0 100
Thyme 9.0 0.0 2.8 68.88 0.0 100 0.0 100 0.0 100 0.0 100

Where; (a): represents the diameter of linear growth (cm); while (b): represents the inhibition (%) of mycelial growth. L.S.D. at
5 %: Essential oils (E) = 1, Concentrations (C) = 1.1, Essential oils (E) × Concentrations (C) = 1.8

3.3. Effect of essential oils emulsions on F. and spore germination of F. oxysporum was thyme,
oxysporum sporulation in vitro which completely prevented both of them. This was
followed by basil that reduced the spore number of the
The results presented in Fig. (2) and Table (2) fungus to 40 ×103 spores and inhibited the spore's
demonstrate that the tested oils emulsions significantly germination by 44.8 %, compared with the untreated
suppressed the F. oxysporum sporulation and spore control. On the other hand, marjoram was the least
germination at 3000 µg/ ml, compared with the effective oil on the sporulation (110 × 103 spores) and
control. However, the most effective oil on sporulation spore germination inhibition (32.5 %) of the fungus.

2143
Novel Research in Microbiology Journal, 2023
 Ali et al., 2023

Fig. 2. Effect of essential oil emulsions of basil and marjoram at a concentration of 3000 µg/ ml on the sporulation of F.
oxysporum after 7 d of incubation at 27 °C

Table 2. Effect of essential oils emulsions at a concentration of 3000 µg/ ml on sporulation and spore germination
of F. oxysporum after 7 d of incubation at 27 °C

Tested essential oils Mean sporulation (×103) % Inhibition of spore

emulsions of F. oxysporum germination
Basil 40 44.8
Marjoram 110 32.5
Thyme 0 100.0
Control 295 00.0
L.S.D. at 5 % 20.2 0.1

3.4. Chemical composition of the tested essential of the marjoram essential oil were terpinen-4-ol
oils (35.6 9 %); β-phyllandrene (12.033 %), and sabinene
(11.34 7%); along with lower quantities of Thujon-4-ol
Using GC technique, the results presented in (8.215 %), linalyl acetate (7.923 %), limonene (6.843
Table (3) show that the major components of basil %), α-pinene (5.67 %), and other components.
essential oil were linalool (46.13 %) and eugenol
(18.41 %), in addition to smaller amounts of myrcene 3.5. Evaluation of the biocide and essential oils
(3.94 %); α- erpineol (6. 3 %), and α-pinene (4.76 %). emulsions effectiveness in controlling Fusarium wilt
Among thyme essential oil's main compounds, it of marjoram under greenhouse conditions
contained thymol (27.945 %); 1.8-cineole (16.34 %),
ρ-cymene ( 5.744 %), and α-terpinene (10.983 %). In The results showed that all the tested treatments
addition, little amounts of limonene (5.729 %), decreased the percentages of disease incidence and
myrcene (4.68 %), γ-terpineol (4.184 %), and broneol recorded higher percentages of plant survival (Fig. 3,
(4.179 %) were detected. The three main components Table 4). However, compared with the control; as they

2144
Novel Research in Microbiology Journal, 2023
 Ali et al., 2023

Table 3. Chemical composition of the essential oils (EO) detected using GC

Basil oil Thyme oil Marjoram oil

Component Area % Component Area % Component Area %

α-pinene 4.76 α-pinene 1.356 Thujene 0.974

Myrcene 3.94 Camphene 2.187 α-pinene 5.67
β-pinene 2.76 Sabinene 3.122 Sabinene 11.347
Linalool 46.13 Myrcene 4.682 β-phyllandrene 12.033
Camphor 0.964 α-terpinene 10.983 Limonene 6.843
γ-terpineol 1.023 ρ-cymene 15.744 Linalool 2.512
Terpinen-4-ol 1.94 Limonene 5.729 Linalyl acetate 7.923
α-terpineol 6.23 1.8-cineole 16.34 Terpinen-4-ol 35.619
Methyl chavicol 2.45 γ-terpineol 4.184 α-terpineol 2.107
Fenchyl acetate 2.189 Broneol 4.179 Thujon-4-ol 8.215
Geraniol 2.776 Thymol 27.945 β-caryophyllene 3.737
Eugenol 18.41 β-caryophyllene 1.628
β-caryophyllene 2.56
Total 96.132 Total 98.079 Total 96.98

significantly reduced the disease incidence, Actamyl The results listed in Fig. 4 reveal that all treatments
70 % wp and Bio Cure F were the most effective increased the activity of the POD and PPO enzymes,
evaluated treatments; recording 6.7 % and 20 % which ranged from 0.91 to 1.27 (units/ ml enzyme)
disease incidence, respectively. On the contrary, thyme and from 0.24 to 0.57(units/ ml enzyme); respectively,
essential oil was the least effective treatment in compared with the untreated control that recorded 0.38
reducing the disease incidence (33.3 %). Compared and 0.11 (units/ ml enzyme), respectively. However,
with the controls, Table (4) demonstrates that all Actamyl 70 % (wp) and Bio Cure F biocide treatments
treatments increased the plant growth parameters, but resulted in the highest increase in activities of the
Actamyl 70 % (wp) and Bio Cure F showed the POD, which recorded 1.27 and 1.23 (units/ ml
highest increases in plants height (27 and 24.6 cm, enzyme); respectively, and PPO that recorded 0.57 and
respectively); roots length (12 and 9.3 cm, 0.51 (units/ ml enzyme), respectively.
respectively), plants fresh weight (7.8 and 7.8 g,
respectively), and in the plants dry weight (4.1 and 4.4 4. Discussion
g, respectively). In contrast, thyme essential oil
Fusarium oxysporum filtrate significantly
expressed the least efficacy in this respect; recording
decreased the percentage of marjoram seed
23.7 cm, 7.7 cm, 6 g, and 3.5 g, respectively.
germination; with a positive correlation between the
3.6. Effect of the tested treatments on the activity of fungal filtrate concentration and the percentage
certain defense-related enzymes reduction of seed germination. Furthermore, the fungal

2145
Novel Research in Microbiology Journal, 2023
 Ali et al., 2023

Fig. 3. Impact of dipping the marjoram seedling roots in various treatments on the disease incidence (%) 90 d after being
planted in soil infested with pathogenic F. oxysporum

Table 4. Effect of different dipping treatments of marjoram seedling roots on the disease incidence (%) and some
plant growth parameters 90 d after planting in soil infested with F. oxysporum under greenhouse conditions

Disease Survivals Plant height Root length Plant fresh Plant dry
Treatments incidence (%) (cm) (cm) weight (g) weight (g)
(%)
Thyme
emulsion 33.3 66.7 23.7 7.7 6.0 3.5

Bio Cure F 20.0 80.0 24.6 9.3 7.8 4.4

Actamyl 70%
(wp) 6.7 93.3 27.0 12.0 7.8 4.1
Untreated
73.3 26.7 17.0 6.0 4.4 3.2
control
L.S.D. at 5% 19.2 --- 4.4 2.0 0.5 0.1

2146
Novel Research in Microbiology Journal, 2023
 Ali et al., 2023

1.6

Enzyme activity (units/ml enzyme)

1.4 1.27
1.23
1.2
1 0.91

0.8 Peroxidase
0.57 activity
0.6 0.51
Polyphenol
0.38
0.4 oxidase activity
0.24
0.2 0.11

0
Thyme essential Bio Cure F Actamyl 70% wp Control
oil Treatments and control

Fig. 4. Peroxidase (POD) and polyphenol oxidase (PPO) activity detected in the treated marjoram seedlings plants, compared
with the control. The Error bars represent the standard deviation (SD)

filtrate enhanced the percentage of wilted seedlings in addition to their capacity to penetrate the fungal
compared with the control. The fungal filtrate of F. cell, are the major reasons for the fungal growth
oxysporum caused toxic effects on the marjoram seeds inhibition. Additionally, a rise in the permeability of
and seedlings; in accordance with the study reported the fungal cell membrane may be the cause of the
by Hassanin, (2007), who revealed that seed observed antifungal effects of the tested essential oils.
germination (%) of marjoram significantly decreased The antifungal substances present in the medical oils
upon treatment with F. oxysporum filtrate, which was may inhibit the fungal sporulation by affecting the
in line with the fungal filtrate concentration. oxygen uptake of the fungal cell, as reported by
Moreover, the author also reported that marjoram Zambonelli et al., (1996); Šegvić Klarić et al., (2007).
seedlings exhibited wilt symptoms as a result of Furthermore, these authors added that the thyme oil's
growth in F. oxysporum fungal filtrate. Similar results activity might be attributed to penetration of the chitin
were also reported by El-Garhy (1994), who of the cell wall and damaging the lipoprotein of the
demonstrated that F. oxysporum culture filtrate cytoplasmic membrane; resulting in cytoplasmic
softened and browned the veins of lentil leaves; escapes. Similar to our obtained results, Arora et al.,
causing the development of necrotic spots. (2023) reported that thyme essential oil and the cold
extract of licorice expressed a highly antifungal
According to the in-vitro assays, thyme at 2000 potential against the tested fungal pathogens of C.
µg/ ml followed by basil at 4000 µg/ ml totally annuum L. At 1 mg/ ml, thyme essential oil caused
prevented the radial growth of F. oxysporum; making 100 % inhibition of the mycelial growth of F.
them the most effective essential oils against this oxysporum; C. capsici, and Pythium aphanidermatum.
pathogenic fungus. The tested essential oils also Furthermore, Saltos-Rezabala et al., (2022) reveled
significantly suppressed the sporulation and spore that application of the thyme essential oil caused an
germination of F. oxysporum; especially the thyme oil, increa e in he en yme ac ivi y of β-1,3-glucanase;
which entirely prevented it. It's possible that the polyphenol oxidase, and peroxidase, in addition to an
essential oils' phenolic compounds such as linalool in improvement in the accumulation of phenolic
basil; terpinen-4-ol in marjoram, and thymol in thyme, compounds and callose precipitation in the infected

2147
Novel Research in Microbiology Journal, 2023
 Ali et al., 2023

plant tissue. Generally, our findings are in accordance incidence (%). However, the most effective treatments
with those of Hassanin et al., (2017), who reported that were of Actamyl 70 % (wp) and Bio Cure F (biocide),
thyme; lemongrass, and basil essential oils completely which significantly decreased the disease incidence,
suppressed the sporulation of F. oxysporum f.sp. compared with the untreated control, and they
cumini. Similar to the current results, Hassanin, (2013) displayed the highest increases in the plant growth
reported that clove oil at 100 % completely inhibited parameters. Hassanin et al., (2020); Serag El-Din et
the sporulation and spore germination of F. oxysporum al., (2020); El-Kaed et al., (2021) reported similar
and F. semitectum. results on various tested crops under artificially or
naturally infested soils. Using a biocide may reduce
The chemical components of the tested essential the disease incidence (%) by producing plant growth
oils was detected using GC, which were recorded as regulators, which encourage the plant tolerance to the
follows: in the thyme oil, thymol (27.945 %); 1.8- microbial infection, as reported by Constantinescu et
cineole ( 6.34 %), ρ-cymene ( 5.744 %), and α- al., (2009). Furthermore, Bolar et al., (2000) reported
terpinene (10.983 %) were detected. Meanwhile, in the that T. harzianum produces several destructive
basil oil, linalool (46.13 %) and eugenol (18.41 %) enzymes such as chitinase, which degrades the fungal
were recorded; while terpinen-4-ol (35.6 9 %); β- pathogen cell wall. The biocide Bio-Cure F may also
phyllandrene (12.033 %), and sabinene (11.34 7%) have a greater impact on the disease incidence (%) by
were observed in the marjoram oil. These results are inducing resistance in the treated marjoram plants.
somewhat in agreement with the findings of Grigore et Another study conducted by Benhamou and Chet,
al., (2010), who performed a quantitative analysis of (1993) reported that Trichoderma spp. have many
the thyme volatile oil of Thymus vulgaris using GC inhibitory mechanisms against growth of the fungal
and a qualitative analysis using high performance thin pathogens via mycoparasitism. Recently, Rahman et
layer chromatography (HPTLC). The results reported al., (2023) highlighted that application of two isolates
that the volatile oil of Thymus vulgaris contained p- of T. harzianum NBG and T. harzianum MC2 resulted
cymene and thymol in high quantities. Pripdeevech et in a significant reduction in the bacterial wilt disease
al., (2010) highlighted that methyl chavicol (81.82 %); incidence of tomato caused by Ralstonia
β-(E)-ocimene ( .93 %), and α-(E)-bergamotene (2.45 solanacearum, in addition to an increase in the tomato
%) were shown to be the primary components in the yield by 54.49 %. All the tested treatments increased
Thai basil oil, whereas basil oil largely contained the antioxidant enzyme activities (i.e., peroxidase and
linalool (43.78 %); eugenol (13.66 %), and 1,8-cineole polyphenol oxidase); however, Actamyl 70 % (wp)
(10.18 %). According to the previous study conducted and Bio-Cure F were the best treatments, compared
by Kowalski et al., (2020), chromatographic analysis with the untreated control. Both agents expressed the
of the marjoram oil revealed the presence of terpinen- highest increase in the POD and PPO activities. These
4-ol (17.61 %) and trans-sabinene hydrate (22 %). data are in agreement with those obtained by
ddi ionally, γ- erpinene ( . 9 %); α-terpinene (6.61 Mahmoud et al., (2006), who revealed that the induced
%), α-terpineol (5.85 %), sabinene (5.51 %), and cis- resistance increases the activity of the oxidative
sabinene hydrate (5.39 %) were recorded in enzymes (i.e., POD and PPO) in the treated root
considerable amounts in the essential oil fraction. The tissues; along with the phenolic compounds
obtained results of the current study are also in accumulation. In accordance, Hassanin, (2013)
agreement with those of Šegvić Klarić et al., (2007); reported that plants grown from the black cumin seeds
Hassanin et al., (2017). that were treated with some fungicide-alternative
In the greenhouse experiments, all the tested materials may produce the antioxidant enzymes as a
treatments increased the plant survival (%) and plant kind of induced defense; since these treatments
growth parameters, and decreased the disease significantly increased the POD and PPO activities.

2148
Novel Research in Microbiology Journal, 2023
 Ali et al., 2023

Conclusion and characterization of pectin from peels of lemon

(Citrus limon), grape fruit (Citrus paradisi) and sweet
This study evaluated the effectiveness of some orange (Citrus sinensis). British Journal of
essential oil emulsions and a biocide (Bio-Cure F) for Pharmacology and Toxicology. 3(6): 259-262.
controlling the marjoram wilt disease incited by F.
oxysporum. According to the obtained results; thyme Arora, H.; Sharma, A. and Sharma, S. (2023).
essential oil has powerful antifungal properties, and Thyme essential oil fostering the efficacy of aqueous
the Bio-Cur F biocide may be used as an effective bio- extract of licorice against fungal phytopathogens of
fungicide to controlling the Fusarium wilt of Capsicum annuum L. Journal of Bioscience and
marjoram. Bioengineering. 135(6): 466-473.
https://doi.org/10.1016/j.jbiosc.2023.03.003
Acknowledgement
Bellakhdar, J.; Passannanti, S.; Paternostro, M.P.
None. and Piozzi, F. (1988). Constituents of Origanum
compactum. Planta Medica. 54(01): 94.
Conflict of interest https://doi.org/10.1055/s-2006-962358
The authors declare non-existence of any conflict of Benhamou, N. and Chet, I. (1993). Hyphal
interests. interaction between Trichoderma harzianum and
Rhizoctonia solani: Ultrastructure and gold
Ethical approval
Cytochemistry. Phytopathology. 83(10): 1062-1071.
Not applicable.
Bolar, J.P.; Norelli, J.L.; Wong, K.W.; Hayes,
Funding source C.K.; Harman, G.E. and Aldwinckle, H.S. (2000).
Expression of endochitinase from Trichoderma
No fund was received for this study. harzianum in transgenic apple increases resistance to
apple scab and reduces vigor. Phytopathology. 90(1):
Author's contributions 72-77. https://doi.org/10.1094/PHYTO.2000.90.1.72
All authors contributed equally in all parts of this Brent, K.J. and Hollomon, D.W. (1998). Fungicide
study. Resistance: The assessment of risk FRAC Monograph
No 2, Global Crop Protection Federation, Brussels, pp.
5. References
48.
Ahmad, A.; Khan, A.; Yousuf, S.; Khan, L.A. and
Constantinescu, F.; Voshol, G. and Bloemberg, G.
Manzoor, N. (2010). Proton translocating ATPase
(2009). Mechanisms involved in biocontrol activity of
mediated fungicidal activity of eugenol and thymol.
B. subtilis B49b against Fusarium oxysporum f. sp.
Fitoterapia. 81(8): 1157-1162.
radicis-lycopersici. IOBC/WPRS Bulletin. 43: 113-
https://doi.org/10.1016/j.fitote.2010.07.020
116.
Ahmed, M.F.A. (2013). Studies on Non-chemical
De Curtis, F.; Lima, G.; Vitullo, D. and De Cicco,
Methods to Control Some Soil-Borne Fungal Diseases
V. (2010). Biocontrol of Rhizoctonia solani and
of Bean Plants Phaseolus vulgaris L. Ph.D. Thesis.
Sclerotium rolfsii on tomato by delivering antagonistic
Faculty of Agriculture, Cairo University, pp. 137.
bacteria through a drip irrigation system. Crop
Aina, V.O.; Barau, M.M.; Mamman, O.A.; Zakari, Protection. 29(7): 663-670.
A.; Haruna, H.; Umar, M.H. et al. (2012). Extraction https://doi.org/10.1016/j.cropro.2010.01.012

2149
Novel Research in Microbiology Journal, 2023
 Ali et al., 2023

El-Garhy, A.M. (1994). Studies on root-rot and wilt Hassanin, M.M.H. (2007). Pathological studies on
of lentil. M.Sc. Thesis, Faculty of Agriculture Al- some important fungal diseases of marjoram and their
Azhar University, (Egypt). management. M.Sc. Thesis, Faculty of Agriculture Al-
Azhar University, (Egypt), pp. 97.
El-Gebaly, M.M. (1998). Studies on root-rot and wilt
disease of marjoram. Ph.D. Thesis, Faculty of Hassanin, M.M.H.; Nada, M.G. and Halawa, A.E.
Agriculture Al-Azhar University, (Egypt). (2020). Evaluation efficacy of some agents as safe
alternatives to fungicides in management root-rot and
Elhalawany, A.S.; Abou-Zaid, A.M. and Amer, A.I. wilt diseases of Artemisia absinthium and A.
(2019). Laboratory bioassay for the efficacy of santonicum. Egyptian Journal of Phytopathology.
coriander and rosemary extracted essential oils on the 48(1): 94-106.
citrus brown mite, Eutetranychus Orientalis https://doi.org/10.21608/ejp.2020.48284.1014
(Actinidida: Tetranychidae). Acarines: Journal of the
Egyptian Society of Acarology. 13(1): 15-20. Hassanin, M.M.H.; Abd-El-Sayed, M.A. and
https://doi.org/10.21608/AJESA.2019.164149 Abdallah, M.A. (2017). Antifungal activity of some
essential oil emulsions and nanoemulsions against
El-Kaed, S.A.; Mergawy, M.M. and Hassanin, Fusarium oxysporum pathogen affecting cumin and
M.M. (2021). Management of the most destructive geranium plants. Scientific Journal of Flowers and
diseases of chia plant and its impact on the yield. Ornamental Plants. 4(3): 245-258.
Egyptian Journal of Phytopathology. 49(1): 37-48. https://doi.org/10.21608/SJFOP.2017.11326
https://doi.org/10.21608/EJP.2021.61936.1023
Hilal, A.A.; Heweidy, M.A.; El-Deeb, A.A. and El-
El-Rafai, I.M.; Asswah, S.M. and Awdalla, O.A. Ghareeb, L.A. (1990). Root-rot and wilt diseases
(2003). Biocontrol of some tomato disease using some affecting marjoram in Egypt. Proc. 6 th Cong.
antagonistic microorganisms. Pakistan Journal of Egyptian Phytopathological Society, Cairo, Egypt, pp.
Biological Sciences. 6(4): 399- 406. 401-417.
https://doi.org/10.3923/pjbs.2003.399.406
Koga, T.; Hirota, N. and Takumi, K. (1999).
Grigore, A.; Paraschiv, I.N.A.; Colceru-Mihul, S.; Bactericidal activities of essential oils of basil and sage
Bubueanu, C.; Draghici, E. and Ichim, M. (2010). against a range of bacteria and the effect of these
Chemical composition and antioxidant activity of essential oils on Vibrio parahaemolyticus.
Thymus vulgaris L. volatile oil obtained by two Microbiological Research. 154(3): 267-273.
different methods. Romanian Biotechnological Letters. https://doi.org/10.1016/S0944-5013(99)80024-X
15(4): 5436-5443.
Kowalski, R.; Kowalska, G.; Pankiewicz, U.;
Hammer, K.A.; Carson, C.F. and Riley, T.V. Włodarczyk-Stasiak, M.; Sujka, M. and Mazurek,
(2003). Antifungal activity of the components of A. (2020). Effect of rapeseed oil aromatisation with
Melaleuca alternifolia (tea tree) oil. Journal of Applied marjoram on the content of volatile fraction and
Microbiology. 95(4): 853-860. antioxidant properties. Journal of Food Science and
https://doi.org/10.1046/j.1365-2672.2003.02059.x Technology. 57(3): 1138-1149.
Hassanin, M.M.H. (2013). Pathological studies on https://doi.org/10.1007/s13197-019-04149-y
root rot and wilt of black cumin (Nigella sativa) and Lebeda, A. and Cohen, Y. (2011). Cucurbit downy
their management in Egypt. Ph. D. Faculty of mildew (Pseudoperonospora cubensis)-biology,
Agriculture Al-Azhar University, (Egypt), pp. 137. ecology, epidemiology, host-pathogen interaction and

2150
Novel Research in Microbiology Journal, 2023
 Ali et al., 2023

control. European Journal of Plant Pathology. 129: Saltos-Rezabala, L.A.; Silveira, P.R.D.; Tavares,
157-192. https://doi.org/10.1007/s10658-010-9658-1 D.G.; Moreira, S.I.; Magalhães, T.A.; Botelho,
D.M.D.S. et al. (2022). Thyme essential oil reduces
Mahmoud, E.; Shokri, S.Y. and Hussin, Z.N. disease severity and induces resistance against
(2006). Induction of resistance in peanut plans against Alternaria linariae in tomato
root rot diseases under greenhouse conditions by some plants. Horticulture. 8(10): 919.
chemical inducers. Journal of Plant Production. 31(6): https://doi.org/10.3390/horticulturae8100919
3511- 3524. https://doi.org/10.21608/jpp.2006.236041
Šegvić Klarić, M.; Kosalec, I.; Mastelić, J.;
Matei, G.M. and Matei, S. (2008). Research on Piecková, E. and Pepeljnak, S. (2007). Antifungal
isolation, characterization and testing the interaction activity of thyme (Thymus vulgaris L.) essential oil
between Trichoderma harzianum and Botrytis cinerea and thymol against moulds from damp dwellings.
for biological control of grey mold in strawberry. Letters in Applied Microbiology. 44(1): 36-42.
Horticulture. 51: 653-657. https://doi.org/10.1111/j.1472-765X.2006.02032.x
Pathak, V.M.; Verma, V.K.; Rawat, B.S.; Kaur, B.; Serag El-Din, W.M.; Hassanin, M.M.H. and
Babu, N.; Sharma, A. et al. (2022). Current status of Halawa, A.E.A. (2020). Using alternative fungicides
pesticide effects on environment, human health and and fertilizers for management root rot and wilt
i ’ eco-friendly management as bioremediation: A diseases and their effect on productivity of rosemary
comprehensive review. Frontiers in Microbiology. pp. plant. Egyptian Journal of Horticulture. 47(2): 161-
2833. https://doi.org/10.3389/fmicb.2022.962619 180. https://doi.org/10.21608/ejoh.2020.47408.1148
Pripdeevech, P.; Chumpolsri, W.; Suttiarporn, P. Singh, G.; Maurya, S.; DeLampasona, M.P. and
and Wongpornchai, S. (2010). The chemical Catalan, C.A. (2007). A comparison of chemical,
composition and antioxidant activities of basil from antioxidant and antimicrobial studies of cinnamon leaf
Thailand using retention indices and comprehensive and bark volatile oils, oleoresins and their constituents.
two-dimensional gas chromatography. Journal of the Food and Chemical Toxicology. 45(9): 1650-1661.
Serbian Chemical Society. 75(11): 1503-1513. https://doi.org/10.1016/j.fct.2007.02.031
https://doi.org/10.2298/JSC100203125P
Snedecor, G.W. and Cochran, W.G. (1989).
Quiles, A.; Hernando, I.; Pérez‐Munuera, I.; Statistical Methods. 8th Edition. Iowa State University
Larrea, V.; Llorca, E. and Lluch, M.Á. (2005). Press, Ames, Iowa, USA.
Polyphenoloxidase (PPO) activity and osmotic
dehydration in Granny Smith apple. Journal of the Wang, R.; Wang, R. and Yang, B. (2009). Extraction
Science of Food and Agriculture. 85(6): 1017-1020. of essential oils from five cinnamon leaves and
https://doi.org/10.1002/jsfa.2062 identification of their volatile compound compositions.
Innovative Food Science & Emerging Technologies.
Rahman, M.; Borah, S.M.; Borah, P.K.; Bora, P.; 10(2): 289-292.
Sarmah, B.K.; Lal, M.K. et al. (2023). Deciphering https://doi.org/10.1016/j.ifset.2008.12.002
the antimicrobial activity of multifaceted rhizospheric
biocontrol agents of solanaceous crops viz., Zambonelli, A.; d'Aulerio, A.Z.; Bianchi, A. and
Trichoderma harzianum MC2, and Trichoderma Albasini, A. (1996). Effects of essential oils on
harzianum NBG. Frontiers in Plant Science. 14: phytopathogenic fungi in vitro. Journal of
1141506. https://doi.org/10.3389/fpls.2023.1141506 Phytopathology. 144(9-10): 491-
494. https://doi.org/10.1111/j.1439-
0434.1996.tb00330.x

2151
Novel Research in Microbiology Journal, 2023