Journal of Phytopathology and Pest Management 10(1): 14-25, 2023
pISSN: 2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗Corresponding author: Abdallah A. M. Ali,
E-mail: abdallahali@agr.aswu.edu.eg
14
Copyright © 2023
Morpho-molecular identification and management of Erysiphe
heraclei causing dill powdery mildew using a biocide, essential
oils, and organic acids
Mahmoud M. H. Hassanin1, Abdallah A. M. Ali2*, Heba Yousef1, Madian M. Mergawy3
1Plant Pathology Research Institute, Agricultural Research Centre, Giza, Egypt
2Plant Pathology Department, Faculty of Agriculture and Natural Resources, Aswan University, Aswan, Egypt
3Central Laboratory of Organic Agriculture (CLOA), Agricultural Research Center (ARC), Giza, Egypt
Abstract
Keywords: dill, Erysiphe heraclei, powdery mildew, Bio-Cur F, thyme essential oil, salicylic acid.
This study aimed to evaluate the efficacy of Bio-Cure F, essential oils of thyme
and clove, ascorbic and salicylic acids in controlling dill powdery mildew under
greenhouse conditions. The causal pathogen of powdery mildew was isolated from
infected dill plants showing symptoms of the disease during a survey in Fayoum
governorate, Egypt, in 2022. Erysiphe heraclei was morphologically characterized
using a light microscope as the causal pathogen of powdery mildew in infected dill
plants. The nucleotide sequence of the internal transcribed spacer (ITS) of the
causal pathogen DNA was deposited in GenBank (Accession No. OP999071.1),
which showed 98-100% similarity with accession numbers of E. heraclei isolates
obtained from the NCBI database. Under greenhouse conditions, dill plants were
sprayed with biocide (Bio-Cure F), elicitors (ascorbic and salicylic acids), and
essential oil emulsions of clove and thyme three times after inoculation with E.
heraclei. The results showed that Bio-Cure F was the treatment that showed the
highest efficacy in reducing both disease severity and incidence percentage,
followed by thyme essential oil. The highest percentages of increase in dill plant
height (cm) as well as branches/plant were obtained by Bio-Cure F treatment
compared to the control, followed by salicylic acid treatment. The results suggest
that Bio-Cure F, salicylic acid, and thyme oil emulsion could be used to control
dill powdery mildew.
Hassanin et al., 2023
15
1. Introduction
The dill plant (Anethum graveolens L.) is a
medicinal and aromatic plant that has a
prominent place in flavorings and belongs to
the Apiaceae (Umbelliferea) family. All plant
green foliage may be used to make salads,
soups, pickles, and fish meals. Dill plants in
different regions are affected by a significant
foliar disease known as powdery mildew. It is
caused by E. heraclei and results in significant
damage to the fruits and foliage. The disease
was recorded on dill for the first time in Egypt
by El-Sayed Ziedan (2010), who revealed that
the symptoms developed as white irregular
areas on leaves, fruits, and stem inflorescences
of dill. At all stages of growth, from
transplanting to harvesting, the dill plant
exhibits symptoms of powdery mildew, as
reported by Bubici (2015). In spite of the high
efficiency of chemical fungicides, repeated use
of these chemicals has resulted in numerous
problems, including toxicity residues in foods,
environmental pollution, and resistance
development in pathogen populations (Yoon et
al., 2013). Consequently, agro-scientists are
constantly searching for safer ways to manage
plant diseases. Recently, alternative
approaches to plant pathogen control have been
developed, including bioagents, essential oil
emulsions, and organic acids (Pereira et al.,
2011). Different Trichoderma species are
considered effective in controlling powdery
mildew in many plants. Deore and Sawant
(2000) reported that Trichoderma is highly
efficient in managing guar powdery mildew.
Awad et al. (2012) found that Bio-zeid
(Trichoderma album 25 × 106 spores/g) is the
most effective biocide that significantly
reduced conidial production and disease
severity percentages of cucumber powdery
mildew compared to control. Abdel-Kader et
al. (2013) reported that the application of
Bacillus subtilis or Trichoderma harzianum
resulted in a significant reduction in the
powdery mildew incidence of pepper,
cantaloupe, and cucumber in comparison with
the application of the other tested bioagents and
control. Essential oils have been found to be
effective for plant disease management (Lo
Cantore et al., 2009; Groot et al., 2004).
According to Saltos-Rezabala et al. (2022),
thyme essential oil strongly suppressed
conidial germination and Alternaria linariae
growth, the pathogen responsible for tomato
early blight disease. The antimicrobial effects
of bioactive compounds found in thyme
essential oil have been demonstrated in
opposition to several plant pathogens (Mugao
et al., 2021; Doost et al., 2020). Thyme
essential oil, which has multiple mechanisms
against plant pathogens, is increasingly
becoming popular in the search for fungicidal
alternatives (Jamiołkowska, 2020). Salicylic
acid is a chemical that can stimulate plant
resistance, reducing the disease incidence
(Oostendorp et al., 2001). Zhang et al. (2001)
found that salicylic acid is significant in
protecting certain crops from late leaf spot.
Salicylic acid affects several molecular and
biochemical processes related to resistance
stimulation against plant diseases
(Hammerschmidt & Smith-Becker, 1999). The
current study aimed to evaluate the
effectiveness of fungicidal alternatives,
including biocide, essential oil emulsions, and
organic acids, in controlling dill powdery
mildew under greenhouse conditions. Additionally,
the study aimed to identify the pathogen
responsible for dill powdery mildew disease
using morphological and molecular methods.
2. Materials and methods
2.1 Disease survey
Survey of dill powdery mildew disease was
carried out during 2022 growing season in six
regions which covered the most dill
widespread cultivated areas in Fayoum
governorate, Egypt. These areas are located at
Tamiya, Abshaway, Etsa, Sinnoures, Youssef
El-Seddik and El-Fayoum center. In order to
illustrate the spread of this disease during the
Hassanin et al., 2023
16
growing season, the survey was conducted
during February and March 2022. The
obtained survey data was arranged as
percentages of disease severity and incidence.
The percentages of disease incidence were
calculated using the formula described by El-
Helaly et al. (1970) and Ahmed (2013) as
follows:
Disease incidence (%) = (Number of infected plants/Total
plant numbers) × 100
The severity of the disease was calculated
based on the equation and the scale (of 0 to 4)
described by Yan et al. (2002) and Parkunan et
al. (2013), where 0 = no disease, 1 = 1-25%
area covered with lesions/ plant, 2 = 26-50%
area covered with lesions/ plant, 3 = 51-75%
area covered with lesions/ plant, 4 = 76-100%
area covered with lesions/plant. The disease
severity percentages were calculated as
follows:
Disease severity (%) = [∑ (n × c)] / (N × C) × 100
Where: n= Number of infected plants, c=
Category number, N= Total number of
examined plants and C= The highest category
number of infections.
2.2 Isolation and identification of the causal
agent based on microscopic examination
Dill plants infected with powdery mildew were
sampled during the survey of disease in
February 2022. To identify the causal fungus
of powdery mildew, epidermal strips (bearing
the fungal conidiophores and conidia) from the
diseased dill plants were used for microscopic
preparations on glass slide, then examined
using light microscope. Pathogen was identified
according to morphological characteristics
according to Soylu and Soylu (2003) and
confirmed by the staff member of Mycological
Center of Assiut University, Egypt.
2.3 DNA extraction and PCR amplification
The fungal conidia of powdery mildew were
gathered in a 1.5 ml microcentrifuge tube, and
the method of Walsh et al. (1991) was used to
extract DNA. The primer pairs used for
amplification were ITS4 (5′-
TCCTCCGCTTATTGATATGC-3′) and ITS5
(5′-GGAAGTAAAAGTCGTAACAAGG-3′)
(White et al., 1990). The total volume of PCR
mixtures was 20 µl including 2 µl of 10x
buffer, 2 µl of dNTPs, 1 µl of each primer, 2
µl of DNA template (50–100 ng), 0.3 µl Taq
DNA polymerase and the remaining volume
amount of double-distilled water. A thermal
cycler was used for the PCR amplification
with conditions as follows: Initiation for 5 min
at 95 °C followed by 11 cycles included
denaturation for 10 s at 94 °C, annealing for
10 s at 65 °C, extension for 30 s at 72 °C
followed by 30 cycles included denaturation
for 10 s at 94 °C, annealing for 10 s at 55 °C,
extension for 30 s at 72 °C and then, a final
extension cycle at 72 °C for 5 min.
2.4 Sequencing and phylogenetic analysis
Sequencing of amplified PCR products was
carried out at Eurofins Genomics Company
(Germany). As with the PCR amplification,
the same primers were employed for
sequencing. Data acquired from the nucleotide
sequence was deposited in GenBank. A
BLAST analysis was performed at the NCBI
(http://ncbi.nlm.nih.gov/BLAST) to get
homologous sequences. MEGA11 (Molecular
Evolutionary Genetics Analysis Version 11)
software was used for constructing the
phylogenetic tree from the isolate of the
current study and the other isolates which were
obtained from NCBI database.
Hassanin et al., 2023
17
2.5 Greenhouse Experiments
2.5.1 Fungal inoculum preparation
Inoculum of E. heraclei was gained from dill
plants showing symptoms of powdery mildew
from Abshaway, Fayoum, Egypt and
conserved on healthy dill plants in the
greenhouse for further investigation. Young,
virulent conidia were used for the inoculation,
which formed within 24 h after removing the
old conidia from the leaves by agitating.
Inoculation was carried out by gently dusting
powdery mildew infected plants above the
healthy plants. Inoculated plants were then
covered with plastic bags, kept at 25±2°C for
24 hours with enough moisture, and then
checked daily for disease progress.
2.5.2 The efficiency of some alternative
control methods for controlling dill powdery
mildew under greenhouse conditions
All experiments were carried out in the
greenhouse of Ornamental, Medicinal and
Aromatic Plants Diseases Research
Department, Plant Pathology Research
Institute, Agricultural Research Center, Giza,
Egypt, at 25±2°C and 60-70% R.H. Seeds of
dill (Anethum graveolens L.) were kindly
obtained from Medicinal and Aromatic Plants
Department, Horticulture Research Institute,
Agricultural Research Center, Giza, Egypt.
Plastic pots (25 cm in diameter) containing
sandy loam soil were planted with ten seeds.
Essential oil emulsions of thyme or clove
(prepared at Ornamental, Medicinal and
Aromatic Plants Diseases Research
Department, Plant Pathology Research
Institute, Agricultural Research Center, Giza,
Egypt) at a rate of 2 ml/l (Hassanin et al.,
2017), ascorbic or salicylic acids [Sigma
Company] at a rate of 0.5 g/l (Halawa et al.,
2018) and Bio-Cure F 1.15% WP
(Trichoderma viride 1 × 106 cfu/g, M/S.T.
Stanes Company Limit-India) at a rate of 6 g/l
(Hassanin et al., 2020) were examined in this
experiment. Plants were sprayed with the
tested treatments three times after inoculation
with the conidial spores of the causal fungus.
The first spray was given 48 h. after
inoculation, second and third sprays were
given 7 days intervals. Plants were sprayed
with water served as an untreated control. For
each treatment or control, three replicates were
employed. The percentages of disease severity
and incidence were calculated 7 days after
every spray according to the formulas
mentioned before. Additionally, the height of
the plants and their number of branches were
under investigation, and the percentage of
increases in these parameters were calculated
as follows:
Percentage of increase (%) = [(A – B) / B] × 100,
Where, A = Value of treatment and B = Value
of control.
2.6 Statistical analysis
In this factorial experiment, three replicates
were used in an overall random design
according to Snedecor and Cochran (1989).
The L.S.D. test was used at 0.05 for the
statistical analysis using MS-TATEC software.
3. Results
3.1 Disease symptoms of dill powdery mildew
Symptoms of powdery mildew were observed
on dill plants in several fields of Fayoum
governorate during the 2022 growing season
in February and March. Symptoms began as
white powdery areas on the lower parts of
plant, became combined and increase in size to
cover the stems and all leaves (Figure 1).
Hassanin et al., 2023
18
Figure 1: Symptoms of dill powdery mildew. A is a healthy plant. B, C and D
are naturally infected plants.
3.2 Disease survey
A disease survey was conducted in February
and March 2022 to investigate the spread of
powdery mildew in dill. The greatest
percentages of disease severity and incidence
were observed in March in all survey regions.
Youssef El-Seddik and Etsa had the greatest
average percentages of disease incidence
during February and March (85.9% and 81.5%,
respectively), as well as the highest mean
percentages of disease severity for the season
(65.5% and 59.3%, respectively). Sinnoures
had the lowest incidence of disease (60.5%),
while Fayoum had the lowest severity of
disease (40.2%). The mean disease incidence
in Fayoum governorate was 74.0%, and the
mean disease severity was 50.6% (Table 1).
3.3 Identification of the causal pathogen of
powdery mildew using microscopic examination
In symptomatic dill plants, E. heraclei was
examined via microscopy. Erysiphe heraclei
conidiophores were upright, short,
unbranched, and cylindrical to oval, and varied
in length from 25 to 38 µm and width from 9
to 13 µm (Figure 2). Cylindrical foot cells
measuring (7.50- 9 × 21-26 µm) followed by
one or two cells that are somewhat smaller (7-
8.50 × 13-15.50 µm). Conidia were created
individually and ranged in shape from
cylindrical to oblong elliptical, measuring 27
to 42 µm in length and 12 to 16 µm in width,
with an average of 34.50 × 14 µm. It had no
fibrosin bodies and formed a lobed
appressorium (Polygoni type) at the extremities of
their germ tubes, which might be extremely
short or very long. Cleistothecia measures
were 108-122 (115 µm) × 85-114 (99.50 µm),
which appeared as dark brown to black spots
on leaves, inflorescences and stems with a
circular shape. Appendages were brown to
black with forked tips and measured 87-225
(156) µm in length and 3-5 (4) µm in width.
Hassanin et al., 2023
19
Table 1: Incidence and severity of dill powdery mildew in Fayoum governorate, Egypt, during the growing
season 2022.
Regions surveyed
Disease Incidence (%)
Mean
Disease Severity (%)
February
March
February
March
Tamiya
62.7
81.3
72.0
31.3
65.9
Abshaway
69.7
88.0
78.9
31.4
67.7
Etsa
73.0
90.0
81.5
44.6
74.0
Sinnoures
55.0
66.0
60.5
25.2
55.4
Youssef El-Seddik
76.7
95.0
85.9
53.9
77.0
El-Fayoum
58.0
71.7
64.9
27.6
52.7
Mean
65.9
82.0
74.0
35.7
65.5
L.S.D. at 5%
1.7
0.9
-
1.4
1.0
Figure 2: Conidia and conidiophores of Erysiphe heraclei. A is a conidium carried
on conidiophore emerging from dill leaf. B is mycelium and conidia of E. heraclei.
3.4 Molecular Characterization and phylogenetic tree
After PCR amplification of DNA extracted
from fungal conidia, the PCR product (621 bp)
was sequenced and submitted in GenBank with
accession number (OP999071.1). The isolate
sequence of the current study showed 98-100%
sequence homology with sequences of E.
heraclei isolates obtained from GenBank
database with accession numbers; (KR269918.1),
(KP055630.1), (OM856023.1), (AB104513.1),
(OM856020.1), (LC270862.1), (KY073878.1),
(MT703849.1), (AB000942.1) and (LC009917.1),
after being analyzed with BLAST. The
phylogenetic tree, as shown in Figure (3), was
then constructed using MEGA 11 software.
3.5 Efficacy of alternative control methods for dill
powdery mildew in greenhouse environments
Table (2) shows that a higher reduction in
incidence and severity of disease (%)
compared with the control was obtained from
Bio-Cure F treatment (90 and 95.1%,
respectively), followed by thyme essential oil
emulsion (82.5 and 87.4 %, respectively). On
contrast, treatment with ascorbic acid was the
least successful in decreasing percentages of
incidence and severity of disease (67.5 and
72.7%, respectively).
Hassanin et al., 2023
20
Figure 3: A phylogenetic tree generated depending on the sequences of ITS rDNA regions of E.
heraclei isolate from the current study and other isolates obtained from NCBI database. The tree
was constructed through MEGA11 software.
Table 2: Impact of tested treatments on dill powdery mildew incidence and severity in greenhouse through
the 2022/2023 cultivation season.
Treatments
Disease Incidence )%(
Reduction (%)
Disease Severity (%)
Reduction )%(
Bio-cur F
10
90
2.8
95.1
Thyme essential oil emulsion
17.5
82.5
7.2
87.4
Clove essential oil emulsion
20
80
8.3
85.5
Salicylic acid
25
75
12.6
77.9
Ascorbic acid
32.5
67.5
15.6
72.7
Control
100
-
57.1
-
L.S.D. at 5%
-
1. 84
-
As shown in Table (3), Bio-Cure F treatment
resulted in the greatest increase in dill plant
height (60.54%) and the number of branches
(121.09%), in comparison with the control. Salicylic
acid application followed, with increases of
46.26% and 94.79%, respectively. Treatment with
clove oil emulsion resulted in the lowest increase in
plant height (30.72%), while ascorbic acid
treatment resulted in the lowest increase in
number of branches per plant (52.61%).
Table 3: Impact of tested treatments on dill plant height and branching in greenhouse through the
2022/2023 cultivation season.
Treatments
Plant height (cm)
Increase (%)
Number of branches/Plant
Increase (%)
Bio-cur F
86.16
60.54
9.33
121.09
Thyme oil emulsion
77.16
43.77
8.00
89.57
Clove oil emulsion
70.16
30.72
7.22
71.09
Salicylic acid
78.50
46.26
8.22
94.79
Ascorbic acid
73.22
36.43
6.44
52.61
Control
53.67
4.22
L.S.D. at 5%
2.86
0.80
Hassanin et al., 2023
21
4. Discussion
In the present study, morphology and
molecular analysis were used to identify the
powdery mildew causal agent, and the efficacy
of some fungicidal alternatives was also
evaluated against powdery mildew of dill in
addition to their impacts on plant growth
measurements under greenhouse conditions.
Infected dill plants with powdery mildew were
obtained during the survey of disease in
February 2022. The onset of the symptoms
began at vegetative stages, which
progressively increased through pre-maturity
and fruiting stages. These findings are in
agreement with those reported by Soylu and
Soylu (2003), Cho et al. (2012) and Bubici
(2015). The survey trials which were done in
different regions of Fayoum governorate
during February and March indicated that,
powdery mildew in March recorded the
highest mean disease incidence and severity
compared with February, particularly in
Youssef El-Seddik and Etsa. These findings
highlighted that, the appearance and severity of
powdery mildew infection on dill were greatly
impacted by climatic changes as relative
humidity and temperature in 2022. According
to Kolte (1985), cool temperatures and low
relative humidity through crop development
favored inoculum accumulation, resulting in
severe epidemics of powdery mildew on
sunflowers. A study by Aust and Hoyningen-
Huene (1986) found that powdery mildew is
more likely to become severe on crops when
the nights are cool, and the weather is dry.
Based on the characteristics of the anamorph
and teleomorph phases, via light microscope,
E. heraclei was identified as the causal agent
of dill powdery mildew. These results are in
accordance with those mentioned by El-Sayed
Ziedan (2010), Cho et al. (2012) and Bubici
(2015). Braun (1995) found that the
morphological characterization of powdery
mildews was mostly dependent on
teleomorphs, like the number of asci and the
shape of the appendages on the cleistotheium.
Additionally, more than twelve morphological
features, including conidia, conidiophores,
houstoria, appressoria, fibrosin bodies, and
mycelium, are present in the powdery
mildew's anamorphic stage (Boesewinkel,
1980). Cunnington et al. (2003) reported that
using of molecular characterization,
particularly the ITS region, offers encouraging
results for identifying the species of several
powdery mildews. The obtained results
revealed that the isolate of the current study
(Accession No. OP999071.1) showed 98-
100% similarity with accession numbers of E.
heraclei isolates obtained from GenBank
database; (KR269918.1), (KP055630.1),
(OM856023.1), (AB104513.1), (OM856020.1),
(LC270862.1), (KY073878.1), (MT703849.1),
(AB000942.1) and (LC009917.1). Under
greenhouse condition, dill plants were sprayed
with clove or thyme essential oils emulsions,
elicitors: ascorbic or salicylic acids and
biocide: Bio-Cure F, three times after
inoculation with E. heraclei. The obtained
results showed that the higher decrease in
disease severity and incidence percentages
was achieved by using Bio-Cure F, followed
by thyme essential oil. The ability of the
biocide to effectively reduce disease incidence
and severity in dill plants may be attributed to
the production of growth regulators that
encourage the plant to tolerate infection
(Naglot et al., 2015; Kumar et al., 2012).
Pokhrel et al. (2022) mentioned that using of
Trichoderma spp. or its released metabolites
make it as biological control agents to manage
plant diseases caused by pathogenic fungi.
Trichoderma spp. are bioagents that attack
plant pathogens through some mechanisms,
including an effective enzymatic system, the
production of antibiotics, and competition for
nutrients (Shoresh et al., 2010). Trichoderma
Hassanin et al., 2023
22
spp. were capable of producing enzymes that
lysed cell walls as well as secondary
metabolites that were effective against plant
pathogens, as mentioned by Rahman et al.
(2009). Zimand et al. (1996) reported that
Trichoderma spp. suppresses some fungal
enzymes like pectinases, which are essential
for the fungus to penetrate the leaf surface. The
effect of thyme essential oil emulsion against
powdery mildew on dill plants maybe as a
result of some active ingredients in the oil that
have an antifungal impact. The antifungal
properties of thyme essential oil are due to the
presence of Thymol, p-cymene and borneol as
reported by Hammad and Hassanin (2022).
Hassanin et al. (2017) reported that because
thyme volatile oil emulsion contains active
ingredients, such as thymol, p-cymene and
Borneol, it has the best antifungal impact at the
lowest concentrations. Ni et al. (2021)
mentioned that thyme essential oil can damage
proteins of the cell membrane and genetic
material by inhibiting gene expression, as well
as damage cell walls. Also, cellular defenses
such as peroxidase activity can be stimulated
indirectly against pathogens by the bioactive
compounds found in thyme essential oil, as
reported by Jamiołkowska (2020). Also, the
results of present work showed that the highest
percentages of increasing in dill plant height
(cm) as well as branches/plant were obtained
by Bio-cur F treatment compared with the
control, followed by the treatment of salicylic
acid. When a plant becomes infected with a
pathogen, an induced plant resistance or
acquired resistance is created. This resistance
can protect the plant against further infections
and has a positive impact on the plant growth.
These findings are in accordance with those
reported by Abdel-Kader et al. (2013) and
Mergawy (2016). Spletzer and Enyedi (1999)
reported that the addition of 200 mM salicylic
acid to tomato plants could induce the
expression of the pathogenesis-related IB gene
and stimulate systemic acquired resistance.
Salicylic acid is a compound generated from
plants and related to plant defense towards
pathogens through the activation of systematic
acquired resistance (Ryals et al., 1994) and
stimulation of antioxidant enzymes in plants
(Janda et al., 1999). According to Hamada and
Hashem (2003), wheat grains soaked in
salicylic acid before planting resulted in
significant decreases in the mean disease
values that were caused by Fusarium
oxysporum, F. oxysporum, or Bipolaris
sorokiniana. Plant growth promotion and
induction of resistance to plant pathogens were
also reported as benefits of salicylic acid
(Vidhyasekaran, 1990; Nickell, 1983).
5. Conclusion
During the 2022 season, Erysiphe heraclei
was characterized as the causal agent of
powdery mildew in dill plants obtained from
Fayoum governorate, through morphological
and molecular characterization. The efficacy
of several fungicidal alternatives, including
Bio-Cure F (Trichoderma viride), essential oil
emusions (clove and thyme), ascorbic and
salicylic acid, was evaluated against the
powdery mildew of dill. The results showed
that Bio-Cur F, emulsion of thyme volatile oil,
and salicylic acid were the most effective
applications for managing dill powdery
mildew and improving plant growth under
greenhouse conditions. These findings suggest
that these alternatives have the potential to be
used as fungicides.
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