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

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

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

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

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

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.