Journal of Phytopathology and Pest Management 6(1): 66-77, 2020

pISSN: 2356-8577 eISSN: 2356-6507

Journal homepage: http://ppmj.net/

∗

Corresponding author:

Ahmed B. Mohamed,

E-mail: ahmedmohamed.5419@azhar.edu.eg

66

Chemical control of tomato early blight caused by

Alternaria solani

using certain fungicides and

chemical inducers

Elsayed S. Abdou

1

, Ali A. El-Banna

1

, Rafik M. ElSharkawy

2

, Ahmed B. Mohamed

2*

1

Department, Faculty of Agriculture, Minia University, Minia, Egypt

2

Agricultural Botany Department, Faculty of Agriculture, Al-Azhar University (Assiut Branch),

71524 Assiut, Egypt

Abstract

Keywords: Alternaria solani, fungicides, antioxidants, tomato, early blight.

67

1. Introduction

Tomato (

Lycopersicon esculentum

) is one

of the vital solanaceous vegetable crops

of global importance grown in Egypt. The

cultivated area of tomato since 2009 to

2014 growing seasons reached 499.536

feddan in old and newly reclaimed lands,

which produced about 7.964.997 tons

(FAO, 2015). Tomato ranks next to the

potato crop and ranks the first among the

processing crops in the world.

Alternaria

solani

(Ellis and Martin) Jones and Grout

is the most important pathogen causing

severe early blight disease every year in

tomato. The disease causes a drastically

reduction in tomato fruits quantity and

quality. Symptoms of the disease are

characterized by brown to dark brown

colored necrotic spots (Mayee et al.,

1986). Due to this disease, about 80%

yield loss was recorded in experimental

field and severity varies from 15-90%

(Pandey et al., 2003). Most currently

grown tomato cultivars are susceptible to

early blight at various degrees;

consequently, foliar fungicides are used

frequently to manage this disease. The

most effective early blight control

measure consists of excessive fungicides

applications starting early in the growing

season before the first symptom appear

(Pscheidt et al., 1988). Primary methods

of controlling early blight include

preventing long periods of wetness on the

leaf surface, cultural scouting, sanitation

and development of the host plant

resistance with the application of

fungicides

(Kumar et al., 2013).

Chemical inducers of plant resistance

possess quite different modes of action as

compared to synthetic biocides as they

have no direct toxicity to pathogens,

plants and animals, no negative effects on

plant growth, yield development, broad

spectrum of defense, long lasting

protection and low economical cost for

farmers and good profit for producers

(Horsfield et al., 2010; Kessmann et al.,

1994). Salicylic acid (SA) plays an

important role in plant defense, and is

well documented for dicotyledonous

plants, where it’s required for basal

resistance against pathogens as well as

for the inducible defense mechanism and

systemic acquired resistance (SAR)

which confers resistance against a broad-

spectrum of pathogens

(Chaturvedi et al.,

2007). The effect of five antioxidants

(citric acid, salicylic acid, benzoic acid,

ascorbic acid and sodium citrate) on the

resistance of tomato plants (

Lycopersicon

esculentum

Mill.) to early blight disease

incited by

Alternaria solani

was

investigated

in vitro

and

in vivo

(Awadalla et al., 2008). Therefore, the

present study was aimed to determine the

efficacy of different fungicides and

antioxidant doses against

Alternaria

early

blight of tomato.

2. Materials and methods

2.1 Isolation and identification of the

causal organism

Leaves of diseased tomato plants

showing typical early blight symptoms

were collected from different localities of

Luxor, Qena, Sohag, Assiut and Minia

governorates, Egypt during 2013

growing season. Samples were washed

using tap-water surface sterilized with

0.5% sodium hypochlorite solution for

two minutes then washed three times in

sterilized distilled water. Samples were

then dried between two layers of

sterilized filter paper to remove the

excess water. The sterilized spotted leaf

tissues were cut with adjacent healthy

tissues using a sterile scalpel and placed

on plain agar medium in Petri-dishes.

Inoculated dishes were incubated at 27°C

for 9 days. Hyphal tips from the outer

68

ends of the growing colonies were

transferred to plates of potato dextrose

agar (PDA) medium and incubated at

2

7

°C. Pure cultures were obtained for

each of the isolated fungi using the single

spore technique according to (Hansen et

al., 1926) and/or hyphal tip technique

according to (Brown et al., 1924). The

purified fungi were identified at Plant

Pathology Research Institute,

Agricultural Research Center according

to their morphological characters using

the description of (Ellis et al., 1993).

Stock cultures were maintained on PDA

slants and stored in a refrigerator at

7±2°C.

2.2 Pathogenicity tests

Tomato transplants (Super strain B) were

grown in plastic pots filled with sterilized

soil. Thirty days after transplanting,

plants were sprayed with sterile distilled

water before inoculation then covered

with polyethylene bags for 24 hours.

Spores and mycelial suspensions for the

eight isolates obtained from isolation and

identified as

A. solani

were prepared in

sterile distilled water from nine days old

cultures. The spore suspensions were

spread and swabbed with moist cotton

onto leaves after being scratched using

carborundum. Such inoculated plants

were again covered with polyethylene

bags. After 24 hours of incubation,

polyethylene bags were removed and the

plants were kept in greenhouse. Control

was maintained by spraying the plants

with only sterile distilled water. Disease

severity was recorded after 30 days of

inoculation. Re-isolation was made from

infected plants and the cultures thus

obtained were compared with original

cultures to confirm the identity and the

pathogenicity of the pathogens. The two

extreme aggressive and non-aggressive

isolates were selected for further

experiments.

2.3 Disease assessment

Scale from 0 to 5 according to (Mayee et

al., 1986) was used to assess the disease

where: 0 = No symptoms on the leaf.

1 = 0-5 percent leaf area infected and

covered by spots, no petioles and

branches. 2 = 6-20 percent leaf area

infected and covered by spots, some

spots on petioles. 3 = 21-40 percent leaf

area infected and covered by spots, spots

also seen on Petioles and branches. 4 =

41-70 percent leaf area infected and

covered by spots, spots also seen on

Petioles, branches and stems. 5 = 71-100

percent leaf area infected and covered by

spots, spots also seen on petioles,

branches, stems and fruits. Disease

severity was calculated according to the

formula:

Disease severit

Where: n = Number of infected leaves in

each category. V = Numerical values of

infection categories. N = Total number of

leaves examined. 5 = Constant, highest

numerical value.

2.4 Effect of chemical inducers on

A.

solani

2.4.1

In vitro

Different concentrations (500, 1000,

1500, 2000, 2500 and 3000 ppm) of four

chemical inducers

i.e.

ascorbic acid,

catechol, citric acid and salicylic acid

were incorporated in 100 ml PDA

medium and poured in 9 cm Petri dishes.

69

A five mm diameter agar disc containing

fungal mycelium growth of the two tested

fungi was transferred to the test medium

(Three plates for each concentration and

control). Then plates were incubated at

27±1°C in a growth chamber. Colony

diameter was observed daily until control

Petri dishes were covered with the fungal

growth and measured as percentage

reduction of linear growth of pathogenic

fungi comparing with control using this

formula:

 





 

If in a trial 30% of introduced weevils

were found in the EO portion of the disc,

it means that percent repellency by the oil

was 70%. A total of 20 trials were carried

out and percent repellency due to the

essential oil and ethanol was recorded

accordingly.

2.4.2

In vivo

To study the effect of ascorbic acid,

catechol, citric acid and salicylic acid on

the disease severity of

A. solani

isolates,

plastic pots (25 cm in diameter) were

filled with sterilized soil. Tomato

transplants (super strain B) were

transplanted at the rate of 3 transplants

/pot. This experiment was carried out

under greenhouse conditions with three

replicates for each particular treatment.

After 3 weeks from transplanting, tomato

plants were sprayed with the tested

chemical inducers. Plants which were

sprayed with distilled water before

inoculation served as control. After seven

days, plants were inoculated with the

suspensions of

A. solani

isolates

(10

6

cfu/ml). The sprayed plants were

covered with polyethylene bags for 24

hours under greenhouse conditions. After

30 days from inoculation, disease

severity was calculated as mentioned

before. The tested inducers were sprayed

with the best concentration for each

particular compound which gave the best

result in reducing linear growth o

A.

solani

isolates

in vitro

.

2.5 Effect of fungicides on

A. solani

2.5.1

In vitro

The effect of different concentrations (5,

10, 25, 50, 100, 150, 200 and 300 ppm

active ingredient) of four fungicides;

Bellis 38% WG (25.2% Boscalid +

12.8% Pyraclostrobin), Amistar-top

32.5% SC (20% Azoxystrobin + 12.5%

Difenoconazole), Folio gold 53.75% SC

(3.75% Metalaxyl M + 50%

Chlorothalonil) and Luna experience

40% SC (20% Fluopyram + 20%

Tebuconazol) was tested on the linear

growth of

A. solani

isolates on PDA

media. The different concentrations of

each fungicide (according to the active

ingredient) were suspended and added to

PDA medium before solidification. Then

media containing fungicides were poured

in Petri dishes (9 cm in diameter) and

three Petri dishes were used for each

concentration alone. The dishes were

inoculated in the center with equal discs

(5 mm in diameter) of 9 days old culture

of

A. solani

isolates and incubated as

mentioned before. Colony diameter was

observed daily until control Petri dishes

were covered with the fungal growth and

measured as percentage reduction of

linear growth of pathogenic fungi

comparing with control using this

formula:

 





 

70

2.5.2

In vivo

To study the effect of the tested

fungicides on the disease severity, plastic

pots (25 cm in diameter) were filled with

sterilized soil. Tomato seedlings (super

strain B) were transplanted with 3

transplants /pot. This experiment was

carried out under greenhouse conditions

with three replicates for each particular

treatment. After 3 weeks from

transplanting, tomato plants were sprayed

with the tested fungicides. Plants which

were sprayed with distilled water before

inoculation served as control. After seven

days, plants were inoculated with the

suspensions of

A. solani

isolates

(10

6

cfu/ml). The sprayed plants were

covered with polyethylene bags for 24

hours under greenhouse conditions. After

30 days from inoculation, disease

severity was calculated as mentioned

before. The tested fungicides were

sprayed with the best concentration for

each particular compound which gave the

best result in reducing linear growth

in

vitro

of

A. solani

isolates.

2.6 Statistical analysis

Analysis of variance of the data was

carried out on the mean values of the

tested treatments according to the

procedures described by Gomez and

Gomez (1984). The least significant

difference (LSD) at 5% probability was

used for testing of significance of the

differences among the mean values of the

tested treatments for each character.

3. Results and Discussion

3.1 Pathogenicity tests

Data presented in Table (1) showed that

all the tested isolates proved to be

pathogenic to the tested tomato plants

(Super strain B), causing symptoms of

early blight which, first, appear as small

irregular to circular dark brown spots on

the lower (older) leaves which turn

yellow and die, in comparison with the

control. In this regard,

Alternariasolani

isolate Assiut-A3 was significantly the

most aggressive isolate recording the

highest disease severity value (57.38),

followed by Minia-M (50.65), Assiut-A2

(48.46), Assiut-A1(34.66), Sohag-S

(30.44), Luxor-L2 (27.3) and Luxor-L1

(23.3), respectively. While, Qena-Q

(22.6) recorded the lowest disease

severity. Hence, the isolates Assiut-

A3(most aggressive) and Qena-Q (least

aggressive) were chosen for the

following experiments.

Table 1: Pathogenicity tests of Alternaria solani isolates

on susceptible tomato plants (Super strain B) under

greenhouse conditions.

Isolate No.

Governorate

Disease severity (%)

1

Luxor-L1

23.3

2

Luxor-L2

27.30

3

Qena-Q

22.60

4

Sohag-S

30.44

5

Assiut-A1

34.66

6

Assiut-A2

48.46

7

Assiut-A3

57.38

8

Minia-M

50.65

Control

00.0

L.S.D at .05%

3.07

3.2 Effect of chemical inducers on

A.solani

3.2.1

In vitro

Four chemical inducers

i.e.

ascorbic acid,

catechol, citric acid and salicylic acid

were tested for their effect on the

mycelial growth of

Alternaria solani

isolates.

Data in Table (2), showed that, all tested

concentrations (500, 1000, 1500, 2000,

71

2500 and 3000 ppm) of four chemical

inducers reduced the growth of

A. solani

isolates as compared with control.

Salicylic acid gave the highest

A. solani

isolates linear growth decrease, followed

by citric acid and catechol respectively.

While, ascorbic acid gave the lowest

effect.

Table 2: Effect of different concentrations of some chemical inducers on the linear

growth of A. solani isolates.

Antioxidant

Conc. (ppm)

Mycelial growth inhibition (%)

Aggressive

Non Aggressive

Ascorbic acid

500

20.6

55.6

1000

36.3

63.6

1500

48.3

75.6

2000

55.6

85.3

2500

70.3

92.6

3000

79.3

100

Salicylic acid

500

36.6

63.3

1000

49.3

74.3

1500

59.6

91.3

2000

75.3

100

2500

88.3

100

3000

100

Citric acid

500

32.6

53.3

1000

45.6

61.3

1500

57.3

72.6

2000

70.6

84.6

2500

87.3

100

3000

100

Catechol

500

27.3

45.6

1000

35.3

56.6

1500

46.6

66.6

2000

65.3

78.6

2500

84.3

90.3

3000

100

Control

0.00

Treatment A

2.59

1.11

Conc B

1.45

1.05

A×B

3.25

2.36

3.2.2

In vivo

Four chemical inducers

i.e.

ascorbic acid,

catechol, citric acid and salicylic acid

were tested for their effect on the disease

severity at certain concentrations under

greenhouse conditions. Data in Table (3)

showed that, all tested concentrations of

the tested chemical inducers resulted a

significantly reduction of early blight

disease severity, as compared with

control. At all concentrations, salicylic

acid was the most effective, followed by

citric acid and catechol, respectively.

While, ascorbic acid was less effective.

3.3 Fungicides

3.3.1

In vitro

The effects of different concentrations (5,

10, 25, 50, 100, 150, 200 and 300 ppm

active ingredient) of the four fungicides

(Bellis 38% WG, Amistar-top 32.5% SC,

Folio gold 53.75% SC and Luna

experience 40% SC) were tested on the

72

linear growth of

A. solani

isolates on

PDA media. Bellis 38% WG fungicide

was the most effective in decreasing the

linear growth diameter of

A. solani

isolates followed by Amistar-top

32.5%SC and Luna experience 40% SC

respectively. While, Foliogold 53.75%

SC gave the lowest effect. Data in Table

(4) showed that, all concentrations of

tested fungicides significantly decreased

the linear growth of

A. solani

isolates

compared with control.

Table 3: Effect of different concentrations of the four chemical inducers on A. solani isolates under greenhouse

conditions.

Antioxidant

Disease severity (%)

Conc. (ppm)

Aggressive

Conc. (ppm)

Non- Aggressive

2015

2016

Mean

2015

2016

Mean

Ascorbic acid

3000

38.26

36.9

37.58

3000

10.86

11.3

11.08

Salicylic acid

3000

20.13

18.73

19.43

2000

0

Citric acid

3000

31.56

28.16

29.86

2500

2.3

3.4

2.85

Catechol

3000

23.23

25.06

24.14

3000

5.86

7.2

6.53

Control

56.0

53.6

54.8

23.16

24.23

23.16

23.69

L.S.D at 0.5%

4.11

4.37

3.39

2.92

Table 4: Effect of the different concentrations of four fungicides on the linear

growth of A. solani isolates.

Fungicides

Conc. (ppm)

Mycelial growth inhibition (%)

Aggressive

Non-aggressive

Bellis 38%

5

21.3

70.6

10

47.6

87.3

25

68.3

100

50

85.3

100

150

100

200

100

250

100

300

100

Amistar-top

32.5%

5

15.6

57.3

10

23.3

60.6

25

47.3

73.3

50

55.6

83.3

100

64.6

96.3

150

83.3

100

200

95.6

100

250

100

300

100

Foliogold

53.75%

5

12.6

34.6

10

15.3

38.3

25

17.6

43.3

50

21.3

47.3

100

26.3

53.3

150

33.3

56.6

200

38.3

63.6

250

48.6

70.3

300

57.3

78.3

Luna express

40%

5

14.3

42

10

23

54.3

25

39.3

66.6

50

47.3

79.3

100

58.3

90.3

150

66.3

100

200

77.6

100

250

93.6

100

300

100

Control

0.00

Treatment A

0.84

1.14

Conc. B

1.09

0.84

A×B

2.44

1.89

73

3.3.2

In vivo

The most effective concentration of

each of the four fungicides (Bellis 38%

WG, Amistar-top 32.5% SC, Folio gold

53.75% SC and Luna experience 40%

SC) were also tested for its effect on

disease severity of

A.

solani

under

greenhouse conditions. Data presented

in Table (5) showed that all tested

fungicides decreased the disease

severity.

Table 5: Effect of certain concentrations of four fungicides on A. solani isolates under greenhouse conditions.

Fungicides

Disease severity %

Conc.

(ppm)

Aggressive

Conc.

(ppm)

Non- Aggressive

2015

2016

Mean

2015

2016

Mean

Bellis 38% WG

50

8.35

5.66

7

25

0

Amistar-top 32.5% SC

200

13.76

16.13

14.94

150

0

Foliogold 53.75% SC

300

32.33

30.1

31.21

200

11.46

9.7

10.58

Luna experience 40% SC

250

22.63

26.1

24.36

150

3.8

6.6

5.2

Control

56.0

53.6

54.8

23.16

24.23

23.16

23.69

L.S.D at .05%

3.05

4.52

2.29

3.34

Results also showed that disease severity

decreased with the increasing of

fungicides concentrations. Bellis 38%

WG fungicide was the most effective in

decreasing disease severity of

A. solani

isolates followed by Amistar-top 32.5%

SC and Luna experience 40% SC,

respectively. While, Foliogold 53.75%

SC was the lowest effective fungicide.

4. Discussion

Eight

A. solani

isolates (Assiut-A3,

Minia-M, Assiut-A2, Assiut-A1, Sohag-

S, Luxor-L2, Luxor-L1, and Qena-Q)

were isolated from five governorates in

Egypt. Virulence test indicated that all

A.

solani

isolates were pathogenic to tomato

plants (Super strain B) causing typical

symptoms of the early blight disease.

Likewise, data indicated that Assiut-A3

isolate proved to be the most aggressive,

recording the highest disease severity

percentage, followed by isolates Minia-

M, Assiut-A2, respectively. Meanwhile,

the other isolates, Assiut-A1, Sohag-S,

Luxor-L2, Luxor-L1, and Qena-Q gave

the lowest virulent. The previous results

indicated that the eight tested isolates

were probably different physiological

races. Similar results were obtained

about

A. solani

isolates and the variation

between them (Burm et al., 1995).

Differences in the pathogenicity of tested

isolates might be due to one or more

several factors related to genetic makeup

of host variety and pathogen as far as

their interaction (Henning et al., 1959).

In addition, a high genetic diversity was

detected among the

A. solani

isolates

(Van der Waals et al., 2004).

Alternaria

solani

production of non-specific as well

as host specific toxins in the case of

pathogenic species (Thomma et al.,

2003). Germination fluids of

Alternaria

solani

contain alternaric acid as well as a

nontoxic substance that acts as

susceptibility – inducing factor

(Langsdrof et al., 1991). Also, the

disease weakens progressively the plant

and increases susceptibility to infection

by reducing the photosynthetic leaf area

and the imbalance between nutrient in

the fruits and nutrient supply from the

74

leaves (Rowell et al., 1953). Statistical

analysis revealed significance between

linear growth values of the evaluated

antioxidants. The lowest linear growth

value was achieved by salicylic acid

followed by citric acid and catechol,

respectively. While, the lowest effective

evaluated compound in reducing mycelial

growth was ascorbic acid that gave the

highest general mean of linear growth.

The inhibitory effect of some

antioxidants to the growth of

A. solani

were investigated by many researchers

(Abada et al., 2008; Abdel-Sayed et al.,

2006; Tofali et al., 2003). Several

investigators reported that the

antioxidants may control seed and soil-

borne fungal diseases (Dmitrier et al.,

2003; Shahda et al., 2001), as well as

foliar fungal diseases (Hassan et al.,

2006). Organic acids are known for years

for their antibacterial and antifungal

properties which have been widely used

in foodstuff industry and agriculture (Pao

et al., 2008; Sathe et al., 2007; De

Muynck et al., 2004). Also, El-Saidy Aml

and Abd El-Hai (2011)

found that acids

effectively controlled fungi. The

activation of SAR is associated with the

heightened level of expression of the

pathogenesis-related proteins, some of

which possess antimicrobial activity

(Chaturvedi et al., 2007). Salicylic acid

(SA) is an important endogenous

molecule involved in plant defense. The

link between SA production and systemic

acquired resistance (SAR) has been well

established

(

Delaney et al., 1997; Klessig

et al., 1994). Transgenic plants

expressing the salicylate dehydrogenase

(nahG) gene, which converts SA into

inactive catechol, and, do not establish

SAR (Gaffney et al., 1993). Furthermore,

there is a correlation between the increase

in SA levels and plant gene expression.

Pathogenesis-related (PR) proteins show

up a few hours after the SA level begins

to rise (Yalpani et al., 1993). Exogenous

SA can induce simultaneous PR

expression and resistance to pathogens,

even in the absence of pathogenic

organisms (Ward et al., 1991). Results

also showed that disease severity

decreased with the increasing of

fungicides concentrations. Bellis 38 %

WG fungicide was the most effective

fungicide decreasing disease severity and

linear growth diameter of

A. solani

isolates followed by Amistar-top 32.5%

SC and Luna experience 40% SC

respectively. While, Foliogold 53.75%

SC

was less effective. Similar results

were obtained by (Horsfield et al., 2010;

Capriotti et al.,

2005). Tofoli et al.

(2003) who reported that tebuconazole

and difenoconazole provided an

important and partial inhibition of

conidium germination, respectively. In

that study, azoxystrobin and

pyraclostrobin+methiram showed a

moderate inhibitory effect on mycelial

growth and complete inhibition of

conidium germination starting from 1

mg

/

ml, while chlorothalonil and

mancozeb demonstrated minor inhibitory

levels but superior nevertheless to the

control (Pasche et al., 2004).

Pyraclostrobin, which significantly

reduced the early blight and increased the

yield in tomato and potato has reported

by many workers (Ganeshan et al., 2009;

MacDonald et al., 2007; Ivey et al.,

2004). Pyraclostrobin alternated with

maneb and pyraclostrobin + boscalid

alternated with maneb significantly

reduced the anthracnose incidence in bell

pepper as compared to control. Best

disease management with an improved

75

yields and fruit quality was reported in

combination product of

pyraclostrobin+metiram which was

effective against both early blight andlate

blight has reported by (Capriotti et al.,

2005). Horsfield et al. (2010) reported

that boscalid was active in the control of

early blight disease of potatoes.

(Jambhulkar et al., 2012) have reported

that spray of azoxystrobin 23% SC

showed promising results by reducing

disease severity by 38.9% as compared

with control. Among the four fungicides

analyzed in our present study, Bellis 38%

WG and Amistar-top 32.5% SC

demonstrated a highest toxicity against

A.

solani

. The tested isolates were

moderately sensitive to Luna express

40% SC. While, Foliogold 53.75% SC

showed low toxicity against isolates.

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