Assessment on early blight of potato in order to compare the two methods<i> in vitro </i>using pathogenic fungi <i>Alternaria solani</i>

doi:10.4236/ns.2013.511145

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Vol.5, No.11, 1189-1192 (2013) Natural Science

http://dx.doi.org/10.4236/ns.2013.511145

Assessment on early blight of potato in order to

compare the two methods in vitro using pathogenic

fungi Alternaria solani

Hamid Reza Mirkarimi1*, Ahmad Abasi-Moghadam2, Javad Mozafari2

1Department of Plant breeding, Faculty of Agriculture, Science and Research Branch, Islamic Azad University of Tehran, Tehran,

Iran; *Corresponding Author: [email protected]

2Department of Genetics & National Plant Gene-Bank, Seed and Plant Improvement Institute, Karaj, Iran

Received 14 September 2013; revised 14 October 2013; accepted 21 October 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Potato (Solanum tuberosum) early blight, caus-

ed by Alternaria solani is one of the most de-

structive fungal foliar diseases. This research

was done in order to study methods comparison

of evaluation by culture filtrate of A. solani in in

vitro condition for selecting resistance cultivars

to early blight. Plantlets of potato viruse free

were obtained from the National plant gene bank

of Iran, and were inoculated in vitro methods

with a culture filtrate of A. solani. In in vitro se-

lection by droplet of culture filtrate method,

leaflet received a 10 µl droplet of the A. solani

culture filtrate and in in vitro selection by direct

using of culture filtrate method, plantlets were

placed in test tubes that include 5 µl A. solani

culture filtrate. The experimental design was

factorial on basis of completely randomized de-

sign (CRD) with two factors, three replications

and six genotypes. During droplet method assay,

the A. solani symptoms appeared 1 - 2 days until

6 days and during direct method they appeared

2 - 3 days until 6 days. The AUDPC values were

submitted to the analysis of varience (ANOVA)

and AUDPC means were compared by using

Duncan test (α = 0.01%). In each method, sig-

nificant difference among potato cultivars was

observed for disease to early blight (p < 0.01).

Results show that casmos cultivar is suscepti-

ble for resistance to early blight and in vitro

methods experiment had the same result.

Keywords: E arly Blight; AUDPC; Resistance;

Alternaria solani; Potato

1. INTRODUCTION

Early blight is a very common disease of both potato

and tomato. It causes leaf spots and tuber blight on po-

tato, and leaf spots, fruit rot and stem lesions on tomato

[1]. The disease can occur over a wide range of climatic

conditions and can be very destructive if it is left uncon-

trolled. Infection can cause serious yield losses in sus-

ceptible cultivars [2,3]. Potato plants are susceptible to a

wide variety of diseases that can severely reduce yield,

quality and storability of tubers. Diseases can occur in

the field or in storage and are caused by infectious bacte-

ria, fungi, viruses and other related organisms. Early

blight, caused by the A. solani fungus, is one of the main

diseases of potatoes in tropical climates, especially

where potato es are grown under irr igation , causing yield-

losses through defoliation of the plants. The fungicides

used to control the disease are expensive and frequently

inefficient [4]. Potato resistance to early blight is a quan-

titative trait, and obtaining successful resistant cultivars

is not simple [5-7]. It has been observed that resistance to

early blight is age-related: early-maturing cultivars are

more susceptible than late-maturing cultivars. A droplet

inoculation method was used for evaluation of tomato

resistance to early blight, caused by Alternaria solani

(Ellis & Martin) Sorauer. In this experiment method,

leaflets are inoculated with small droplets of a conidial

suspension in water or culture filtrate [8,9]. This method

was first introduced by Locke (1948) to find sources of

resistance to early blight (Locke, 1949). The droplet in-

oculation method has been used to evaluate early blight

resistance components (O’Leary and Shoemaker, 1983).

The direct method was described by [9,10]. Plantlets

were inoculated in a 18 × 2 cm test tube, containing 5 ml

of A. solani culture filtrate. Severity values were plotted

against time and the area under the disease progress

H. R. Mirkarimi et al. / Natural Science 5 (2013) 1189-1192

1190

curve (AUDPC) was calculated [11].

2. MATERIAL AND METHOD

2.1. Plant Material

The experiment was conducted during 2008-2009 un-

der in vitro conditions. Virus free clones of potato culti-

vars were obtained from the National plant gene bank of

Iran. Six cultivars were conducted Ells, Picasso, Mara-

dona, Marfona, Casmos and Desiree that the cultivar

Desiree is used as susceptible reference cultivar when

screening potato genotypes in Brazil [6,8]. Plantlets were

propagated through nodal cutting and kept in growth

chamber at 23˚C ± 2˚C, light with a period of 16 h light

and 8 h dark. After 4weeks-old the plants were trans-

ferred to in vivo conditions that plantlets were planted in

pots (one seedling per pot). The planting bed is include

pit/perlit/turb (2:1 :1), temperature and humid abou t 27˚C

- 33˚C, 75% - 80% respectively.

2.2. Sporulation and Culture Filtrate

The mycelia of an A. solani isolate were grown in

plastic Petri plates on potato carrot agar (PCA) in the

condition (8/16) light/darkness. After 10 days surface

mycelium was removed with 10 ml of sterile distilled

water (SDW) and a clean paintbrush and the suspension

was discarded. Then suspension with105 conidia/ml were

placed in 500 ml glass flasks containing 100 ml of potato

dextrose broth (PDB) medium and maintained in the

dark at 28˚C ± 2˚C. After 12 days the contents of glass

flasks were filtered through the Whatman filter 0.2 µm

and concentrated to centrifuge at 2000 - 2500 g for 10 -

15 min and the samples are centrifuged at a time.

2.3. In Vitro Selection by Droplet of Culture

Filtrate Method

Three replications per cultivar were placed whole in

vitro plantlets in an 18 × 2 cm test tube. The plantlets of

potato into test tube were inoculated by droplet of culture

filtrate method that the leaflet of potato received a 10 µl

droplet of the A. solani culture filtrate. The test tubes

were placed in a growth chamber at a temperature of

20˚C - 25˚C. The leaflets were rated according to Table 1

for reaction to the treatments 1 - 2 days after inoculation

until 6 days.

2.4. In Vitro Selection by Direct Using of

Culture Filtrate Method

Three replication per cultivar were inoculated by

placing whole in vitro plantlets in a 18 × 2 cm test tube

each, containing 5 ml of A. solani culture filtrate. This

study was conducted using factorial based on completely

randomized design (CRD) with 3 replications. The test

Table 1. Scale for evaluation of the damage produced by Al-

ternaria species in potato in v itro and greenhouse plants [12].

Rating of affect at ion Description of symptoms

1 no lesion development

2 lesions < 1-mm diameter

3 lesions 1- to 5-mm diameter

4 lesions > 5-mm diameter

tubes were placed for 6 - 7 days in a growth chamber at

20˚C - 25˚C, with a photosynthetic photon flow density

of 100 µE/m/s and a day length of 16 h [10]. During in

vitro assay the A. solani symptoms appear 1 - 3 days un-

til 6 days. For evaluation of the damage produced by A.

solani using the scale described in Table 1.

2.5. Pathogenicity Test

At the end of each of the above tests to ensure the ab-

sence of pathogens and other fo reign pathogenicity tests,

infected leaves after washing with tap water, placed in

sterile distilled water for one minute. Then by sodium

hypochlorite solution (% 0.5) for 35 seconds and re-steri-

lization were washed with sterile distilled water. Finally,

the pieces are placed on sterile filter paper (for drying)

and then transferred to the culture medium.

2.6. Statistical Analysis

The statistical analyses were accomplished using

MSTATC. AUDPC values were submitted to analysis of

variance (ANOVA) and treatment means were compared

using Duncan test (% 0.01).



 

AUDPC0.5 Yi1YiTi1Ti











Y = the response of plants based on Pryor & Michal-

ides.

i = shift notes – T = date of the Inoculation.

3. RESULT

Variance analysis square shows that significant differ-

ence between methods, cultivars and interaction methods

× cultivars (Table 2).

3.1. In Vitro Selection by Droplet of Culture

Filtrate Method

Significant different had between cultivars (Ta bl e 3 ).

Mean comparison showed that potato cultivars were

grouped to two classes. Ells, Marfona, Casmos and De-

siree cultivars were grouped at same class, and these

cultivars had a low resistance (Table 4). Picasso and

Maradona cultivars had a high lev el of resistance in com-

parison with other cultivars (p < 0.01).

H. R. Mirkarimi et al. / Natural Science 5 (2013) 1189-1192 1191

Table 2. Variance analysis square for AUDPC mean in in vitro

selection.

Source Degree of FreedomMean square F-value

Methods (A ) 1 1018.674 4731.9032**

Cultivars ( B ) 5 113.774 528.4968**

A × B 5 1.207 5.6065**

Error 24 0.215

total 35

CV% 1.69%

Table 3. Va riance analysis square for AUDPC mean in methods

pf evaluation.

Source Degree of

Freedom F-value Droplet

method F-value

Direct method

cultivars 5 317.827** 219.450**

Error 12

Total 17

CV % 1.39% 2.13%

Table 4. Mean comparison in in vitro method.

cultivars AUDPC droplet AUDPC direct

Ells 35.66 A 24 B

Picasso 27 B 16.66 C

Maradona 26.50 B 17.33 C

Marfona 35.83 A 24.66 AB

Casmos 36.16 A 25.33 A

Desiree 35.50 A 25 AB

3.2. In Vitro Selection by Direct Using of

Culture Filtrate Method

In direct method was observed significant difference

among potato cultivars (Ta bl e 3 ). Mean comparison in-

dicated that potato cultivars were grouped to four class

(Table 4). Result showed that Casmos cultivar was the

most sensitive in other cultivars. The other cultivars had

a high level of resistance in comparison with other culti-

vars.

4. DISSCUSION

Disease severity is a valuable component for studying

resistance to early blight. Disease severity assessments

could was done on lower, middle and upper leaves but

middle leaf assay is a useful factor for potato cultivars

evaluation [1,6]. Therefore in this research was used

from middle leaf for resistance level selection. Mean

comparison in vitro method (Table 4) showed that in

droplet method Desiree cultivar had low resistance level,

however in direct method Desiree cultivar had high level

the symptoms of early blight in vitro with were taken 1 -

3 days after inoculation that results is a similar too de-

scribed by [6]. Culture filtrate was used in in vitro condi-

tion and caused leaf necrotic, were similar to those

caused by infection through spores as was described by

[9,10,13].

Rodriguez et al. showed that in vitro direct method is

an effective in the evaluation of disease resistance of

potatoes wave spots [10]. While Locke showed that drip

into the glass, is a useful technique [14]. Our experi-

ments show that the efficiency of both methods is almost

identical. Thus, the results of both cover together.

5. CONCLUSION

Given the diversity of Alternaria species in the wor ld,

sources of resistance to the early blight disease are very

small, and sometimes can be found in the wild plant. The

transfer of genes from wild species is associated with

many problems. Accordingly, resistance to diseases is

considered as an advantage.

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