Vol.5, No.11, 1189-1192 (2013) Natural Science
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: rezamirkarimi21@gmail.com
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
Copyright © 2013 Hamid Reza Mirkarimi et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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
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
Copyright © 2013 SciRes. OPEN ACCESS
H. R. Mirkarimi et al. / Natural Science 5 (2013) 1189-1192
curve (AUDPC) was calculated [11].
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-
i = shift notes – T = date of the Inoculation.
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).
Copyright © 2013 SciRes. OPEN ACCESS
H. R. Mirkarimi et al. / Natural Science 5 (2013) 1189-1192 1191
Table 2. Variance analysis square for AUDPC mean in in vitro
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-
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
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.
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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