Vol.4, No.9, 473-476 (2013) Agricultural Sciences
In vitro and greenhouse evaluation for resistance to
early blight of potato isolated from Alternaria
Hamid Reza Mirkarimi1*, Ahmad Abasi-moghadam2, Javad Mozafari2
1Department of Plant Breeding, Science and Research Branch, Islamic Azad University, Tehran, Iran;
*Corresponding Author: rezamirkarimi21@gmail.com
2Department of Genetics & National Plant Gene-Bank of Iran, Seed and Plant Improvement Institute, Karaj, Iran
Received 23 June 2013; revised 25 July 2013; accepted 10 August 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.
Early blight of pot a to is caused by the fungus Al-
ternaria alternata, one of the most destructive
foliar diseases, especially in hot climates under
irrigation. In this study, the virus free potato
seedlings were obtained from the National Plant
Gene Bank of Iran and were inoculated in vitro
with a culture filtrate of A. alternate. The leaflets
received a 1000-µl droplet of the A. alternata
culture filtrate and were inoculated by spraying
with a suspension of 105 conidia/ml of isolate A.
alternata in the greenhouse method. The ex-
perimental design w as a completely randomized
design (CRD) with three replications and seven
genotypes, which have been infected with the
two leaves of each iteration. In vitro selection of
fungal isolates of A. alternata, chlorotic and ne-
crotic symptoms began 1 to 2 days after inocu-
lation, but the assessment of greenhouse symp-
toms appeared 6 - 10 days after inoculation. The
area under the disease progress curve values
were presented by analysis of variance (ANOVA),
and they were compared using Duncan’s test (a
= 0.01%). In both methods, there was a signifi-
cant difference between the potato genotypes (P
< 0.01). For In vitro selection and evaluation
greenhouse, Casmos were resistant to at least
figure and Marfona genotype had the highest
Keywords: Potato; Early Blight; Alternaria;
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.
The disease can occur over a wide variety of climatic
conditions and can be very critical if left uncontrolled.
Potato plants are susceptible to a wide diversity of dis-
eases that can severely reduce yield, qu ality and storabil-
ity of tubers. Diseases can occur in the field or in storage
and are caused by infectious bacteria, fungi, viruses.
Early blight, caused by the A. alternata fungus, is one of
the main diseases of potatoes in tropical climates, espe-
cially where potatoes are grown under irrigation. He de-
scribed the new report of A. alternata that was caused by
leaf blight of tomato in Pakistan [1]. The fungicides used
to control the disease are expensive and frequently inef-
ficient [2]. Potato resistance to early blight is a quantita-
tive trait, and obtaining successful resistant cultivars is
not simple [3-5]. It has been observed that resistance to
early blight is age-related: early-maturing cultivars are
more susceptible than late-maturing cultivars. A. alter-
nata is a well-known pathogen on many crops but a few
records report this fungus as a causal agent of leaf spot
on deciduous trees. Glasshouse tests using spray inocula-
tion of a conidial suspension on leaves are widely used
for conidial inoculum production techniques [6]. In vitro
selection is caused by the direct method that plantlets
were inoculated in an 18 × 2 cm test tube each, contain-
ing 5 ml of A. solani culture filtrate [7]. Severity values
were plotted against time and the area under the disease
progress curve (AUDPC) was calculated [8].
2.1. Plant Material
The experiment was conducted during 2008-2009 un-
der in vitro and ex vitro conditions. Virus free clones of
potato cultivars were obtained from the National plant
Copyright © 2013 SciRes. OPEN A CCESS
H. R. Mirkarimi et al. / Agricultural Sciences 4 (2013) 473-476
gene Bank of Iran. Seven cultivars were conducted Ells,
Picasso, Maradona, Marfona, Delta, Casmos and Desiree
that were propagated through nodal cutting every three
month and kept in growth chamber at 25˚C ± 1˚C light
with a period of 16 h light and 8 h dark.
2.2. Tissue Culture
In vitro plantlets of potato were multiplied routinely
by subculturing single node cuttings. Single node cut-
tings were propagated MS basal medium with 3% su-
crose and 0.7% ag ar in petri dishes (25 × 100 mm). Cul-
tures were placed in tissue culture growth room at 16
hour photoperiod and 25˚C ± 1˚C temperature system for
4 weeks. Fore week-old plantlets (4 - 6 cm long) were
transplanted in a plastic cover into a sterile mixture of
peat moss, perlite and turb (2:1:1) in pots (one seed- ling
per pot), temperature and humid about 27˚C - 33˚C, 75%
- 80% respectively. The plantlets were irrigated three
times at every day that was increased moist and tem-
2.3. Sporulation and Culture Filtrate
The mycelial (1 cm2) of an A. alternata isolate were
grown in plastic Petri plates on potato carrot agar (PCA)
in the condition (8/16) light/darkness. After 10 days sur-
face mycelium was removed with 10 ml of sterile dis-
tilled water (SDW) and a clean paintbrush and the sus-
pension was discarded. Then suspension with 105 co-
nidia/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 what
man filter 0.2 µm and concentrated to centrifuge at 2000
- 2500 g for 10 - 15 min and the samples are centrifuged
at a time.
2.4. In Vitro Selection
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. alternata culture filtrate. This
study was conducted using factorial based on completely
randomized design (CRD) with 3 replications. The test
tubes were placed for 72 h in a growth chamber at 22˚C
± 2˚C. with a photosynthetic photon flow density of 100
µE/m/s and a day length of 16 h [7]. During in vitro as-
say the A. alternata symptoms appear 2 - 3 days until 6
day. For evaluation of the damage produced by A. alter-
nata using the scale described in Table 1.
2.5. Greenhouse Evaluation
Three plant of each cultivar were inoculated by sporu-
lation of conidial suspension with 105 conidial/ml of A.
Table 1. Scale for evaluation of the damage produced by Al-
ternaria species in potato in vitro and greenhouse Plants [9].
Rating of affectation Description of symptoms
1 no lesion development
2 lesions < 1-mm diameter
3 lesions 1 - 5-mm diameter
4 lesions > 5-mm diameter
alternata isolate on leaflet in plant. After inoculation,
plants were kept for 24 h in a plastic at 25˚C, 12 h pho-
toperiod. After this time, plants were transferred to
greenhouse conditions. Early blight was allowed to de-
velop in the greenhouse through inoculation, recording
the intensity of affectation using the scale described in
Table 1.
2.6. Statistical Analysis
The statistical analyses were accomplished using Spss.
AUDPC values were submitted to analysis of variance
(ANOVA) and treatment means were compared using
Duncan test (% 0.01).
3.1. In Vitro Selection
During in vitro assay, the A. alternata symptoms ap-
pear 1 - 2 days after inoculation. Disease severity as-
sessments were taken every day beginning at 1 day until
6 day (Figure 1). Severity value observed in the disease
development curve. The severity value to area under the
disease progress curve (AUDPC) was calculated. Sig-
nificant different was observed amongst potato cultivars
(Ta b le 2). Mean comparison among the potato cultivars
indicated the cultivars were grouped into five class (Ta-
ble 3). Casmos had a high level of pathogenecity in
comparison with other cultivars and Marafona cultivar
was the highest resistant.
3.2. Greenhouse Evaluation
A. alternata chlorotic an d necrotic symptoms ap pear 6
- 10 days after inoculation in the greenhouse plantlets.
Disease severity assessments were taken every 2 days
beginning at 3 day until 21 day. Severity value in ob-
served the disease development curve (Figure 2). Early
blight severity was calculated as AUDPC varied accord-
ing to cultivar. Statistical analysis determined significant
differences between cultivars (Table 2). Cultivar resis-
tance levels was grouped into foure class. Results show
that Casmos was the most sensitive cultiv ar and Marfona
was the most tolerance cultivar.
Copyright © 2013 SciRes. OPEN A CCESS
H. R. Mirkarimi et al. / Agricultural Sciences 4 (2013) 473-476 475
Figure 1. Disease development curve in in vitro condition.
Tab le 2. Variance analysis square for AUDPC maen in in vitro
selecton and greenhouse evluation Obtained, on 10 notes.
Source Degrees of
Freedom F-value
(in vitro) F-value
Genotype 6 34.157** 106.043**
error 14
Total 20
Cv% 3.82% 4.41%
** = Significant at leve l 1%.
Table 3. Mean comparison in in vitro and greenhouse condition
for assessment of resistance level to early blight of potato iso-
lated from Alternaria alternate.
Cultivars of
potato Greenhouse
eveluation In vitro selection
ELLS 40 c 5.58 cd
PICASSO 32.83 d 5.41 cd
MARADONA 44.16 bc 5.91 c
MARFONA 25.83 e 5.25 d
DELTA 29 de 5.41 cd
CASMOS 54 a 7.41 a
DESIREE 45.66 b 6.5 b
The letter “a” is most sensitive, and the move to the next character, resis-
tance is greater.
This study was done on middle leaves with a result
similar to the observation [4] and middle leaf assay is a
useful factor for potato cultivars evaluation. Results and
disease symptom of A. alternata by infection culture
filterate in in vitro plantlets of potato cultivars were
similar to the symptom by infection sporulation in
gr eenhou se method as was described by [7,10,11]. Lesion
expansion rate has been used for assay observation and
disease severity and lesion expansion rate can be evalu-
ated with due scale. Disease severity assessments in in
vitro were taken every 2 days beginning at 6 until 21
Figure 2. Disease development curve in greenhouse condition.
days post inoculation that were described [4]. Lack of
resources or lack of resistan ce to Alternaria was declared
among potato lines, as well as among commercial potato
[12]. However, different levels of resistance have been
observed in the wild diploid potato [13]. Thus, the possi-
ble genotypes are safe, with a high level of resistance to
this disease, which can greatly affect wave spot disease
resistance and production of resistan t crop cultivars used.
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