<i>In vitro</i> and greenhouse evaluation for resistance to early blight of potato isolated from <i>Alternaria alternata</i>

doi:10.4236/as.2013.49063

Paper Menu >>

Journal Menu >>

Vol.4, No.9, 473-476 (2013) Agricultural Sciences

http://dx.doi.org/10.4236/as.2013.49063

In vitro and greenhouse evaluation for resistance to

early blight of potato isolated from Alternaria

alternata

Hamid Reza Mirkarimi1*, Ahmad Abasi-moghadam2, Javad Mozafari2

1Department of Plant Breeding, Science and Research Branch, Islamic Azad University, Tehran, Iran;

*Corresponding Author: [email protected]

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

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

ABSTRACT

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

resistance.

Keywords: Potato; Early Blight; Alternaria;

Inoculation

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.

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. MATERIAL AND METHOD

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

H. R. Mirkarimi et al. / Agricultural Sciences 4 (2013) 473-476

474

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-

perature.

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. RESULT

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.

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

(Greenhouse)

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.

4. DISCUSSION

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.

REFERENCES

[1] Akhtar, P.K., Saleem M.Y., Asghar, M. and Haq, M.A.

(2004) New report of Alternaria alternate causing leaf

blight oftomato in Pakistan. Plant Pathology, 53, 816.

doi:10.1111/j.1365-3059.2004.01099.x

[2] Cassells, A.C. and Kowalski, B. (1998) Strategies for the

evaluation of variation as a source of resistance to early

and late blight of potato. In: Khurana, P., Chandra, R. and

Mahesh, D., Eds., Comprehensive Potato Biotechnology,

Malhotra Publishing House, New Delhi, 50-60.

[3] Christ, B.J. and Haynes, K.G.V. (2001) Inheritance to

early blight disease in a diploid potato population. Plant

Breeding, 120, 169-172.

doi:10.1046/j.1439-0523.2001.00565.x

[4] Dita Rodriguez, M.A., Brommonschenkel, S.H., Matsu-

oka, K. and Mizubuti, E.S.G. (2006) Components of re-

sistance to early blight in four potato cultivars: Effect of

leaf position. Journal of Phytopathology, 154, 230-235.

doi:10.1111/j.1439-0434.2006.01089.x

[5] Herriott, A.B., Haynes, J.F.L. and Shoemaker, P.B. (1990)

Inheritance of resistance to early blight disease in tetra-

ploid x diploid crosses of potatoes. Hortscience, 25, 224-

226.

[6] Barksdale, T.H. (1969) Resistance of tomato seedlings to

early blight. Phytopathology, 59, 443-446.

[7] Rodriguez, N.V., Kowalski, B., Rodriguez, L.G., Cara-

balloso, B., Suarez, M.A., Perez, P.O., Quintana, C.R.,

Gonzalez, N. and Ramos, R.Q. (2007) In vitro and ex vi-

tro selection of potato plantlets for resistance to early

blight. Journal of Phytopathology, 155, 582-586.

doi:10.1111/j.1439-0434.2007.01282.x

H. R. Mirkarimi et al. / Agricultural Sciences 4 (2013) 473-476

476

[8] Shaner, G. and Finney, R.E. (1977) The effect of nitrogen

fertilization in the expression of slow-mildewing resis-

tance in Knox wheat. Phytopathology, 67, 1051-1056.

doi:10.1094/Phyto-67-1051

[9] Pryor, B.M. and Michailides, T.J. (2002) Morphological,

pathogenic, and molecular characterization of Alternaria

isolates associated with Alternaria late blight of pistachio.

Phytopathology, 92, 406-416.

doi:10.1094/PHYTO.2002.92.4.406

[10] Martinez, P.R. and Sinclair, M. (1994) Selection in vitro

of resistance to Early Blight (Alternaria solani Sorauer.)

in Creole Potat oes (Solanum phureja Junz). Fitopatol Co-

lomb, 18, 90-100.

[11] Veitia, N., Dita, M.A., Garcia, L., Herrera, L., Bermu dez,

I., Acosta, M., Clavero, J., Orellana, P., Romero, C. and

Garcia, L. (2001) Use of tissue culture and the mutagene-

sis in vitro for the improvement of the resistance to Al-

ternaria solani (Solanum tuberosum Lin.), to var Desiree.

Biotecnología Vegetal, 1, 43-47.

[12] Rotem, J. and Feldman, S. (1965) The relation between

the ratio of yield to foliage and the incidence of early

blight in potato and tomato. Israeli Journal of Agricul-

tural Research, 15, 115-122.

[13] Thompson, P.G.. and Mendoza, H.A. (1984) Genetic vari-

ance estimates in a heterogenous potato population pro-

pagated from true seed. American Potato Journal, 61,

697-702. doi:10.1007/BF02852933