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Effect of Salicylic Acid, DL-beta-amino-n Butyric Acid and Acibenzolar-s- methyl + metalaxyl on Mycelial Growth and Spore Germination of Alternaria mali in vitro and on Young Apple Seedlings.

Byline: Hulya Ozgonen and Ayse Karatas

Abstract

In order to determine the effects of chemical elicitors used for inducing resistance in plants against necrotic leaf spot caused by Alternaria mali, Salicylic acid (SA) with 0-700 ppm, DL-beta-amino-n-butyric acid (BABA) and Acibenzolar-s-methyl + metalaxyl (ASM) with 0-1500 ppm concentrations were tested on PDA. A. mali isolated from Red Jim apple variety and having high virulence was used. The spore germination tests were conducted at 24 C for 12 h on water agar medium included different concentrations of chemicals and the effects on hyphal length were determined. SA decreased the mycelial growth of A. mali with increasing concentration and it was inhibited at 700 ppm completely. SA had the same effect on spore germination inhibition at 300 ppm concentration. BABA had no significant effect on mycelial growth with increasing concentration. However, the hyphal development was decreased with increasing concentration and hyphal lysis occurred just after spore germination at 800 ppm concentration.

Mycelial growth of A. mali was decreased by increasing ASM concentration as well, but it was not inhibited completely. However, spore germination decreased by increasing concentration and hyphal lysis occurred at 500 ppm. In vivo tests, plant activators were found effective with increasing concentration. SA, BABA and ASM at 100 ppm concentration reduced disease severity by 89.5%, 87.6% and 87.6% on young apple seedlings, respectively. Plant activators that induce host resistance have potential for the control of necrotic leaf spot disease. (c) 2012

Friends Science Publishers

Keywords: Salicylic acid; DL-beta-amino-n-butyric-acid; Acibenzolar-s-methyl; Alternaia mali; Apple

Introduction

Necrotic leaf spot caused by Alternaria mali Roberts is an important disease after apple scab and shows common symptoms in some cultivars (Ozgonen and Karaca, 2006). The first signs of symptoms of the disease are small, round, brown spots on the leaves. Spots are 2-5 mm in diameter and surrounded by purple margin and sometimes become darker and more irregular showing secondary expansion. Severe infection results in defoliating on apple trees. Chemical control of A. mali provided through using of fungicides such as iprodione, mancozeb and captan (Lee and Kim, 1986; Osanai et al., 1987).

Induced resistance to plant diseases has been a method used as an alternative to the fungicides against plant pathogens in recent years. To induce systemic and local resistance against diseases, biotic such as bacterial and fungal cell wall fragment, weakened or dead spore cultures, non-pathogenic strains and abiotic inducers such as UV, heavy metals, herbicides, ethylene and other chemicals are used. A wide range of special compounds such as salicylic acid, butyric acid isomers and acibenzolar-s-methyl were used effectively as abiotic agents and these chemicals applications resulted in systemic acquired resistance in plants (Kuc, 1995; Yang et al., 1997; Cohen, 2002). Chemical applications activate natural defense mechanisms against pathogens in plants that provide protection from the stress conditions and external factors (Dempsey and Klessig, 1995). Salicylic acid, Beta amino butyric acid and Acibenzolar-s-methyl are important signaling molecule in promoting resistance in plants.

Active substances are rapidly taken from leaves and act as a systemic signal in the plant (Jakab et al., 2001). Induced resistance and its mechanisms have been investigated for the control of many plant diseases (Baysal et al., 2005; Alkahtani et al., 2011).

Present study aimed to evaluate the antimicrobial action of inducers including salicylic acid, DL-B-amino-n butyric acid and acibenzolar-s-methyl + metalaxyl on mycelial growth and spore germination of A. mali in vitro and on young apple seedlings under controlled conditions.

MATERIALS AND METHODS

Materials

In this study, virulent A. mali isolated from Red Jim apple

cultivar was used. Salicylic acid (Merck, S4404331), DL-B- amino-n-butyric acid (Fluka, GA13766), Acibenzolar-s- methyl + metalaxyl (BION, Syngenta) were used as inducers.

Effect of Chemicals on Mycelial Growth and Spore

Germination of A. mali In Vitro

To determine the chemical effects on A. mali, different concentrations of salicylic acid (SA), DL-B-amino-n-butyric acid (BABA) and acibenzolar-s-methyl + Metalaxyl (ASM) were tested. Mycelial growth retardation were tested for SA at the concentration of 0-700 ppm and for BABA and ASM between 0-1500 ppm adjusted from stock solutions; while concentrations were selected as 0.1-300 ppm for SA and 0.1-800 for BABA and 0.1-500 ppm for ASM for spore germination.

Potato dextrose agar (PDA) composed of potato infusion from 200 g, D(+)-glucose (2%) and agar (1.5%) per liter was prepared (pH 5.6). Then, PDA was divided into 10 mL poured into the 15 mL glass test tubes and sterilized at 121oC and 1 kPA. After cooling, different concentrations of chemicals from stock solutions were added into the medium then poured into the petri dishes. Media without addition of chemicals was prepared for comparison. The fungus was cultured on PDA plates at 24oC for 7 days. Chemicals added to petri dishes were inoculated with 6 mm diameter of mycelial disc of A. mali using cork borer and incubated at 24degC. After 5 days, colony diameters were measured. Experiment was conducted in a completely randomized design with 5 replicates.

Water agar (WA) was prepared at amount of 10 ml in glass test tubes. After autoclaving and cooling, different concentrations of chemicals were added and poured into the petri dishes. A. mali was cultured on PDA for 10 days and sterilized water was added to the cultures and spores scraped with a spatula. Then, mixtures of spores and mycelium were suspended through two layers cheesecloth. Spore suspension concentration was adjusted to 106 spore mL-1 using haemocytometer and 100 mL of spore suspension was spread over the surface of the media using a rod then incubated at 24degC. Hyphal length of germinated spores was measured after 12 h under light microscope with ocular micrometer. Experiment was conducted with 5 replicates. Measurements of hyphal length of germinated spores were performed measuring 10 hyphal length in 3 microscopic field of view totally 30 measurement from each replicates.

Effect of Chemicals on A. mali in Apple Seedlings under

Controlled Conditions

Experiment was conducted on young seedlings cv Red Jim and plants were maintained under controlled conditions at 24+-2oC, RH 70+-5%, 14 h photoperiod. Stock solutions of SA, BABA and ASM were prepared at 25, 50 and 100 ppm concentration and sprayed to leaves of young apple seedlings using hand sprayer. After 24 h, spore suspension at 106 conidia mL-1 of A. mali were sprayed to leaves. Control plants were sprayed with distilled water before inoculation. Disease severity was evaluated using 0-5 scale by Horsfall (1986): where: 0: No signs of symptoms, 1: lesions covered leaf surface by 0-3%, 2: lesions covered leaf surface by 4-6%, 3: lesions covered leaf surface by 7-12%, 4: lesions covered leaf surface by 13-25%, 5: lesions covered leaf surface by 26-50%.

Statistical Analysis

Data were subjected to analysis of variance and the differences were compared by LSD multiple comparison tests. The effects of chemicals on the development of the mycelial growth and spore germination of pathogen (% effect) were calculated by the Abbott formula (Karman, 1971).

RESULTS

Effect of Chemicals on Mycelial Growth of A. mali

The effects of different concentrations of SA, BABA and ASM on mycelial growth of A. mali were determined. The results of the effect of SA at 0-700 ppm were summarized in Table I. SA decreased the mycelial growth with increasing concentration. The mean colony diameter was 84.7 mm in control while it was decreased to 19.0 mm at 600 ppm concentration. SA inhibited the mycelial growth at 700 ppm completely.

Concentrations of 100-1500 ppm BABA did not affect the mycelial growth of A. mali and colony diameter did not differ significantly (Table II). Although BABA reduced the mycelial growth of A. mali with increasing concentration up to 1500 ppm, it was not inhibited completely. The colony diameter was found 49.0 mm in control and 44.3 mm at 1500 ppm, respectively. Similarly, ASM did not inhibit the mycelial growth of A. mali completely, although it reduced the mycelial growth with concentration up to 1500 ppm. The colony diameter in control petri dishes was found as 62.0 mm but it was reduced to 44.3 mm at 1500 ppm (Table 3).

Effect of Chemicals on Spore Germination of A. mali In Vitro

The effects of different concentrations of SA, BABA and ASM on hyphal growth of A. mali were determined. SA decreased spore germination of A. mali with increasing concentration (Table IV). Hyphal length of germinated spore was 58.1 um in control, while it decreased to 3.2 um at 200 ppm concentration and completely inhibited at 300 ppm concentration. Similarly, BABA and ASM decreased spore germination of A. mali with increasing concentration, also (Table V and VI). Hyphal length of germinated spore was decreased to 2.5 um at 800 ppm for BABA and 2.0 um

Table 1: The effects of SA on mycelial development of A. mali

SA concentrations (ppm)###Means colony diameter (mm) % Effect

100###83.0 e###2.0

200###73.3 d###13.4

300###55.3 d###34.6

400###36.3 c###57.1

500###36.0 c###57.5

600###19.0 b###77.6

700###0.0 a###100.0

Control###84.7 e###-

Table 2: The effects of BABA on mycelial development of A. mali

BABA concentrations (ppm)###Means colony diameter (mm)###% Effect

100###49.0 ab###10.9

200###49.3 ab###10.3

300###49.0 ab###10.9

400###49.0 ab###10.9

500###48.7 ab###11.5

600###46.0 a###16.4

700###46.0 a###16.4

800###46.0 a###16.4

900###46.3 a###15.8

1000###45.3 a###17.6

1100###45.3 a###17.6

1200###45.3 a###17.6

1300###45.3 a###17.6

1400###45.3 a###17.6

1500###44.3 a###19.4

Control###55.0 ab###-

Table 3: The effects of ASM on mycelial development of A. mali

ASM concentrations (ppm)###Means colony diameter (mm)###% Effect

100###61.7 cd###0.5

200###61.3 cd###1.1

300###60.0 c###3.2

400###60.0 c###3.2

500###59.3 c###4.3

600###55.0 c###11.3

700###54.7 c###11.8

800###55.7 c###10.2

900###49.0 ab###21.0

1000###49.0 ab###21.0

1100###49.7 ab###19.9

1200###45.7 a###26.3

1300###46.0 a###25.8

1400###45.7 a###26.3

1500###44.3 a###28.5

Control###62.0 e###-

Means within the column was following by different letters are significantly different (P=0.05) according to Fisher's LSD test % effect was calculated using Abbott formula

at 500 ppm for ASM, respectively and completely inhibited at these concentrations.

Effect of Chemicals on Disease Severity of A. mali In Vivo

Disease severity (%) of A. mali on leaves was reduced by all tested chemicals with increasing concentration (Table VII). The disease severity was 70% at control plants, while it was decreased to 7.3% at 100 ppm of SA concentration. BABA

Table 4: The effects of SA on spore germination of A. mali

SA concentrations (ppm)###Means hyphal length (mu) m)###% Effect

0.1###56.9 f###2.2

1###56.7 f###2.5

5###56.3 f###3.2

10###43.2 e###25.6

50###33.1 d###43.1

100###12.4 c###78.7

200###3.2 b###94.5

300###0.0 a###100

Control###58.1 f###-

Table 5: The effects of BABA on spore germination of A. mali

BABA concentration(ppm)###Means hyphal length (mu)m)###% Effect

0.1###57.3 f###1.5

1###57.2 f###1.6

5###55.8 f###4.1

10###34.8 e###40.1

50###24.0 d###58.7

100###23.7 d###59.2

200###19.0 d###67.3

400###12.6 c###78.3

600###7.3 b###87.4

800###2.5 a###100.0

Control###58.1 f###-

Table 6: The effects of ASM on spore germination of A. mali

ASM concentration (ppm)###Means hyphal diameter (mu) m)###% Effect

0.1###56.8 f###2.4

1###56.3 f###3.2

5###52.2 f###10.3

10###41.3 e###29.0

50###17.2 d###70.4

100###13.3 cd###77.1

200###9.3 c###84.0

300###9.1 c###84.3

400###5.5 b###90.5

500###2.0 a###100.0

Control###58.1 f###-

Table 7: The effects of plant activator on A. mali in vivo

Treatments###Disease severity (%)###% Effect

Control###70.0 a###-

SA 25 ppm###25.3 e###63.8

SA 50 pmm###15.3 f###78.1

SA 100 ppm###7.3 g###89.5

BABA 25 ppm###34.0 d###51.4

BABA 50 ppm###14.0 f###80.0

BABA 100 ppm###8.7 fg###87.6

ASM 25 ppm###50.7 b###27.6

ASM 50 ppm###39.3 cd###43.8

ASM 100 ppm###8.7 fg###87.6

Means within the column was following by different letters are significantly different (P=0.05) according to Fisher's LSD test

and ASM showed similar effect on disease and the disease severity was found 8.7% at 100 ppm concentration for BABA and ASM, respectively.

DISCUSSION

All tested compounds reduced the mycelial development of pathogen and/or hyphal length from germinated spores in vitro and provided control by inducing resistance in vivo.

In our study, SA, decreased the mycelial growth of A. mali with increasing concentration and completely inhibited at 700 ppm. Ozgonen et al. (2001) reported that SA inhibited the mean colony diameter of F. oxysporum f.sp lycopersici at 0.6 mM completely in vitro. El-Moughy (2002) revealed that the inhibitory effect of SA and acetyl salicylic acid to the growth and sporulation of some plant pathogenic fungal disease including Fusarium solani f.sp pisi, Rhizoctonia solani, Sclerotium rolfsii. In present study, BABA had no effect on the mean colony diameter of A. mali between 100-1500 ppm concentrations. Similarly, ASM did not reduce the mycelial development of A. mali in vitro. In some studies reported that despite the ineffectiveness on the development of mycelial growth of pathogens in vitro, it prevent the growth of pathogen in plants by promoting plant resistance against diseases following applications (Cohen et al., 1994; Sunwoo et al., 1996; Tosi et al., 1998; Agostini et al., 2003; Ozgonen, 2004).

According to the results of microscopic examination, SA, BABA and ASM showed a decreasing effect on spore germination of A. mali with increasing concentration. SA had a similar effect on the germinated spores and it was inhibited at 300 ppm completely. The hyphal growth from germinated spore reduced compared to control up to 50 ppm of BABA, significantly. ASM reduced the mean hyphal length of germinated spores up to 10 ppm concentration, also. In another study, SA inhibited spore germination of F. oxysporum over 5 mM completely (Mahdy et al., 2009). Similarly, Porat et al. (2003) tested concentrations of BABA between 1 to 100 mM to Penicillium digitatum in grapefruit and revealed that increasing concentration exhibited direct antifungal activity and inhibited spore germination and germ tube elongation in vitro.

In the present study, SA, BABA and ASM reduced the disease severity by 89.5%, 87.6% and 87.6% at 100 ppm concentration, respectively. Induced-resistance compounds evaluated for the control of diseases of other agricultural crops and provided significant reduction in disease. BABA applied at 50 mM concentration for 6 h as seed treatment provided the maximum seedling vigor and protected the plants by 75% compared to control. After the second inoculation to plants, disease severity in control and BABA treated plants were 71-76% and 10-12%, respectively (Shailasree et al., 2001). Ishii et al. (1999) tested the different concentrations of ASM on Cladosporium cucumerinum, Colletotrichum lagenarium, Fusarium oxysporum f.sp. cucumerinum and Corynespora cassiicola in cucumber; Venturia nashicola in pear, A. alternata pathotype Japan pear and Gymnosporangium asiaticum; Botrytis cinerea in grapevine and Didymella bryoniae in melon.

ASM did not inhibit the mycelial development and conidial germination of all tested fungal pathogens in vitro. However, ASM at 100 ug.mL-1, controlled the C. lagenarium and C. cucumerinum in cucumber; G. asiaticum in pear (75.9%) under pot conditions effectively. Brisset et al. (2000) reported that ASM induced the systemic acquired resistance in pear against fire blight of pear caused by Erwinia amylovora. ASM at 100 and 200 mg/l a.i was applied before artificial inoculation of Golden Delicious seedlings was provided protection against disease. The level of protection against seedling disease in the greenhouse and garden trees were 50% and 69%, respectively. Smith- Becker et al. (2003) reported that ASM provided protection via the systemic acquired resistance against fungal pathogen Colletotrichum lagenarium and cucumber mosaic virus of melon.

ASM at 50 or 100 mg/mL concentrations provided complete protection against fungal pathogens and the spread of cucumber mosaic virus was delayed effectively under greenhouse conditions. In another study, Alkahtani et al. (2011) reported that abiotic elicitors including oxalic acid, potassium oxalate, salicylic acid, Bion, Fungastop and Photophor) induced resistance against powdery mildew (Sphaerotheca fuliginea) disease of cucumber via biochemical change in both pathogenesis-related proteins (PR) and phytoalexin accumulation in treated plants comparing with the control. Pretreatment of cucumber plants with all tested elicitors recorded a decrease in powdery mildew disease severity but Bion recorded the most effective inducers (63.8-72.4%).

This study presents the practical relevance on the use of SA, BABA and ASM by single application of young seedlings before pathogen attack for controlling necrotic leaf disease in apple caused by A. mali.

CONCLUSION

SA, BABA and ASM have been widely investigated for the control of disease of agricultural crops. The induced- resistance products evaluated for control of diseases have provided significant reduction. Current results confirmed the effectiveness of these products for control of foliar disease of apple. SA showed maximum inhibitory effect on radial growth of fungus. SA and BABA were highly active on spore germination in vitro and in vivo as pure active substance. Also, ASM showed similar effect in vivo and could be used beside the natural compounds as a potent resistance activator against A. mali. In conclusion, it would be better to use abiotic inducers as alternatives to the fungicides against necrotic leaf spot of apple.

REFERENCES

Agostini, J.P., P.M. Bushong and L.W. Timmer, 2003. Greenhouse evaluation of products that induce host resistance for control of scab, melanose and Alternaria brown spot of citrus. Plant Dis., 87: 69-74

Alkahtani, M., S.A. Omer, M.A. El-Naggar, E.M. Abdel-Kareem and M.A. Mahmoud, 2011. Pathogenesis-related Protein and Phytoalexin Induction against Cucumber Powdery Mildew by Elicitors. Int. J. Plant Pathol., 2: 63-71

Baysal, O., C. Turgut and G. Mao, 2005. Acibenzolar-S-methyl induced resistance to Phytophthora capsici in pepper leaves. Biol. Plant., 49: 599-604

Brisset, M.N., S. Cesbron, S.V. Thomson and J.P. Paulin, 2000. Acibenzolar-S-methyl

induces the accumulation of defence related enzymes in apple and protects from fire blight. Eur. J. Plant Pathol., 106: 529-536

Cohen, Y.R., 2002. beta-Aminobutyric acid-induced resistance against plant pathogens. Plant Dis., 86: 448-457

Cohen, Y., T., Niderman, E. Mosinger and R. Fluhr, 1994. -Aminobutyric acid induces the accumulation of pathogenesis-related proteins in tomato (Lycopersicon esculentum L.) plants and resistance to late blight infection caused by Phytophthora infestans. Plant Physiol., 104: 59-66

Dempsey, D.A. and D.F. Klessig, 1995. Signals in plant disease resistance. Bull. Inst. Pasteur., 93: 167-186

El-Moughy, N.S., 2002. In vitro studies on antimicrobial activity of salicyclic acid and acetylsalicyclic acid as pesticidal alternatives against some soilborne plant pathogens. Egypt. J. Phytopathol., 30: 41-55

Horsfall, J.G. 1986. This week's citation classic: Horsfall J.G. and R.W. Barratt. An improved grading system for measuring plant disease. Phytopathology, 35: 655. 1945. Agric. Biol. Environ. Sci., 15: 14

Ishii, H., Y. Tomita, T. Horio, Y. Narusaka, Y. Nakazawa, K. Nishimura and S., Iwamoto, 1999. Induced resistance of acibenzolar-S-methyl (CGA 245704) to cucumber and Japanese pear disease. Eur. J. Plant Pathol., 105: 77-85

Jakab, G., V. Cottier, V. Toquin, G. Rigoli, L. Zimmerli, J.P. Metraux and B. Mauch-Mani, 2001. b-Aminobutyric acid-induced resistance in plants. Eur. J. Plant Pathol., 107: 29-37

Karman, M., 1971. Bitki Koruma Arastirmalarinda Genel Bilgiler, Denemelerin Kurulusu ve Degerlendirme Esaslari, p: 279. Bornova Izmir, Turkey

Kuc, J., 1995. Sytemic induced resistance. Asp. Appl. Biol. 42: 235-242.

Lee, C.V and K.H. Kim, 1986. Cross tolerance of Alternaria mali to various fungicides. Kor. J. Mycol., 14: 71-78

Mahdy, A.M.M., A.O. Sagitov and G.A. Ahmed. 2009. Efficacy of some Antioxidants for Control of Cucumber Fusarium wilt Disease under Greenhouses, p: 7. Republican Scientific-theoretical conference from 23-25 in Republic of Kazakhstan

Osanai, M., N. Suzuki, C. Fukushima and Y. Tanaka, 1987. Reduced Sensitivity to Captan of Alternaria mali Roberts, Vol. 38, pp: 72-73. Annual Report of the Society of Plant Protection of North Japan

Ozgonen, H., M. Bicici and A. Erkilic, 2001. The Effect of Salicyclic Acid and Endomycorrhizal Fungus Glomus etunicatum on Plant Development of Tomatoes and Fusarium Wilt Caused by Fusarium oxysporum f.sp. lycopercisi. Turk. J. Agric For., 25: 25-29

Ozgonen, H., 2004. Biberde Kokbogazi Yanikligi (Phytophthora capsici Leonian)'na Karsi Mikorizal Funguslar, Salisilik Asit ve B-amino-n-butirik asit ile Dayanikliligin Tesvik Edilmesi, p: 122. Doktora Tezi.

Cukurova Universitesi, Fen Bilimleri Enstitusu, Bitki Koruma Anabilim Dali, Adana, Turkey

Ozgonen, H. and G. Karaca, 2006. First report of Alternaria mali causing necrotic leaf spot of apples in Turkey. Plant Pathol., 55: 578

Porat, R., V. Vinokur, B. Weiss, L. Cohen, A. Daus, E.E. Goldschmidt and S. Droby. 2003. Induction of resistance to Penicillium digitatum in grapefruit by beta-aminobutyric acid. Eur. J. Plant Pathol., 109: 901-907

Smith-Becker, J., N.T. Keen, and J.O. Becker, 2003. Acibonzolar-S-methyl induces resistance to Coletotrichum lagenarium and cucumber mosaic virus in cantaloupe. Crop Prot., 22: 769-774

Shailasree, S., B.R. Sarosh, N.S. Vasanthi and H.S. Shetty, 2001. Seed treatment with beta-aminobutyric acid protects Pennisetum glaucum sistemically from Sclerospora graminicola. Pest Manage. Sci., 57: 721-728

Sunwoo, J.Y., Y.K., Lee and B.K. Hwang, 1996. Induced resistance against Phytophthora capsici in pepper plants in response to DL-b-amino-n- butyric acid. Eur. J. Plant Pathol., 102: 663-670

Tosi, L., R. Luigetti and A. Zazzerini, 1998. Induced resistance against Plasmopara helianthi in sunflower plants by DL-b-amino-n-butyric acid. J. Phytopathol., 146: 295-299

Yang, Y., J. Shahand and D.F. Klessig, 1997. Signal perception and transduction in plant defense responses. Genes Dev., 11: 1621-1639

1University of Suleyman Demirel, Agricultural Faculty, Department of Plant Protection, 32260 Isparta, Turkey

For correspondence: hozgonen@yahoo.com; hulyaozgonen@sdu.edu.tr

To cite this paper: Ozgonen, H. and A. Karatas, 2013. Effect of salicylic acid, DL-B-amino-n butyric acid and acibenzolar-s-methyl + metalaxyl on mycelial growth and spore germination of Alternaria mali in vitro and on young apple seedlings. Int. J. Agric. Biol., 15: 165-169
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