Plant Defence Activators Inducing Systemic Resistance in...

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Indian Journal of Biotechnology Vol 2. October 2003, pp 591-595 Plant Defence Activators Inducing Systemic Resistance in Zingiber officinale Rose. against Pythium aphanidermatum (Edson) Fitz. N C Karmakar, Rajyasri Ghosh and R P Purkayastha* Department of Botany, University of Calcutta, Kolkata 700 019, India Received 15 January 2002; accepted 17 March 2003 Selected plant defence activators were employed to induce systemic resistance in a susceptible cultivar of ginger against Pythium aphanidermatum (Edson) Fitz., a severe rhizome rot causing pathogen. Among the 6 cultivars of ginger tested, Varada was most susceptible, followed by Suprabha and Maran. Prior to sowing, soaking of rhizome seeds for 1 hr in 5 mM salicylic acid (SA), DL-I3-aminobutyric acid (BABA) or 2,1,3-benzothiadiazole (BTH), significantly reduced the disease. Systemic protection against disease was evident even after 8 weeks of treatment. Analysis of leaf proteins of non-inoculated, inoculated and activator-treated both non-inoculated and inoculated plants (cv. Suprabha) by SDS-PAGE (Sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and Image Master VDS-ID gel analysis version 3.0 showed 18 protein bands including 3-5 defence related proteins having molecular masses 67, 56, 32, 20 and 14 kDa. These defence proteins increased in activator-treated inoculated plants and enhanced the resistance against P. aphanidermatum. In vitro growth response of P. aphanidermatum to different conc. of SA, BABA and BTH was tested. SA and BABA,respectively were the most and least inhibitory to the fungus. Keywords: ginger, Pythium aphanidermatum, rhizome rot, defence activators, systemic resistance Introduction Induction of systemic resistance in plants against a broad spectrum of microorganisms by various plant defence activators and role of pathogenesis related proteins (PR-proteins) have been discussed earlier in detail by several workers (Kessmann et al, 1994; Sticher et al, 1997; Shetty & Kumar, 1999; Dow et al, 2000). It was demonstrated that salicylic acid, 2-6, dichloroisonicotinic acid, DL-~-aminobutyric acid and benzo-(1,2,3)-thiadiazole-7-carbothioic acid S- methyl ester could induce resistance against rust of groundnut (Sathiyabama & Balasubramanian, 1999), powdery mildew of barley (Kogel et al, 1994), late blight of tomato (Cohen & Gisi, 1994) and bacterial speck of tomato (Thaler, 1999). Production/accumulation of PR-proteins in host plants in response to infection or treatment with chemical elicitors/activators has also been related to induce resistance in plants (Shewry & Lucas, 1997). One of the approaches to utilize PR- proteins/defence related proteins for enhancing natural resistance is an effective manipulation of endogenous pathways leading to expression of such proteins. It may be possible to exploit the natural * Author for correspondence: Tel: 033~24753681, ext. 304 E-mail: [email protected] signalling mechanisms that alert plants to the attack of pathogens (Ryals et al, 1992; Shewry & Lucas, 1997). Previous studies indicate that SA is synthesized via shikimate-phenyl propanoid pathway and acts as a natural signal that triggers the systemic induction of PR-proteins and disease resistance in plants (Sticher et al, 1997). Therefore, an attempt has been made to induce systemic resistance in a susceptible ginger cultivar against rhizome rot disease caused by Pythium aphanidermatum and to detect defence related proteins in ginger leaves after induction of resistance by different plant defence activators. The purpose of this communication is to focus a non- conventional, alternative (to fungicides) approach to control rhizome rot of ginger through exploitation of defence proteins and it can be made possible by chemical or herbal activation of defence protein (DP) synthesis, direct use of DP or transgenic plants having high levels of DP. Materials and Methods Rhizome seeds of 6 cultivars (Pulpally, Kunduli, Himachal, Maran, Varada and Suprabha) of ginger (Zingiber officinale Rosc.) were disinfected, sown in earthenware pots [one rhizome seed (5-6 em long)/pot -20 ern diam] containing sterilized sandy soil and kept under ordinary conditions of light and

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Indian Journal of BiotechnologyVol 2. October 2003, pp 591-595

Plant Defence Activators Inducing Systemic Resistance in Zingiber officinaleRose. against Pythium aphanidermatum (Edson) Fitz.

N C Karmakar, Rajyasri Ghosh and R P Purkayastha*Department of Botany, University of Calcutta, Kolkata 700 019, India

Received 15 January 2002; accepted 17 March 2003

Selected plant defence activators were employed to induce systemic resistance in a susceptible cultivar of gingeragainst Pythium aphanidermatum (Edson) Fitz., a severe rhizome rot causing pathogen. Among the 6 cultivars ofginger tested, Varada was most susceptible, followed by Suprabha and Maran. Prior to sowing, soaking of rhizomeseeds for 1 hr in 5 mM salicylic acid (SA), DL-I3-aminobutyric acid (BABA) or 2,1,3-benzothiadiazole (BTH),significantly reduced the disease. Systemic protection against disease was evident even after 8 weeks of treatment.Analysis of leaf proteins of non-inoculated, inoculated and activator-treated both non-inoculated and inoculatedplants (cv. Suprabha) by SDS-PAGE (Sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and ImageMaster VDS-ID gel analysis version 3.0 showed 18 protein bands including 3-5 defence related proteins havingmolecular masses 67, 56, 32, 20 and 14 kDa. These defence proteins increased in activator-treated inoculated plantsand enhanced the resistance against P. aphanidermatum. In vitro growth response of P. aphanidermatum to differentconc. of SA, BABAand BTH was tested. SA and BABA,respectively were the most and least inhibitory to the fungus.

Keywords: ginger, Pythium aphanidermatum, rhizome rot, defence activators, systemic resistance

IntroductionInduction of systemic resistance in plants against a

broad spectrum of microorganisms by various plantdefence activators and role of pathogenesis relatedproteins (PR-proteins) have been discussed earlier indetail by several workers (Kessmann et al, 1994;Sticher et al, 1997; Shetty & Kumar, 1999; Dow et al,2000). It was demonstrated that salicylic acid, 2-6,dichloroisonicotinic acid, DL-~-aminobutyric acidand benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester could induce resistance against rust ofgroundnut (Sathiyabama & Balasubramanian, 1999),powdery mildew of barley (Kogel et al, 1994), lateblight of tomato (Cohen & Gisi, 1994) and bacterialspeck of tomato (Thaler, 1999).Production/accumulation of PR-proteins in host plantsin response to infection or treatment with chemicalelicitors/activators has also been related to induceresistance in plants (Shewry & Lucas, 1997).

One of the approaches to utilize PR-proteins/defence related proteins for enhancingnatural resistance is an effective manipulation ofendogenous pathways leading to expression of suchproteins. It may be possible to exploit the natural

*Author for correspondence:Tel: 033~24753681, ext. 304E-mail: [email protected]

signalling mechanisms that alert plants to the attack ofpathogens (Ryals et al, 1992; Shewry & Lucas, 1997).Previous studies indicate that SA is synthesized viashikimate-phenyl propanoid pathway and acts as anatural signal that triggers the systemic induction ofPR-proteins and disease resistance in plants (Sticheret al, 1997). Therefore, an attempt has been made toinduce systemic resistance in a susceptible gingercultivar against rhizome rot disease caused byPythium aphanidermatum and to detect defencerelated proteins in ginger leaves after induction ofresistance by different plant defence activators. Thepurpose of this communication is to focus a non-conventional, alternative (to fungicides) approach tocontrol rhizome rot of ginger through exploitation ofdefence proteins and it can be made possible bychemical or herbal activation of defence protein (DP)synthesis, direct use of DP or transgenic plants havinghigh levels of DP.

Materials and MethodsRhizome seeds of 6 cultivars (Pulpally, Kunduli,

Himachal, Maran, Varada and Suprabha) of ginger(Zingiber officinale Rosc.) were disinfected, sown inearthenware pots [one rhizome seed (5-6 em long)/pot-20 ern diam] containing sterilized sandy soil andkept under ordinary conditions of light and

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592 INDIAN J BIOTECHNOL, OCTOBER 2003

temperature (28-32°C) of the experimental garden ofthe department. Usually 4-week-old plants wereinoculated with Pythium aphanidermatum (Edson)Fitz. The inoculum was prepared by growing thepathogen in sand-maize meal medium (9:1) for 10-14days and subsequently mixed with the top soil in eachpot (50 g/pot). Disease severity was assessed after 4weeks of inoculation. Percentage loss in fresh weightof rhizomes in relation to control was noted. Cultivarsshowing <10%, up to 50% and above 50% loss infresh weight of rhizomes were categorized as least,moderate and highly susceptible. Yellowing of leavesis one of the important symptoms of rhizome rotdisease of ginger. Yellowing Index (Y.!.) per plantwas calculated as follows:

Y I / I_Total No. of leaves showing yellowing symptom.. pant-------------------~----~~~---

Total No. of test plants per treatment

To induce resistance in ginger plants, 5 mM ofthree plant defence activators, namely salicylic acid(SA), 2,1,3-Benzothiadiazole (BTH) and DL-~-aminobutyric acid (BABA) were used separately forsoaking rhizome seeds for 1 hr prior to sowing.Rhizome seeds soaked in water for 1 hr prior tosowing were treated as control. Treated and untreatedplants (28-day-old) were inoculated with P.aphanidermatum and disease intensity was measuredafter 4 weeks of inoculation. The cultivar, Suprabhawas used throughout the experiment instead of Varadabecause of insufficient availability of its plantmaterial.

For extraction and estimation of proteins, leaftissues of ginger (Suprabha) were kept for 1 hr at-20°C, homogenised (4 ml gol) in prechilled mortarand pestle with 0.1 M Tris-HCl buffer (pH 7.5)supplemented with 1 mM PMSF (Bera &Purkayastha, 1999; Stadnik & Buchenauer, 2000).The extracts were strained through muslin cloth,centrifuged at 10,000 rpm for 20 min at 4°C. Thesupernatant was concentrated by a lyophilizer andconcentration of protein was adjusted to 3 mg ml'following the method of Lowry et al (1951), usingBovine Serum Albumin (BSA) as standard.

Analysis of leaf proteins was carried out by sodiumdodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) using 13.5% (w/v) acrylarnide gel.Sigma wide range molecular weight markers (range:205 kDa-6.5 kDa). were used as standard. Afterelectrophoresis, gels were stained overnight with0.03% Coomassie brilliant blue and destained in a

methanol: acetic acid:water (40:7:53 and 5:7:88)mixture. For further analysis, Image Master VDS-JDGel Analysis version 3.0 was used.

Growth response of P. aphanidermatum to plantdefence activators was tested by growing theorganism in glucose-asparagine medium (g/I -glucose, 30 g; asparagine, 1 g; KH2P04, 1.5 g;MgS04.7H20, 0.5 g) supplemented with appropriatequantity of desired activator for obtaining 0.5, 0.05and 0.005 mM concentrations. Flasks containingmedium (50 m1I250 ml flask) were inoculated withagar block (3 mm diam) containing 4-day-old myceliaof the pathogen and incubated for 12 days at 30±1°C.Mycelia were collected after 3, 6, 9 and 12 days ofincubation, dried at 60°C for 96 hrs, cooled andweighed.

Results and DiscussionSusceptibility of 6 cultivars (cvs.) of ginger against

P. aphanidermatum was tested and the results aregiven in Table 1. All test cvs. except Kunduli weremoderately susceptible to rhizome rot pathogen.Among the six cvs., Kunduli appeared to be leastwhile Varada was the most susceptible (about 38%loss in fresh wt of rhizome), followed by Suprabha(about 32%) and Maran.

The results in Table 2 show that treatment withdefence activators reduced the rhizome rot disease butSA and BABA were more effective than BTH. SAwas also reported to protect the cucumber plants fromCladosporium cucumarinum (Narusaka et al, 1999)and tobacco from Botrytis cinerea (Murphy et 0/,2000). Prachi et al (2002) reported that SA (104 flM)induced insensitivity to culture filtrate of Fusariumoxysporum f. sp. zingiberi in the calli of Z. officina/e.Quintanilla and Brishammar (1998), however,reported that when SA at two different concentrationswas injected before planting into potato seed tubers of

Table I-Susceptibility of different cultivars of ginger againstP. aphanidermatum

Cultivars % loss in fresh wt ofrhizomes in relation to control

(4 weeks)*

Reaction ofcultivars

Pulpally 15.95 MSKunduli 4.32 LSHimachal 10.14 MSMaran 18.56 MSVarada 37.84 MSSuprabha 31.50 MS

Average of 4 replicate plants/treatmentMS: Moderately susceptible; LS: Least susceptible* 4 weeks after inoculation

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KARMAKAR et at: PLANT DEFENCE ACTIVATOR INDUCED SYSTEMIC RESISTANCE 593

Treatment

Table 2 - Effect of soaking rhizome seeds in plant defence activators on disease development in ginger (cv. Suprabha)

Yellowingindex/plant(4 weeks)

Untreated(Control)

Non Inoculatedinoculated

o 1.75

Treated withSA 0 0.81BTH 0 1.40BABA 0 1.00

CD at5%CD at 1%

SA: Salicylic acid (5mM)BTH: 2,1,3 - Benzothiadiazole (5 roM)BABA: DL - ~ -Aminobutyric acid (5mM)*: Average of 4- replicate rhizome seeds/treatment**: After 4 weeks of inoculation and 8 weeks of sowing

Average* fresh wt (g) ofrhizomes

(4 weeks)**

% loss in wt of rhizomesin relation to control

(4 weeks)Non Inoculated

inoculated41.82 ±3.00 28.45 ±2.26 31.97 ± 4.72

34.00±1.8229.10±1.3232.00±1.25

31.30±2.7425.20±1.7829.10±1.60

7.94 ± 1.0313.40 ± 1.929.06 ± 2.00

8.5011.92

a genetically resistant cultivar, higher concentrationincreased the resistance to leaf blight, while lowerconcentration enhanced the susceptibility. Effectiveconcentrations of exogenously applied SA are veryclose to phytotoxic mark as reported earlier for othercrops (Neuenschwander et at, 1996). One of thereasons may be that exogenously applied SA becomesrapidly conjugated to the ~-glucoside form, which isphysiologically inactive and phloem immobile(Enyedi & Raskin, 1993). Although different viewshave been expressed about phytotoxicity of SA butlower concentrations are not phytotoxic. In thepresent investigation, rhizome seeds of ginger weresoaked in 5 rnM SA for 1 hr because seed treatmentfor 4 hr caused phytotoxicity. Thus, toxicity dependsnot only on concentration but also on the duration oftreatment.

Like SA, BABA can also provide systemicprotection to tomato, potato and tobacco againstPhytophthora infestans and Peronospora tabacina(Cohen, 1994; Cohen & Gisi, 1999). However,Zimmerli et at (2001) demonstrated that BABAtreated Arabidopsis was less sensitive to two differentstrains of Botrytis cinerea. Actually, the compoundprotected those mutants which were defective in thejasmonate and ethylene pathways but was inactive inthe plants having impaired systemic acquiredresistance transduction pathway.

Analysis of leaf proteins of ginger revealed thatdefence proteins (molecular masses 67, 56, 32, 20, 14kDa) increased in BABA-treated inoculated anduntreated inoculated plants (Table 3, Fig. 1). A totalof 18 protein bands were detected in leaf-extracts of

Table 3-Comparison of protein patterns in leaves of healthy.inoculated and activator-treated ginger plants (cv. Suprabha)"

Treatment No. of defencerelated

protei ns/stressproteins

Molecular wt ofdefence relatedprotei ns/stressproteins (kDa)

UntreatedNon-inoculatedInoculated

o3**

o67,56. 14

Treated with SANon-inoculatedInoculated

23

41, 1256,14,12

Treated with BTHNon-inoculatedInoculated

33

20. 14, 1267,56,20

Treated with BABANon-inoculated 3 56, 32. 22Inoculated 4 67,32,20. 14

*Rhizome seeds soaked in activator for 1 hr as described. plantsinoculated after 4 weeks of sowing and leaves collected after 4weeks of inoculation**Total protein bands 18 in all cases

ginger (cv. Suprabha) and involvement of specificproteins was confirmed in activator-inducedresistance. Results in Table 3 show that 56 kDaprotein increased in the leaves of untreatedinoculated, and SA- and BTH-treated inoculatedplants but not in BABA-treated inoculated plants.However, 56 kDa protein also increased in leaves ofBABA-treated non-inoculated plants (compared tountreated control) but there was no further increaseafter inoculation. This may be due to the inactivationof any specific enzyme involved in this particular

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594 INDIAN J BIOTECHNOL, OCTOBER 2003

protein synthesis or degradation of this protein.Reduction in pixel intensity of 56 kDa protein bandalso indicates that there is no increase of this proteinin leaves of BABA-treated inoculated plants (Fig. 1).

co I Aso ~I Untreated non - inoculated

'01 ;J" I'-- 14 1\:: F ~ --J \)'0 i\10-"~o:-~~__~,-~~ __~~: ~ Untreated inoculated ~

:: Y ~6)\ r. r jli1 ~I~jlj'v ~ V 'V~i~1~T'~OO~-7,,' ~ , ~

10 ~

50 '00 150 200 250 300 350Pixel Position

Fig. l-Comparison of protein profiles of ginger leaves (cv.Suprabha). Graph showing pixel intensity of protein bands ofSOS-PAGE through Image Master VOS-ID gel analysis version3.0. A-untreated non-inoculated; B-untreated inoculated; C-BABA-treated non-inoculated, and O-BABA-treated inoculated

Recently, it has been reported that intercellularfluid (IF) from SA-treated tobacco plants inhibited thein vitro hyphal growth of B. cinerea. However, whencomponents with a molecular weight greater than3000 Da were removed from IF, it was no longerable to inhibit the mycelial growth (Murphy et al.2000). It was suggested that the inhibitory factor wasone or more extra-cellular pathogenesis relatedproteins. Although a number of defenceproteins/stress proteins have been detected in gingerleaves, their proper identification and characterisationyet remains to be worked out. These defence proteinsmay be used in disease management of ginger infuture.

Although high concentration (5 mM) of activatorswas applied on ginger plants as seed treatment, muchlower concentrations (0.5, 0.05, 0.005 mM) were usedto test the growth response of P. aphanidermatum inliquid medium since fungal pathogens are moresensitive than higher plants. It is clear from Table 4that 0.5 rnM cone. was inhibitory to fungal growth buthigher concentration applied on plants had no directeffect on the pathogen as they were applied 28 daysbefore the inoculation. Plant defence activators wereemployed in this study as an alternative to fungicidesfor controlling rhizome rot of ginger throughactivation of natural defence of plants.

'00

AcknowledgementThe authors are grateful to the Director, IISR,

Calicut, Kerala for providing the fungal culture andrhizome seeds of ginger. Thanks are due to Prof P KSircar, University of Calcutta for his help and

Treatment

Table 4-Effect of plant defence activators on the growth of P. aphanidermatum

Average mycelial dry wt (mg) with SE*Concentration(mM) 3 days** 12 days

Basal medium(BM)

o 89.30±2.03

BM+SA0.5 00.05 4.23±0.50

0.005 53.26±2.55

0.5 45.8Q±2.330.05 57.46±2.37

0.005 65.43±3.04

0.5 50.70±1.480.05 62.20±3.02

0.005 74.1O±1.41

BM+BTH

BM+BABA

*Average of 3 replicates/treatment**incubation periodTreatment: 30o± I°C

6 days 9 days

215.70±1.21 296.83±0.84 312.90±1.21

o1O.90±1.22

181.33±2.54

o40.30±1.61260.63±2.14

o63.20±2.54

287.30±3.00

122.30±0.87159.16±2.24216.76±2.25

191.61±2.76254.7Q±3.43278.26±2.42

248.60±1.2l274.40±2.09293.73±J.54

131.43± 1.14177.50±2.14220.46±1.49

266.32±1.l9292. 16±0.85304.50±1.47

231.60±J.37256.90±1.63294.67±2.63

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KARMAKAR et al: PLANT DEFENCE ACTIVATOR INDUCED SYSTEMIC RESISTANCE

comments. The financial support received from theDepartment of Biotechnology, Government of India isgratefully acknowledged.

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