Terpene Profile, Leaf Anatomy, and Enzyme Activity of ... Perkebunan/29(3)/4. AAP_197-209F.pdf ·...

Terpene profile, leaf anatomy, and enzyme activity related with cocoa clones resistance to VSD

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PELITA PERKEBUNAN, Volume 29, Nomor 3, Edisi Desember 2013

Pelita Perkebunan 29(3) 2013, 197-209

Terpene Profile, Leaf Anatomy, and Enzyme Activityof Resistant and Susceptible Cocoa Clones

to Vascular Streak Dieback Disease

Profil Terpena, Anatomi Daun, dan Aktivitas EnzimKlon Tahan dan Rentan Terhadap Penyakit Pembuluh Kayu

A. Adi Prawoto1*), Teguh Iman Santoso1), Marifah2), L. Hartanto2), and Sutikno2)

1)Indonesian Coffee and Cocoa Research Institute, Jl. PB. Sudirman 90 Jember, East Java, Indonesia2)Biology Faculty, Gadjah Mada University, Jl. Sicio Yustisio, Bulaksumur, Yogyakarta, Indonesia

*)Corresponding author (alamat penulis): [email protected] (diterima) 03 September 2013, accepted (disetujui) 01 Desember 2013

Abstract

Vascular-streak dieback (VSD, Oncobasidium theobromae) is the mostprevalent disease of Theobroma cacao L. in Indonesia. This study aims toanalyze resistance mechanism to VSD based on terpene profile, leaf anatomy,chitinase, and peroxidase study. Resistant clones of Sulawesi 1 and Sca 6 andsusceptible clones of ICS 60 and TSH 858 were used for terpene profile,leaf anatomy analysis, chitinase, peroxides, polyphenol, lignin, and celluloseanalysis. Those clones and KEE 2, KKM 22 and ICS 13 were used forperoxides analysis. For trichome study, the resistant clones of Sulawesi 1,Sca 6, KEE 2, and KKM 22, and susceptible clones of ICS 60 and TSH 858were used. GCMS analysis showed that chromatogram pattern of resistant andsusceptible groups were quite similar, but resistant clones contained 22% morecomponents than the susceptible ones. Resistant clones contained groups of pinene,decane, myrcene, and octadecanoic acid, while those substances on susceptibleclones were absent. Trichome was thicker on younger leaf, and its density onthe basal was higher than that on the middle and tip leaf parts. Trichomedensity of resistant clone was not always thicker than that of susceptible ones.On resistant clones, stomatal density was lower and width of stomate pits wasnarrower, while thickness of epidermis layer and pallisade parenchym were higher.Polyphenol content of resistant clones were higher but lignin and cellulose ofboth groups were similar. Chitinase activity which has a role in hydrolysis ofmycelia cell wall was higher on the resistant clones, but peroxides which has arole in polymeration of lignin biosynthesis was similar between both groups.It is concluded that groups of terpene pinene, decane, myrcene, and octadecanoicacid, thickness of leaf epidermis, density and width of stomata pit, and chitinaseactivity plays important role in cocoa resistance to VSD.

Key words: Theobroma cacao L., clone, vascular-streak dieback, resistance, leaf anatomy,chitinase, polyphenol

Abstrak

Penyakit pembuluh kayu [vascular-streak dieback (VSD), Oncobasidiumtheobromae] merupakan penyakit kakao yang berbahaya karena tanaman yang diserangdapat mati terutama pada fase muda. Penelitian ini bertujuan untuk mengetahuimekanisme ketahanan terhadap VSD ditilik dari aspek kandungan metabolit sekunder

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INTRODUCTION

Vascular-streak dieback (VSDOncobasidium theobromae Talbot & Keane)is a serious constrain of cocoa cultivation inIndonesia. Infection on young plants causeddeath, while on mature plants it can reducehealthy pods and yield. Yield loss due to thisdisease can reach 70% (Varghese et al., 1992).VSD is spread by spores in night, and infectyoung leaves (flush). The mycelia grow andspread to xylem vascular bundle of leaf,invade to petiole and then to branch anddownward to stem (Keane, 2000).

The most effective and efficient methodto control this diseases is by using resistantclones. Nowadays, several resistant clonesto VSD has been detected, i.e. Sulawesi 1,Sca 6, KEE 2, and KKM 22 on the other

hand ICS 60, ICS 13, TSH 858, aresusceptible (Halimah & Sri-Sukamto, 2007).In Malaysia, PBC 123 is resistant to VSDand used as an comparison in testing resis-tance clones (Kamil et al., 2005).

VSD resistance mechanisms is still notknown, and thought related with structuraland biochemical resistance. Antixenoxis isa type of rejection of plant to pest anddisease (Shodiq, 2009), and it can be chemi-cally or physically in action. Physicalantixenoxis is caused by the structure ormorphology that inhibits the growth ofdisease, while chemical antixenoxis is causedby allelochemical compounds, typicallysecondary metabolites. Terpene is anexample of secondary metabolite, withbuilding block of isoprene C5H8. Terpenecan be considered as condensation in form

terpena, polifenol dan lignin daun, anatomi daun, dan aktivitas enzim peroksidasedan khitinase. Klon tahan VSD Sulawesi 1 dan Sca 6 serta klon rentan ICS 60dan TSH 858 digunakan untuk kajian metabolit sekunder, anatomi daun dan aktivitasenzim. Klon tersebut ditambah KEE 2, KKM 22, dan ICS 13 untuk dianalisiskandungan peroksida. Untuk kajian trikoma daun, digunakan klon tahanSulawesi 1, Sca 6, KEE 2 dan KKM 22, dan klon rentan ICS 60 dan TSH 858.Kromatogram GC MS menunjukkan bahwa klon tahan VSD mengandung 22%metabolit lebih banyak daripada klon rentan. Klon tahan mengandung kelompokpinene, decane, myrcene, dan asam oktadekanoid, sementara senyawa tersebutdalam klon rentan tidak terdeteksi. Pada klon tahan, densitas stomata lebih sedikitdan lebar celah bukaan stomata lebih sempit dibandingkan pada klon rentan. Densitastrikoma pada kedua kelompok klon relatif sama. Trikoma lebih padat pada daunyang lebih muda, kepadatan pada bagian pangkal daun lebih tinggi dibandingkanpada bagian tengah dan ujung daun. Kandungan polifenol pada klon tahan cenderunglebih tinggi tetapi kandungan lignin dan selulosa relatif sama. Aktivitas peroksidaseyang berperan dalam polimerasi biosintesis lignin, sama pada kedua kelompokklon, akan tetapi aktivitas khitinase yang memiliki peran hidrolisis dinding selmiselia jamur, lebih tinggi pada klon tahan. Disimpulkan bahwa kelompok metabolitsekunder terpene yakni pinen, decane, myrcene dan asam oktadekanoid, densitasdan lebar celah stomata dan aktivitas khitinase, berperan penting dalam mekanismeketahanan kakao terhadap penyakit VSD.

Kata kunci: Theobroma cacao L., klon, vascular-streak dieback, ketahanan, anatomi daun,

khitinase, polifenol


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of monoterpene, sesquiterpenes, andditerpenes. Monoterpene examples arepinene, nerol, citral, camphor, menthol,limonene, have a characteristic of colorless,insoluble in water (Robinson, 1991), andused as an antiseptic, expectorant, spas-molytic and sedative. Sesquiterpenes hascharacters of antifeeding, antimicrobes,antibiotic and toxin. Groups of sesquiter-penes are farnesol, metilen -lacton asfungicide (Robinson, 1991).

Structural antixenoxis can be in formof plant cell wall lignification, injuredtissue recovery, leaf hair (trichomes), andwax accumulation in epidermis. Leaf hairaffect on laying/fixing of spores, eggs,dehydrated eggs or spores, allelochemicalby hair glands (Norris & Kogan, 1980).Lignification is one mechanisms of plantresistance to fungi, incorporation of lignininto cell wall provides mechanical strengthand allow cell wall more resistant todegradation enzymes secreted by pathogens(Goodwin & Mercer, 1990).

Another chemically antixenoxis is byincreasing the synthesis of various proteinsthat inhibit development of pathogens, suchas chitinase and peroxides. Chitinase ispathogen-related protein which will increasewith infection in plant tissue. Peroxidesinvolved in processes of oxidation andprecursors polymerization of lignin bio-synthesis, are compounds that serve as physi-cal barriers to impede growth of fungi. Highperoxides activity in plants infected withpathogens is correlated with higher resis-tance to Sclerotium rolfsii in peanuts(Pudjihartati et al., 2006a).

This study aims to determine theresistance mechanism of cocoa to VSD,based on terpenoid content, leaf anatomy,activity of peroxides and chitinase, andcontent of some secondary metabolites.

MATERIALS AND METHOD

Determination of resistant and susceptibleclones to VSD was based on Halimah &Sri-Sukamto (2007) and on field observation.

Resistant clones of Sulawesi 1, Sca 6,and susceptible clones of ICS 60 andTSH 858 were used for terpene analysis.Samples of youngest fully grown leaves weredried in oven at 57OC for 24 hours. Afterdrying, 0.5 g of crushed and weighed dryleaf was extracted with hexan 25 mL, stirredand allowed to stand overnight. The nextextract was filtered three times with filterpaper then the precipitate was redissolvedin 5 mL of hexane and then each 3L ofthem was ready to be injected to Chroma-tography Gas. The equipment wasGCMS-QP2010S Shimadzu. Column wasAGILENTJ%W DB-1, 30 m length,ID 0.25 mm, carrying gas helium, detec-tor FID, column oven temperature 80OC,injector temperature 310OC, injection modesplit, flow control mode pressure, pressure16.5 kPa, total flow 40.0 mL/min, columnflow 0.50 mL/min, linear velocity 26.1 cm/sec, purge flow 3.0 mL/min, split ratio 73.0.Chromatogram profile was grouped basedon similarity of the retention times, and kindof chemicals were analyzed based on libraryWiley 229.

Leaf Anatomy Analysis

Samples of greenish youngest leaves ofICS 60, TSH 858, Sulawesi 1, and Sca 6were used for leaf anatomy observation. Leafanatomy variables including the thicknessof leaf, epidermis, palisade layer, spongylayer and stomatal density, was conductedin Plant Embryology and Micro techniqueLaboratory, Biology Faculty of Gadjah MadaUniversity. Transversal section was madeby rotary microtome, observation by using

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light microscope at 100 and 400 magnifi-cations.

For trichome density, the observedclones were resistant clones of Sulawesi 1,Scavina 6, KEE 2 and KKM 22, andsusceptible clones of ICS 60 and TSH 858clones. Leaf samples were very young (25%of fully-grown area), young (50% of fully-grown area), full (100% fully-redness), weretaken from three plants for each clones.Because each clone had specific leaf size,therefore in this research the leaf size or ageof leaf flush was based on the percentageto the full growth of leaf. Trichome prepa-ration by embeding of nail polish on theabaxial and adaxial of leaf samples on thebasal, medium and apex part of leaf. Af-ter dry, the nail polish was uncovered withtransparant cellotape. Observation of thetrichomes used light microscope at100 magnifications. Trichome densitywas observed 10 times of field of view, thenthe data was presented as average andstandard deviation.

Biochemical Analysis

Peroxides and protein content analysiswas observed on Sulawesi 1, Sca 6,KEE 2, and KKM 22 for resistant clonesand ICS 60, TSH 858 and ICS 13 forsusceptible ones. Youngest and greenishfully-grown leaves were used. Those clonesexcept KEE 2, KKM 22, and ICS 13 werealso used for polyphenol, lignin and cellu-lose content analysis.

Chitinase enzyme activity analysis wascarried out by modification method ofMitshuhiro & Motoo (1992), peroxidesenzyme according to Hammerschmidt et al.(1982), polyphenols, lignin and cellulosecontent according to Anderson & Ingran(1993). Samples of peroxides and proteincontent analysis was observed on youngest

and greenish fully-grown leaves, replicatedthree plants, while for polyphenol, ligninand cellulose content analysis werecomposites of three plants. Data ofperoxides and protein was analyzed usingcomplete randomized design with threereplications.

RESULT AND DISCUSSION

Terpenoid Profile

Terpenes is one of the most importantcomponents of resin and essential oils ofmany types of plants. This compound istoxic, and has smell of fragrant andusually used for pharmaceutical industryespecially for antibiotics, anti-fungal, andanti-tumor. GCMS results showed that peaknumber of resistant clones was more thanthat of susceptible ones. On ICS 60 andTSH 858 (both susceptible) showed 51and 57 peaks, respectively, while onSulawesi 1 and Sca 6 (resistant) showed65 and 67 peaks, respectively (Figure 1).Resistant clones contained pinene anddecane chemicals, while those chemicals onsusceptible clones were absent (Table 1).Beta-pinene is known as constituent of es-sential oil and efficacious and enzymecoagulator (Moghtader, 2012). Terpenoidchemicals which is lipophilic may interferecell membrane of fungi and dissolve lipidspresent in cell membrane. Anti-fungalmechanism of terpenoid is supposed bydamaging cell membranes and cell walls offungi. As a result the cell membrane is notfully formed and interfered with theprocess of osmosis and lead to cell death.

Resistant clones contained octadecanoicacid which is efficaceous the growth ofmicrobes (Warsinah et al., 2011) and insynergistic with other terpenoid compoundsto increase its activity of anti-fungal (Padmini


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Figure 1. Terpenoid chromatogram of Sca 6 and Sulawesi 1 (resistant); ICS 60 and TSH 858 (susceptible)Gambar 1. Kromatogram terpenoid Sca 6 dan Sulawesi 1 (tahan); ICS 60, dan TSH 858 (rentan)

Sca 6

ICS 60

TSH 858

Sulawesi 1

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et al., 2010). Myrcene or -myrcene ismonoterpene detected only in the resistantclones. Myrcene is one of the most importantchemicals used in the perfume industry andas anti-fungal characters.

Leaf Anatomy

Thickness of epidermis and cuticle ofresistant clones was greater than that onsusceptible clones (Table 2). The presence ofcuticle deposits on the walls limiting theinvasion of fungi into the mesophyll tissue(Nyadanu et al., 2012). Cuticle layer ofcutin is a hydrophobic substance that reducesthe attachment of water on the leaf surface andfacilitate drying of the leaf surface. Becausegermination of spores require moist, leaves thatremain dry was suspected to help the inhibi-tion of germination of VSD spores. Cuticle onsome plants contain fungistatic chemical that

are beneficial prevent flow of nutrients fromthe host plant (Alcerito et al., 2002).

Another epidermis derivate was stomata,formed by two guard cells. Cocoa stomatawas only found on the lower epidermis (abaxialside), kidney-shaped of cell guard, andcryptophore type. Resistant clones had lowerstomata density and narrower opening pit widththan the susceptible ones (Table 2). Thisphenomena was similar with resistance ofArachis hypogea to Phaeoisariopsis personatathat stomatal width was narrower than thesusceptible varieties (Kusumo, 1996). Cocoastomata was thought to be the structuralbarrier against penetration of VSD pathogens,but the presence only in the bottom surface,then it was doubtful the role in the mecha-nism of resistance to VSD. The process ofsome diseases infection was reported throughsome organs such as hydathoda, lenticel,

Sulawesi 1 ICS 60 TSH 858 Sca 6

Table 1. Terpene compounds on resistant but absent on susceptible clones based on Willey library 229

Tabel 1. Senyawa terpen pada klon tahan tetapi tidak terdapat dalam klon rentan berdasar Willey library 229

5.901 Alpha-pinene no no 6.685 Sabinene6.817 Beta-pinene no no 7.008 Myrcene7.017 Beta-myrcene no no 7.583 Trans-beta-Ocimene7.958 Limonene no no 16.300 1-pentadecene8.067 Indene no no 16.475 Pentadecane Very high

Sangat tinggi9.317 Citronellol, dihydro- no no 18.717 8-heptadecene

Geraniol, perhydro- no no 18.770 8-heptadecene10.383 1-methylindene no no 18.992 Heptadecene-n-

heptadecene1-methyl-indene no no n-eicosane

11.017 Azulene, no no 23.161 9-Octadecanoic acid, HighCyclopentacycloheptane methyl ester Hinggi

11.675 Dodecane no13.433 Tridocane, Dodecane no16.422 Heptadecane, no Low

Hexadecane, Tridecane rendah19.258 Cyclohexane,

Germacrane-A22.083 Ethyl ester of High

docosanoic acid tinggiEthyl ester ofbehenic acidEthyl ester palmitate

RemarkCatatan

CompoundSenyawa

Retention TimeWaktu retensi

RemarkCatatan

RemarkCatatan

CompoundSenyawa

Retention TimeWaktu retensi


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ectodesmata (Guest & Brown, 1997) there-fore for Oncobasidium theobromae infectionstill needs more observation.

Palisade is a networking venue forphotosynthesis because chlorophyll accumu-lated in its cells. Palisade cells of resistantclones were thicker than on susceptible clones(Table 2). The cell thickness affect thehardness of leaf mesophyll tissue and oncotton crop it was reported that the cellthickness affect resistance to Empoascadevastant (Shodiq, 2009). Spongy tissuelocated below the palisade tissue, composedof parenchyma cells in irregular shape.In this tissue, there is air space andchloroplasts but the number is not as muchon the palisade. Thickness of spongytissue in both groups of cocoa, was notdifferent.

The other leaf organs that thought tobe closely linked with the catchment of VSDspores is trichome. Trichome is epidermisderivate and has a role in reducing evapo-ration. It is an adaptation responce of aclone to dryness and has a role in reducinginfection of plant disease (Woelaningsih,1984). Cocoa trichome is stellate type,variate in tentacle number but in averagehas eight tentacles, mostly grow from leafvenae. Trichome on young cocoa leavesincluding glandular and non-glandular,type of branched hair or star-shaped.Beside reduce evapotranspiration, trichomeshas a role in preventing pests and diseasesattack (Gairola et al., 2009).

However, resistant clones had similarnumber of trichomes as in susceptible ones(Figure 2). Among four resistant clones, onlySulawesi 1 showed more trichomes than thesusceptible clones TSH 858 and ICS 60.Trichomes on abaxial epidermis was denserthan that on adaxial. Thichomes on imma-ture leaves was denser than on mature ones.In association with resistance to pests anddiseases, trichomes affect egg or sporelaying. Its motion, stick, and toxic effectsare allelochemical signs, and barriers (Norris& Kogan, 1980). This statement is quitelogical that the denser trichome inhibit sporelaying to the epidermis and inhibit penetra-tion of mycelia into leaf organs. Thisresult was not similar to those expressed byGairola et al. (2009) in Tetradenia riparia.Trichome has a role as physical barriersfor spore trapping and inhibiting spore ger-mination of Uromyces (Martin & Glover,2007). Therefore, the more trichome, theless contact frequency of germinated sporesto initiate penetration (Chattopadhyay et al.,2011). Young leaves of cocoa were denselycovered with trichomes; however, the densityof trichomes progressively decreases withleaf maturity. This suggests that thetrichomes were established early in leafdifferentiation and their density decreaseswith leaf development and age.

It was suspected that cocoa trichomeswas glandular and non glandular types.Secondary metabolite terpene was synthe-sized and accumulated on secretory organs

Notes (Catatan): data ± standard deviation (data ± simpangan baku).

Table 2. Leaf anatomy of four cocoa clones different in resistance to VSD

Tabel 2. Anatomi daun empat klon kakao berbeda ketahanannya terhadap VSD

Thickness of, Ketebalan, µmClonesKlon

EpidermisEpidermis

PalisadePalisade

Spongy parenchymParenkim bunga karang

Number stomataJumlah stomataper 0.69 mm2

Width of stomata pitLebar celah stomata

µm

ICS 60 25±3.5 80.4±4.3 75.0± 9.8 221.8 ± 2.2 9.2± 1.2TSH 858 24.2±0.7 47.5±9.4 91.3± 16.3 191.6 ± 7.4 7.8± 2.9Sulawesi 1 20.8±3.7 104.3±8.2 81.0± 12.2 163.8 ± 3.5 5.7± 0.6Sca 6 23.7±2.1 40.6±2.2 88.2± 7.4 107.2 ± 4.2 7.0± 1.1

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i.e. trichomes (Sangwan et al., 2001),although it still needs to be studied moredeeply if cocoa has glandular trichomes.However, epidermis of young cocoa leavescontained cells which has a role as secre-tory organ and colored violet or transparantin fresh preparations. Those difference indensity between resistant and susceptibleclones to VSD is still unknown, hence it isinteresting to be explored.

Chitinase and Peroxides

Chitinase is expressed as antifungalprotein (enzymes) (Wang et al. cit. Pudjihartatiet al., 2006b). Chitinase activity on resistantclones was higher than that on susceptibleones (Figure 3). Chitinase is one of pathogen-related (PR) protein which expression wouldbe increased by infection of the plant tissue.Increasing of chitinase activity has been reportedon citrus which was inoculated by Pichiamembranefaciens in order to become moreresistant against Penicillium italicum and Peni-cillium digitatum (Luo & Zeng, 2012). Inmango, leaf chitinase and -1,3-glucanasemaybe contributing towards resistance to

malformation caused by Fusarium mangiferae(Ebrahim et al., 2011). Chitinase mechanismsin enhancing pathogen resistance was reportedin two ways (i) inhibit the growth of fungalhyphae with directly hydrolyze wall offungal mycelia, and (ii) release of endogenouselicitor which can increase systemic resistanceresponse in the host (Oku, 1994).

Meanwhile, peroxides activity was notdifferent between two groups of cocoa clones,even on resistant ones the activity was lowerthan that on susceptible ones (Table 3).Although peroxides expression werecorrelated with the onset of inducedresistance (Luo et al., 2012) also in peanutsinfected with Sclerotium rolfsii (Pudjihartatiet al., 2006a), but for VSD it seeminglydid not contribute significantly. Peroxidesrevealed a role in oxidation process andinclude cross-linking cell wall polysaccha-rides lignifications. These compounds havea role as physical barrier to inhibit growthof pathogens (Lagrimini et al., 1993). Inaddition, this enzyme catalyzes oxidationphenol compounds to quinone with H2O2

to produce toxic compounds (Do et al. cit.Pudjihartari et al., 2006a).

Figure 2. Trichome density per 0.69 mm2 of cocoa clones different in resistance to VSD. Error bar isstandard deviation

Gambar 2. Densitas trikoma per 69 mm2 klon kakao berbeda ketahanan terhadap VSD. Balok erroradalah simpangan baku

Num

ber

of t

rich

omes

Jum

lah

trik

hom

a

3 5

3 0

2 5

2 0

1 5

1 0

5

0

-5Basal Middle Apex Basal Middle Apex Basal Middle Apex Basal Middle Apex

25% max 50% max 100% max redness Youngest greenesh

Sulawesi 1 Sca 6 KEE 2 KKM 22 ICS 60 TSH 858


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Polyphenol, Lignin and Cellulosa

Cocoa polyphenols in form of fla-vonoid, consist of 37% monomer of (-)epicatechin and (+) catechin, 4% anthocya-nin, and 58% polymer of procyanidin. Fla-vonoid is secondary metabolite, protectsplant tissue damage caused by ultraviolet,pest and disease attack. Asparagus enhancedresistance against Fusarium oxysporum f. sp.Asparagi might be associated with theincrease of anti-oxidative ability, andtotal contents of polyphenol and ascorbic

acids (Nahiyan & Matsubara, 2012). Inthis study, total polyphenol was higher inresistant clones (Figure 4). Polyphenolmaybe even higher when the plant wasinfected with VSD, because most ofsecondary metabolites were inducible resis-tance mechanism.

Lignification on cell wall is a formof plant resistance against penetration ofpathogens. Lignin located on the middlelamella, primary and secondary cell walls.Increasing of lignin content may inhibit

Table 3. Protein content and peroxides activity

Tabel 3. Kadar protein dan aktivitas peroksidase

Note (catatan): Figures in the same column followed by the same letter was not significantly different at 5% levelaccording to DMRT (Data diikuti huruf yang sama tidak berbeda nyata pada taraf 5% menurut ujiDuncan).

Clones (Klon) Protein, mg/gPeroxides (Peroksidase),

unit/mg protein

Sulawesi 1 0.47 bc 93.94 b

Sca 6 0.51 b 63.07 c

KEE 2 0.38 bc 86.50 bc

KKM 22 0.45 bc 37.98 d

ICS 60 0.31 c 68.11 c

TSH 858 0.33 c 144.72 a

ICS 13 0.71 a 101.18 ab

Figure 3. Chitinase activity of resistant and susceptible cocoa clones to VSD. Error bar is standard ofdeviation

Gambar 3. Aktivitas khitinase klon tahan dan rentan VSD. Balok error adalah simpangan baku

Chi

tinas

e, u

nit/m

g pr

otei

n

4

3,5

3

2,5

2

1,5

1

0,5

0Resistant Susceptible

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Figure 4. Polyphenol, lignin, and cellulose content of susceptible and resistant cocoa clones to VSDGambar 4. Kandungan polifenol, lignin, dan selulosa dalam daun klon kakao tahan dan rentan VSD

Lign

in,

%

2 5

2 0

1 5

1 0

5

0ICS 60 TSH 858 Sca 6 Sulawesi 1

Poly

phen

ol,

%

8

6

4

2

0ICS 60 TSH 858 Sca 6 Sulawesi 1

4.66

5.53 5.57

7.751

18.92

21.56

16.7517.99

ICS 60 TSH 858 Sca 6 Sulawesi 1

Cel

lulo

se,

%

3 0

2 5

2 0

1 5

1 0

5

0

27.02

21.46 22.47

26.01


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penetration physically to pathogens invading,blocking the spread of toxins and enzymesreleased by pathogens, as well as inhibit thesupply of nutrients needed by pathogens (Vanceet al. cit. Pudjihartati et al., 2006a).

The result suggested that lignificationwas not resistance mechanism on cocoaclones against VSD. Lignin content ofsusceptible clones was higher than that onresistant clones (Figure 4). The tendencywas similar with peroxides activity(Table 3). This result was contrast withPudjihartati et al. (2006a) in peanutswhich concluded that infection of S. rolfsiiimproved peroxides activity and lignincontent in the infected tissues, indicatingthe more resistant of the genotype, the moreperoxide activity in the tissue. Besidelignin, cellulose content in susceptibleand resistant clones to VSD were similar,therefore lignin and cellulose content werenot important variables in determiningresistance of cocoa clones to VSD.

CONCLUSION

Cocoa clones resistant to VSD have22% more terpenoid components thanthat of susceptible ones. Resistant clonescontain groups of pinene, decane, myrcene,and octadecanoic acid, while thosesubstances were absent on susceptibleclones. Trichome was thicker on youngerleaf, and its density on the basal was higherthan that on the middle and tip of leafparts. However, the density on resistant andsusceptible clones were quite similar. Onresistant clones, stomata density was lowerand width of stomata pits was narrower.Thickness of cuticula and epidermis layer,and pallisade parenchym were higher onresistant clones. Polyphenol content ofresistant clones was higher but lignin andcellulose of both groups were similar.

Peroxides which has a role in polymerationof lignin biosynthesis was similar, butchitinase which has a role in hydrolysis ofmycelia cell wall was significantly higheron the resistant clones. It is concluded thatgroups of terpene pinene, decane, myrcene,and octadecanoic acid, density and widthof stomata pit, thickness of epidermis andcuticula layer, and chitinase activity playimportant roles in cocoa resistance to VSD.

ACKNOWLEDGEMENTS

Thank you very much was addressed toDirector of ICCRI for the supporting andfunding, the similar appreciation deliveredto Mrs. Suprapti and Mr. Sudomo for theirhelp in anatomy and GCMS analysis.

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