Analysis of Secondary Metabolites as Potential

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PELITA PERKEBUNAN, Volume 33, Number 1, April 2017 Edition

Pelita Perkebunan 33 (1) 2017, 10—23

Analysis of Secondary Metabolites as Potential Phytoalexins,Their Secretion Sites and Proposed Resistance Markers to

Vascular Streak Dieback in Theobroma cacao L.

Teguh Iman Santoso1*), M. Miftahudin2), Yohana C. Sulistyaningsih2) and Suryo Wiyono3)

1)Indonesian Coffee and Cocoa Research Institute, Jl. PB. Sudirman 90, Jember, Indonesia.2)Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Bogor, Indonesia.

3)Department of Plant Protection, Faculty of Agriculture, Bogor Agricultural University, Bogor, Indonesia.*) Corresponding author: [email protected]

Abstract

Study on resistance mechanism to vascular-streak dieback (VSD) diseasein cacao (Theobroma cacao L.) is limited due to the lack of fungal spores forartificial inoculation. This research was conducted to study the production ofsecondary metabolites that appear to be evidence of defense signaling inresistant clone of Sca 6 and susceptible clone of TSH 858 to Ceratobasidiumtheobromae natural infection. A fungal staining method was employed to detectC. theobromae hyphae at early infection stages, before VSD symptoms appear.Metabolite profiling was analyzed using pyrolysis gas chromatography massspectrometry (Py-GCMS) at pre-, early and late stages of C. theobromae infection.Histochemical and anatomical characteristics of both healthy and infected leaveswere also observed to identify the accumulation sites of secondary metaboliteson and in cocoa leaf tissues. The results confirmed that fungal staining using trypanblue can detect early stages of C. theobromae infection; at the 14th week (onsusceptible seedlings) and the 18th week (on resistant clones), following placementof the seedlings under infected cacao plants. Phenylpropanoid biosynthesis,terpenoid biosynthesis, environmental information processing signal transductionpathways, and aromatic biodegradation were detected as important metabolitepathways during defense mechanism. I-limonene (terpenoid), p-ethylguaiacol(phenols) and 2.3 dihidrobenzofuran (heterocyclic compounds) were proposed asan active defense produced by the host after infected by pathogen mainly on lateinfection of C. theobromae. Terpenoid and phenol compounds were accumulated onthe glandular trichomes, idioblast of upper and bottom epidermis, phloem vesseland cortex idioblast of cacao leaves. Epidermis thickness of resistant clone wassignificantly greater than that of susceptible clone on both surfaces. Leaf epidermistissue and the accumulated compounds in epidermis idioblast may act as the physicaland biochemical markers of cocoa resistance to VSD.

Keywords: Theobroma cacao, fungal staining, histochemical, Py-GCMS, resistances, VSD

ISSN: 0215-0212 / e-ISSN: 2406-9574

INTRODUCTION

Vascular streak dieback (VSD) is a majorconstraint of cacao (Theobroma cacao L.)production in Southeast Asia and Melanesia

(Samuel et al., 2012). In Southeast Asiancountries, which include Indonesia as theworld third largest cocoa producer (ICCO,2014), VSD caused 40-60% losses of thenational cacao production. The occurrence

mailto:[email protected]

11PELITA PERKEBUNAN, Volume 33, Number 1, April 2017 Edition

Secondary metabolites as potential phytoalexins, secretion sites and proposed resistance markers to VSD in cocoa

of VSD had been firstly reported in PapuaNew Guinea (PNG), and the disease hadexpanded northwestward to Kerala State inIndia, and Hainan Island, the southernmostof China (Keane & Prior, 1991). Wind-dispersedbasidiospores of C. theobromae are releasedduring periods of continously heavy rainfalland high humidity. After penetrating cacao leaf,the fungal hyphae colonize xylem vessels,causing browning of the veins of lamina;which spreads to leaf midrib and petioles,and eventually reaches the branch. Coloni-zation by C. theobromae is found only inthe xylem vessels of cacao (Talbot & Keane,1971); as a characteristic that distinguishesit from other diseases caused by fungi incacao (Samuel et al., 2012). The recogniz-able symptoms of VSD include chloro-sis of the leaves, with scattered spots onthe lamina remaining green (Guest & Keane,2007).

VSD was caused by the basidiomycetefungus of C. theobromae, which is a memberof the genus Ceratobasidium (Talbot & Keane,1971). This fungus is an obligate parasiteand cannot be grown in pure culture. The onlyexperiment that successfully induced thesexual stage of the fungus was conductedin Malaysia using solid Corticium culturemedia (CCM) and liquid coconut water media,incubated at 25OC under 10/14 hours of light/dark (Lam et al., 1988), but this experi-ment could not be repeated in PNG (Dennis,1991). Therefore, the challenge is to studythe plant response to the fungus without theoption of artificial inoculation.

Cacao resistance mechanism in responseto C. theobromae infection is still unclear.Resistance evaluation and selection ofplants for evaluation can only be done in aVSD epidemic area due to the lack of sporesthat could be used for artificial inoculation(Guest & Keane, 2007). The resistanceresponse to VSD varies among cacao clones.

Halimah & Sri-Sukamto (2007) classifiedsome cacao clones of Sulawesi 1, Sulawesi 2,and Sca 6 as VSD-resistant, based on aVSD-symptom-scoring method; whereasother cacao clones of ICS 60 and TSH 858,were classified as VSD-susceptible (Susilo& Anita-Sari, 2009). Other potentially VSD-resistant clones of DRC 15, KA 2-10 andVSD2Ldg were also found in Sulawesi(McMahon et al., 2010).

The epidemiology of VSD is controlledby a strong association between the fungussurvival and environmental factors, andtherefore it is necessary to study the hostresponse to C. theobromae infection undernatural conditions. However, it is difficultto determine early infection by C. theobromaebased on obvious VSD symptoms. Histo-chemical staining of C. theobromae hyphaefrom infected leaves can be used to detectthe presence of the fungus during early naturalinfection. The presence of C. theobromaehyphae may also be an indication of secondarymetabolite production; which acting asphytoalexins, in response to defense signalingas part of cocoa resistance mechanism responseto the pathogen.

The objectives of the research were toanalyze profiles of secondary metabolitesproduced by C. theobromae-infected cacaoleaves, to identify secondary metaboliteproduction sites in the infected leaf tissues,and to identify any leaf anatomical differencesas characteristics of VSD-resistant and VSD-susceptible cacao clones. This paper reportedthe secondary metabolites produced duringC. theobromae infection may be as phyto-alexins, histochemical analysis of secondarymetabolite accumulation in leaf tissues, andleaf anatomical data. Information gatheredfrom this study can be used for pathogenesisstudies and selection tools to supportbreeding and development of VSD-resistantcacao.

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Santoso et al.


MATERIALS AND METHODS

Two cacao clones, i.e. Sca 6 (resistant)and TSH-858 (susceptible) were used in theexperiment. Seven-month-old seedlingspropagated through top grafting, were grownin and outside VSD epidemic area.

The study was conducted in a VSDepidemic area, Kaliwining ExperimentalStation of Indonesian Coffee and CacaoResearch Institute (ICCRI), Jember, Indonesia.Histo-chemical and anatomical analyses werecarried out in Microtechnique Laboratory,Department of Biology, Bogor AgriculturalUniversity, Bogor, Indonesia. Secondarymetabolite analysis using pyrolysis gas chro-matography mass spectrometry (Py-GCMS)was carried out at the Chemical and Proxi-mate Integrated Laboratory, Forestry Researchand Development Center, Gunung Batu,Bogor, Indonesia.

The experiment used a randomizedcomplete block design in factorial with threereplications. The first factor was two cacaoclones, i.e. VSD-resistant (Sca 6) and VSD-susceptible (TSH 858). The second factor wastwo VSD infection treatments, i.e. naturallyVSD-infected cacao seedlings, placed underinfected cacao trees, and non-infected cacaoseedlings, placed outside the area of cacaoplants at a distance of approximately 3000 m.Observation for the presence of C. theobromaeinfection and VSD symptoms was performedevery two weeks using three plant samplesper experimental unit. Metabolite profilingwas carried out at pre-, early and late infectionstages using three biological replications withtwo technical replications per biologicalreplication. Phenolic, terpenoid and alkaloidhistochemical staining was done using threebiological replications with five observationviews per replication.

Pre-infection is the pre-exposure condition,one day before seedlings are placed underinfected plants, and when neither C. theobromae

nor VSD symptoms have yet been detected.Early infection is defined as the first timeC. theobromae hyphae have been detectedin the xylem vessel of the infected leaf,but VSD symptoms have not yet appeared.Late infection is the condition when bothC. theobromae and VSD symptoms havebeen detected.

The presence of C. theobromae hyphaewas checked every two weeks using a fungalstaining method based on Liberato et al.(2005). Leaf midrib samples (lamina containingmidrib) of 1 cm2 were soaked in a 20 mL1:1 (v/v) solution of glacial acetic acid andabsolute ethanol in a closed container at roomtemperature for 24 hours. Leaf samples werecross-sectioned using a razor blade, thenmounted on glass slide and given a drop of85% lactic acid and 0.1% trypan blue. Anypresence of the fungus was observed underlight microscope Olympus CX21 (Olympus,Japan), and identified using species descrip-tion of C. theobromae as done by Junianto& Sri-Sukamto (1986). The hyphae ofC. theobromae are typically characterizedby right-angle branching, and colonize onlyxylem vessels of cacao.

Secondary metabolite profiles were iden-tified using a Shimadzu QP2010 (Shimadzu,Japan) Pyrolysis GCMS, and the samplepreparation method was based on Chaves& Gianfagna (2007). Leaf samples werehomogenized using a pestle and mortar, anda 0.002 g homogenized sample was then putinto the GCMS instrument. The chemicalcomponents of the sample were identifiedunder the following settings/conditions:pyrolysis temperature at 400OC; GC oventemperature at 50OC; injector temperatureat 280OC; and interface temperature ionsource at 280OC. Helium gas was used as thecarrier.

The mass spectrograms were identifiedby matching them against those from theexisting mass spectrum database provided



in the GCMS instrument, which cross-refersto the National Institute of Standard andTechnology (NIST) Library, Maryland,USA. Analysis of peak area chromatogramdata was performed using the One-WayAnalisis of variance (Anova) and Tukey testat 5% of confidencial level. The types ofmetabolite were identified using the PubChem(http://pubchem.ncbi.nlm. nih.gov/) database.Metabolite pathways were developed usingthe pathway maps of the KEGG database(http://www.genome.jp/kegg/pathway).

Metabolite accumulation sites on and inleaf tissues were identified by histochemicaltest. The leaf used for sampling in the analysiswas the sixth leaf back from the tip in eachcase. Fresh samples were cross-sectioned usinga freezing microtome: a Yamato RV-240(Yamato Kohki Ind Co, Japan) with 30 mrazor-blade thickness. Terpenoids were iden-tified using 5% copper acetate, followinga procedure suggested by Harborne (1987);phenolic compounds were identified usinga 5% ferric trichloride procedure suggestedby Johansen (1940). Anatomical characteris-tics of the cacao leaf were also investigated,based on both transversal and paradermalsections. Samples were fixed in 70% alcohol,and then cross-sectioned using a freezingmicrotome: a Yamato RV-240 (Yamato KohkiInd Co, Japan) in 20 m thickness. Paradermalsections of each leaf were prepared as semi-permanent samples using a whole-mountmethod first suggested by SASS (1951). Thealcohol-fixed leaves were rinsed with distilledwater and soaked in 50% nitric acid solution,before being rinsed again with distilled waterthree times. Adaxial and abaxial surface layersof leaves were peeled using a razor blade.Samples were observed under an OlympusCX-21 light microscope (Olympus, Japan),and photographed using an Optilab Advancecamera (Miconos, Indonesia). Measurementof anatomical characteristics, i.e. epidermisthickness, stomatal size, stomatal density,

stomatal index, palisade thickness, glandulartrichome density, glandular trichome lengthand glandular trichome width, was performedusing Image Raster Software (Miconos,Indonesia). Stomatal index was measuredbased on number of epidermis divided bynumber of stomata.

RESULTS AND DISCUSSION

Plant disease resistance mechanismsinvolve activation of a series of defensepathways as part of the defense mechanismagainst pathogen attack. Plants have a regula-tory mechanism to activate particular defenseresponses according to the specific stimulireceived (Chaves & Gianfagna, 2007). Phyto-alexin is a chemically diverse group ofsecondary metabolites with strong antimi-crobial actions, and these metabolites accu-mulate around the infection site. They aregenerally undetectable in the plant beforeinfection, but are synthesized very rapidlyafter pathogen attack (Taiz & Zeiger, 2010).

Fungal staining successfully detectedthe early infection stage of C. theobromaein VSD-susceptible and VSD-resistant plants,at the 14th and 18th weeks, respectively, aftercacao seedlings had been placed under VSD-infected cacao plants. Susceptible clone(TSH 858) was infected faster than resistantclones (Sca 6). Although the hyphae hadbeen detected in the xylem vessels, the leavesappeared normal and healthy, exhibiting novisible symptoms of VSD (Figure 1 & 2).The VSD symptoms of leaves used in thisstudy were interpreted according to observedleaf chlorosis with green spots, as describedby Guest & Keane (2007).

During onset of C. theobromae infection,cacao leaves produced 20 different metabo-lites. Chromatogram areas from Py-GCMSanalysis of both VSD-resistant (Sca 6) andVSD-susceptible clones (TSH 858) were notstatistically different at the pre-infection

http://pubchem.ncbi.nlm.

http://www.genome.jp/kegg/pathway).

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Santoso et al.


Figure 1. Morphological symptom on VSD-susceptible clone of TSH 858 (A) and VSD-resistant cloneof Sca 6 (B) at the three infection stages of C. theobromae

Figure 2. Fungal staining by trypan blue for detection of C. theobromae presence. Midrib cross-sectionsat pre-infection stage (A), early infection stage (B), and late infection stage (C). Midrib longitudinalsection: typical branching of C. theobromae hyphae at right angles (D) (xy = xylem, Ot = C. theobromaehyphae, br = hyphae typical branching). Scale bars = 50 µm (A-C); 30 µm (D)



stage (Table 1). There was then an increasein production of I-limonene (a terpenoidcompound) and p-ethylguaiacol (a phenoliccompound) in both clones at the early stageof C. theobromae infection, and again, therewas no significant difference between theVSD-resistant and VSD-susceptible clones(Table 1). However, at the late infection stage,production of I-limonene, p-ethylguaiacol, and2,3 dihidrobenzofuran (terpenoids), weresignificantly higher in resistant clones thanin susceptible clones (Table 1).

Early infection defined as the first timeC. theobormae hyphae detected in the xylemvessel of infected leaf, but VSD symptomsnot yet appear. Early infection occurred inweek 14th (TSH 858) and week 18 th (Sca 6)following placement of cacao seedlings underVSD-infected cacao plants. Control wascacao seedlings of the same age, placedoutside area of infected cacao plants.

Late infection is defined as when bothC. theobormae and VSD symptom have beendetected. Late infection in TSH 858 and Sca 6occurred at 15th and 19th weeks respectively,after cacao seedlings placed under VSDinfected cacao plants. Control was cacaoseedlings of the same age placed outside areaof infected cacao plants.

In the early stage of C. theobromaeinfection there was increasing productionof I-limonene and p-ethylguaiacol in bothVSD-resistant and VSD-susceptible clones,with no significant difference between thetwo types of clone at this stage (Table 1).This might be due to similar basic responsesof both VSD-resistant and VSD-susceptiblecells to the infection, and similar rates ofphytoalexin formation (Kuc, 1992). Increasedproduction of I-limonene and p-ethylguaiacol,as well as 2,3 dihidrobenzofuran, occuredmore significantly in the VSD-resistant clonesat the late infection stage (Table 1). It issuggested that I-limonene, p-ethylguaiacol

and 2,3 dihidrobenzofuran could be consideredas indicators of a triggered defense signalingmechanism, and are produced by host plantsinduced by C. theobromae, mainly in lateinfection stage. I-limonene, p-ethylguaiacol,and 2,3 dihydrobenzofuran were synthesizedduring C. theobromae infection stages throughdifferent metabolite pathways. I-limoneneis a monoterpenoid that is synthesizedthrough methyl erythritol phosphate (MEP)in the terpenoid backbone metabolism.

Limonene has been reported as a mono-terpenoid that exhibits an antifungal actionthat is similar to the commercial fungicide,carbendazim. Limonene and carbendazimhave been tested against Rhizoctonia solani,Fusarium oxysporum, Penicillium digitatumand Aspergilus niger (Marei et al., 2012).Fungi produce a cellulase enzyme that candegrade cell walls during pathogenesis (Milling& Richardson, 1995), and it was reportedthat limonene has a strong inhibitory effect,similar to that of commercial fungicides, oncellulase activity (Marei et al., 2012).

Monoterpenoid is a type of metaboliteproduct synthesized in large quantities througha signal transduction pathway, as a result ofjasmonic acid activity (KEGG, 2015). Jasmonicacid is a signal molecule that induces a signaltransduction pathway, which leads to the produc-tion of phytoalexins as part of the resistancemechanism of Glycine max, in response toRhizoctonia solani infection (Aliferis et al.,2014).

Phenolic compound p-ethylguaiacol issynthesized through phenylpropanoid path-ways. Secretion of the compound fromplants is induced by wounds and is involvedin the communication and defense systemsduring Agrobacterium and Rhizobium infection(Bhattacharya et al., 2010). Accumulation ofphenolic compounds associated with a hyper-sensitive response by a plant to pathogeninfection has been found in coffee plant

16

Santoso et al.


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defense mechanism against Colletotrichumkahawae (Loureiro et al., 2012).

2,3-dihydrobenzofuran is a heterocycliccompound that is synthesized through degra-dation of certain aromatic compounds (KEGG,2015). Asperfuran is an example of an anti-fungal 2,3 dihydrobenzofuran produced bya strain of Aspergillus oryzae. Asperfuraninhibits chitin synthase produced byCoprinus cinereus (Pfefferle et al., 1990).Chitin is a major component of fungal cellwalls, which play a vital role in hyphal tipgrowth and fungal morphogenesis. Chitinis synthesized by chitin synthase and it iswell known that the latter is inhibited by com-mercial fungicides (Kong et al., 2012). The2,3 dihydrobenzofuran compound has alsobeen reported to be produced by Bauheniapurpurea and shows antifungal activityagainst Candida albicans (Abyaneh & Rai,2013).

Role of terpenoid and phenolic compoundsin cacao resistance mechanism to pathogenshave been reported as phytoalexins in respectof Verticillium dahlia infection. Productionof triterpenoid arjunolic acid and the phenoliccompounds 3,4 dihydroxyacetophenone and4-hydroxyaceto-phenone, significantly increasesin cacao infected by Verticillium dahlia(Resende et al., 1996). In addition to theseterpenoid and phenolic phytoalexins, elementalsulphur is produced as an antifungal substance,in cacao during Verticillium dahlia infection(Cooper & Williams, 2004; Resende et al., 1996).

Metabolite Accumulation Sites

Metabolite accumulation sites were foundin healthy leaves of both VSD-resistant andVSD-susceptible clones at pre-infection stage.Both clones had leaf laminae consisting ofsingle-layered epidermis tissue with idioblastcells containing phenolic and terpenoidcompounds in the upper and lower epidermises

(Figures 3B & 3D). Phenolic compounds werealso detected in palisade tissue (Figure 3A).Glandular trichomes containing phenolic andterpenoid compounds were distributed acrossboth cacao leaf surfaces, although mainly onthe leaf midrib (Figures 3C & 3E). Phenoliccompounds also appeared in numerousidioblast cells in the cortex of the midrib(Figures 4A, 4B, & 4C).

Similar accumulation sites were foundin the leaves of VSD-resistant and VSD-susceptible clones during the infection stagesof O. theobromae. Histochemical stainingshowed that phenolic and terpenoid contentof the phloem in VSD-resistant clones washigher than that of susceptible clones. Thiswas indicated by increasing intensity of darkbrown color of the phloem at the time ofinfection (Figure 5, Table 4).

The secretion of terpenoid and phenoliccompounds during C. theobromae infectioninvolves phloem tissues. The role of phloemin the secretion of phenolic compounds hasbeen reported in the case of Picea abiesinfected by Ophiostoma polonicum (Brignolaset al., 2007). It is understandable that phenolicand terpenoid contents in the phloem ofVSD-resistant clones were higher than insusceptible clones. This might be related tothe secretion of the compounds during theinfection stage. Cacao leaves have glandularand non-glandular trichomes (Nakayama et al.,1996). In some plants, glandular trichomeshave a role as physical and chemical barriersto spores (Martin & Clover, 2007; Chattopadyayet al., 2011). However, there is lack of studyfindings regarding the metabolite content ofcacao glandular trichomes. This researchshowed that glandular trichomes of cacao,containing phenolic and terpenoid com-pounds, were distributed on both surfacesof cacao leaves, mainly on the leaf midribs.However, the trichomes were generally moreabundant on the abaxial than on the adaxial

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Glandular trichomes + + + + + + + + (+) (+) (+) (+)Idioblast of upper epidermis + + + + + + + + + + + +Idioblast of lower epidermis + + + + + + + + + + + +Palisade tissue + - + - + - + - + - + -Phloem vessels ++ + ++ + +++ +++ + ++ +++ +++ ++ +++Idioblast of cortex + + + + + + + + + + + +

Note: P = phenolic compounds; T = terpenoids; - = not detected, + = fairly detected, ++ = moderately detected, +++ =strongly detected; (+) = lysis.

Tabel 2. Secondary metabolite production sites during O. theobromae infection, based on histochemical staining

Pre infection Early infection Late infectionCell/tissue Sca 6 TSH 858 Sca 6 TSH 858 Sca 6 TSH 858

P T P T P T P T P T P T

Figure 3. Histochemical staining of cacao leaves. Phenolic compounds detected in palisade tissue (A),idioblast epidermis (B) and glandular trichomes (C). Terpenoids compounds detected in idioblastepidermis (D) and glandular trichomes (E). Glandular trichomes in water as control (F). (pl =phenolic compounds, tr = terpenoids). Scale bar: 50 m (A), 20m (B - F)

leaf surface. Cacao glandular trichomes areof the multiseriate capitate type: composedof a basal cell, two-cell uniseriates and eightcells making up the secretory head. The dis-tribution of glandular trichomes on both VSD-

resistant (Sca 6) and VSD-susceptible clones(TSH 858) did not differ statistically, sug-gesting that the glandular trichome is notclosely related to cacao resistance mechanismsto VSD.



Figure 4. Histochemical staining of phenolic compounds (A-F) and terpenoids (G-L) in cacao leaf midribof Sca 6 (A-C and G-I) and TSH 858 (D-F and J-L) showing positive results in phloem duringpre-infection (A, D, G, J), early infection (B, E, H, K) and late infection (C, F, I, L). (xy =xylem; ph = phloem; id = idioblast; Ot = O. theobromae hyphae). Scale bar: 50 m (A-L)

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Epidermis as a Barrier

The leaf epidermis tissue of VSD-resistantclones was significantly thicker than that ofsusceptible clones, for both leaf surfaces(Table 3). However, size and density of stomata,as well as the distribution of glandular trichomes,were not significantly different betweenVSD-resistant and VSD-susceptible clones.Interestingly, stomatal pores on the VSD-susceptible clones were found to be widerthan those of resistant clones.

Pathogenesis caused by infectious agentsbegins when the pathogen contacts host plants.Certain anatomical characteristics of leaves canact as physical barriers to pathogens penetratinga host plant. Most pathogens gain access tothe plant interior through natural openings suchas stomata (Agrios, 1988).

The VSD-resistant and VSD-susceptibleclones were not significantly different interms of stomatal density, length and width.The study suggested that these stomatalcharacteristics were not directly related to cacaoresistance mechanisms to VSD. This findingdiffers from a previous study that reported apositive association between stomatal densityand cacao resistance to VSD (Susilo & Anita-Sari, 2009). However, our research foundthat the stomatal pores of susceptibleclones were wider than those of resistant

clones. We suspect that pathogens havemore opportunities to penetrate susceptiblethan resistant plants through the formerwider stomatal openings.

A previous study explained that hyphaepenetrated young leaves by growing directlythrough cuticle layers into mesophyll tissues(Prior, 1979). This study found that cacaoleaves have a very thin cuticle layer, evenon the leaves of resistant clones. Our resultoffers the possible explanation that physicaland biochemical resistance mechanisms toVSD infection may involve leaf epidermistissue characteristics. The leaf epidermal tissue,on upper and lower surfaces, of resistantclones was significantly thicker than that ofsusceptible clones on both surfaces. In addi-tion, our results also confirmed that phenolicand terpenoid compounds accumulated onboth surfaces of the leaf epidermis. Phenoliccompounds that accumulated in the idioblastof epidermis tissue may be involved in biologicalinteraction between cacao and pathogens.Epidermis tissue plays a role in the penetrationby pathogens of the host plant through cellwall degradation (Agrios, 1988). Therefore, wepropose that epidermis characteristics and thecompounds accumulated in the epidermis, beconsidered as anatomical and biochemicalmarkers of cacao resistance to VSD.

Upper epidermis thickness, µm 0.000 22.616 a 16.953 bLower epidermis thickness, µm 0.000 10.448 a 8.679 bPalisade tissue thickness, µm 0.000 22.335 b 27.018 aDensity of adaxial glandular trichomes, mm-2 0.271 1.897 a 1.743 aDensity of abaxial glandular trichomes, mm-2 0.278 5.282 a 4.974 aLength of glandular trichomes head, µm 0.314 28.705 a 32.688 aWidth of glandular trichomes head, µm 0.308 26.700 a 29.057 aStomatal density, mm-2 0.184 1138.5 a 1076.9 aStomatal index 0.007 14.842 a 13.380 bLength of stomata, µm 0.063 12.410 a 12.730 aWidth of stomata, µm 0.083 13.764 a 14.120 aLength of stomatal pore, µm 0.000 4.950 b 5.972 a

Note: *)Number in the same row followed by the same letter are not significantly different, based on T-test = 5%.

Table 3. Anatomical characters of the leaves of Theobroma cacao clones Sca 6 and TSH 858

Anatomical characteristics P ValueSca 6 TSH 858

Cocoa clone



CONCLUSIONS

I-limonene, p-ethylguaiacol and 2,3 dihi-drobenzofuran may act as potential phyto-alexins produced by the host after infectedby pathogen mainly on the late infection ofC. theobromae. Terpenoid and phenolcompounds were accumulated on idioblastof upper and bottom epidermis of cacao leaves.Epidermis thickness of resistant clone wassignificantly greater than that of susceptibleclone on both surfaces. Leaf epidermis tissueand the accumulated compounds in epidermisidioblast are proposed as a physical and bio-chemical markers of cocoa resistances againstVSD disease.

ACKNOWLEDGEMENT

The authors thank to Director of ICCRIfor supporting funding this research throughInsinas Research Grant 2015 and also to Ministryof Research, Technology and Higher Educationof the Republic of Indonesia.

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