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Pelita Perkebunan 33 (1) 2017, 10—23 Santoso et al. ISSN: 0215-0212 / e-ISSN: 2406-9574

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

Abstract

Study on resistance mechanism to vascular-streak dieback (VSD) disease in cacao (Theobroma cacao L.) is limited due to the lack of fungal spores for artificial inoculation. This research was conducted to study the production of secondary metabolites that appear to be evidence of defense signaling in resistant clone of Sca 6 and susceptible clone of TSH 858 to Ceratobasidium theobromae natural infection. A fungal staining method was employed to detect C. theobromae hyphae at early infection stages, before VSD symptoms appear. Metabolite profiling was analyzed using pyrolysis gas chromatography mass spectrometry (Py-GCMS) at pre-, early and late stages of C. theobromae infection. Histochemical and anatomical characteristics of both healthy and infected were also observed to identify the accumulation sites of secondary metabolites on and in cocoa tissues. The results confirmed that fungal staining using trypan blue can detect early stages of C. theobromae infection; at the 14th week (on susceptible seedlings) and the 18th week (on resistant clones), following placement of the seedlings under infected cacao . biosynthesis, biosynthesis, environmental information processing signal transduction pathways, and aromatic biodegradation were detected as important metabolite pathways during defense mechanism. I- (terpenoid), p-ethylguaiacol (phenols) and 2.3 dihidrobenzofuran (heterocyclic compounds) were proposed as an active defense produced by the after infected by pathogen mainly on late infection of C. theobromae. Terpenoid and phenol compounds were accumulated on the glandular trichomes, idioblast of upper and bottom epidermis, phloem vessel and cortex idioblast of cacao leaves. Epidermis thickness of resistant clone was significantly greater than that of susceptible clone on both surfaces. Leaf epidermis tissue and the accumulated compounds in epidermis idioblast may act as the physical and biochemical markers of cocoa resistance to VSD.

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

INTRODUCTION (Samuel et al., 2012). In Southeast Asian countries, which include Indonesia as the Vascular streak dieback (VSD) is a major world third largest cocoa producer (ICCO, constraint of cacao (Theobroma cacao L.) 2014), VSD caused 40-60% losses of the production in Southeast Asia and Melanesia national cacao production. The occurrence

10 PELITA 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 Papua Halimah & Sri-Sukamto (2007) classified New Guinea (PNG), and the disease had some cacao clones of Sulawesi 1, Sulawesi 2, expanded northwestward to Kerala State in and Sca 6 as VSD-resistant, based on a India, and Hainan Island, the southernmost VSD-symptom-scoring method; whereas of China (Keane & Prior, 1991). Wind-dispersed other cacao clones of ICS 60 and TSH 858, basidiospores of C. theobromae are released were classified as VSD-susceptible (Susilo during periods of continously heavy rainfall & Anita-Sari, 2009). Other potentially VSD- and high humidity. After penetrating cacao leaf, resistant clones of DRC 15, KA 2-10 and the fungal hyphae colonize xylem vessels, VSD2Ldg were also found in Sulawesi causing browning of the veins of lamina; (McMahon et al., 2010). which spreads to leaf midrib and petioles, The epidemiology of VSD is controlled and eventually reaches the branch. Coloni- by a strong association between the zation by C. theobromae is found only in survival and environmental factors, and the xylem vessels of cacao (Talbot & Keane, therefore it is necessary to study the host 1971); as a characteristic that distinguishes response to C. theobromae infection under it from other diseases caused by fungi in natural conditions. However, it is difficult cacao (Samuel et al., 2012). The recogniz- to determine early infection by C. theobromae able symptoms of VSD include chloro- based on obvious VSD symptoms. Histo- sis of the leaves, with scattered spots on chemical staining of C. theobromae hyphae the lamina remaining green (Guest & Keane, from infected leaves can be used to detect 2007). the presence of the fungus during early natural VSD was caused by the basidiomycete infection. The presence of C. theobromae fungus of C. theobromae, which is a member hyphae may also be an indication of secondary of the genus Ceratobasidium (Talbot & Keane, metabolite production; which acting as 1971). This fungus is an obligate parasite phytoalexins, in response to defense signaling and cannot be grown in pure culture. The only as part of cocoa resistance mechanism response experiment that successfully induced the to the pathogen. sexual stage of the fungus was conducted The objectives of the research were to in Malaysia using solid Corticium culture analyze profiles of secondary metabolites media (CCM) and liquid coconut water media, produced by C. theobromae-infected cacao O incubated at 25 C under 10/14 hours of light/ leaves, to identify secondary metabolite dark (Lam et al., 1988), but this experi- production sites in the infected leaf tissues, ment could not be repeated in PNG (Dennis, and to identify any leaf anatomical differences 1991). Therefore, the challenge is to study as characteristics of VSD-resistant and VSD- the plant response to the fungus without the susceptible cacao clones. This paper reported option of artificial inoculation. the secondary metabolites produced during Cacao resistance mechanism in response C. theobromae infection may be as phyto- to C. theobromae infection is still unclear. alexins, histochemical analysis of secondary Resistance evaluation and selection of metabolite accumulation in leaf tissues, and plants for evaluation can only be done in a leaf anatomical data. Information gathered VSD epidemic area due to the lack of spores from this study can be used for pathogenesis that could be used for artificial inoculation studies and selection tools to support (Guest & Keane, 2007). The resistance breeding and development of VSD-resistant response to VSD varies among cacao clones. cacao.

PELITA PERKEBUNAN, Volume 33, Number 1, April 2017 Edition 11 Santoso et al.

MATERIALS AND METHODS nor VSD symptoms have yet been detected. Early infection is defined as the first time Two cacao clones, i.e. Sca 6 (resistant) C. theobromae hyphae have been detected and TSH-858 (susceptible) were used in the in the xylem vessel of the infected leaf, experiment. Seven-month-old seedlings but VSD symptoms have not yet appeared. propagated through top grafting, were grown Late infection is the condition when both in and outside VSD epidemic area. C. theobromae and VSD symptoms have The study was conducted in a VSD been detected. epidemic area, Kaliwining Experimental The presence of C. theobromae hyphae Station of Indonesian Coffee and Cacao was checked every two weeks using a fungal Research Institute (ICCRI), Jember, Indonesia. staining method based on Liberato et al. Histo-chemical and anatomical analyses were (2005). Leaf midrib samples (lamina containing carried out in Microtechnique Laboratory, midrib) of 1 cm2 were soaked in a 20 mL Department of Biology, Bogor Agricultural 1:1 (v/v) solution of glacial acetic acid and University, Bogor, Indonesia. Secondary absolute ethanol in a closed container at room metabolite analysis using pyrolysis gas chro- temperature for 24 hours. Leaf samples were matography mass spectrometry (Py-GCMS) cross-sectioned using a razor blade, then was carried out at the Chemical and Proxi- mounted on glass slide and given a drop of mate Integrated Laboratory, Forestry Research 85% lactic acid and 0.1% trypan blue. Any and Development Center, Gunung Batu, presence of the fungus was observed under Bogor, Indonesia. light microscope Olympus CX21 (Olympus, The experiment used a randomized Japan), and identified using species descrip- complete block design in factorial with three tion of C. theobromae as done by Junianto replications. The first factor was two cacao & Sri-Sukamto (1986). The hyphae of clones, i.e. VSD-resistant (Sca 6) and VSD- C. theobromae are typically characterized susceptible (TSH 858). The second factor was by right-angle branching, and colonize only two VSD infection treatments, i.e. naturally xylem vessels of cacao. VSD-infected cacao seedlings, placed under Secondary metabolite profiles were iden- infected cacao trees, and non-infected cacao tified using a Shimadzu QP2010 (Shimadzu, seedlings, placed outside the area of cacao Japan) Pyrolysis GCMS, and the sample plants at a distance of approximately 3000 m. preparation method was based on Chaves Observation for the presence of C. theobromae & Gianfagna (2007). Leaf samples were infection and VSD symptoms was performed homogenized using a pestle and mortar, and every two weeks using three plant samples a 0.002 g homogenized sample was then put per experimental unit. Metabolite profiling into the GCMS instrument. The chemical was carried out at pre-, early and late infection components of the sample were identified stages using three biological replications with under the following settings/conditions: two technical replications per biological pyrolysis temperature at 400OC; GC oven replication. Phenolic, terpenoid and temperature at 50OC; injector temperature histochemical staining was done using three at 280OC; and interface temperature ion biological replications with five observation source at 280OC. Helium gas was used as the views per replication. carrier. Pre-infection is the pre-exposure condition, The mass spectrograms were identified one day before seedlings are placed under by matching them against those from the infected plants, and when neither C. theobromae existing mass spectrum database provided

12 PELITA PERKEBUNAN, Volume 33, Number 1, April 2017 Edition Secondary metabolites as potential phytoalexins, secretion sites and proposed resistance markers to VSD in cocoa in the GCMS instrument, which cross-refers stomatal index, palisade thickness, glandular to the National Institute of Standard and trichome density, glandular trichome length Technology (NIST) Library, Maryland, and glandular trichome width, was performed USA. Analysis of peak area chromatogram using Image Raster Software (Miconos, data was performed using the One-Way Indonesia). Stomatal index was measured Analisis of variance (Anova) and Tukey test based on number of epidermis divided by at 5% of confidencial level. The types of number of stomata. metabolite were identified using the PubChem (http://pubchem.ncbi.nlm. nih.gov/) database. Metabolite pathways were developed using RESULTS AND DISCUSSION the pathway maps of the KEGG database Plant disease resistance mechanisms (http://www.genome.jp/kegg/pathway). involve activation of a series of defense Metabolite accumulation sites on and in pathways as part of the defense mechanism leaf tissues were identified by histochemical against pathogen attack. Plants have a regula- test. The leaf used for sampling in the analysis tory mechanism to activate particular defense was the sixth leaf back from the tip in each responses according to the specific stimuli case. Fresh samples were cross-sectioned using received (Chaves & Gianfagna, 2007). Phyto- a freezing microtome: a Yamato RV-240 alexin is a chemically diverse group of (Yamato Kohki Ind Co, Japan) with 30 mm secondary metabolites with strong antimi- crobial actions, and these metabolites accu- razor-blade thickness. were iden- mulate around the infection site. They are tified using 5% copper acetate, following generally undetectable in the plant before a procedure suggested by Harborne (1987); infection, but are synthesized very rapidly phenolic compounds were identified using after pathogen attack (Taiz & Zeiger, 2010). a 5% ferric trichloride procedure suggested by Johansen (1940). Anatomical characteris- Fungal staining successfully detected tics of the cacao leaf were also investigated, the early infection stage of C. theobromae based on both transversal and paradermal in VSD-susceptible and VSD-resistant plants, sections. Samples were fixed in 70% alcohol, at the 14th and 18th weeks, respectively, after and then cross-sectioned using a freezing cacao seedlings had been placed under VSD- microtome: a Yamato RV-240 (Yamato Kohki infected cacao plants. Susceptible clone Ind Co, Japan) in 20 mm thickness. Paradermal (TSH 858) was infected faster than resistant sections of each leaf were prepared as semi- clones (Sca 6). Although the hyphae had permanent samples using a whole-mount been detected in the xylem vessels, the leaves method first suggested by SASS (1951). The appeared normal and healthy, exhibiting no alcohol-fixed leaves were rinsed with distilled visible symptoms of VSD (Figure 1 & 2). water and soaked in 50% nitric acid solution, The VSD symptoms of leaves used in this before being rinsed again with distilled water study were interpreted according to observed three times. Adaxial and abaxial surface layers leaf chlorosis with green spots, as described of leaves were peeled using a razor blade. by Guest & Keane (2007). Samples were observed under an Olympus During onset of C. theobromae infection, CX-21 light microscope (Olympus, Japan), cacao leaves produced 20 different metabo- and photographed using an Optilab Advance lites. Chromatogram areas from Py-GCMS camera (Miconos, Indonesia). Measurement analysis of both VSD-resistant (Sca 6) and of anatomical characteristics, i.e. epidermis VSD-susceptible clones (TSH 858) were not thickness, stomatal size, stomatal density, statistically different at the pre-infection

PELITA PERKEBUNAN, Volume 33, Number 1, April 2017 Edition 13 Santoso et al.

Figure 1. Morphological symptom on VSD-susceptible clone of TSH 858 (A) and VSD-resistant clone of 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-sections at pre-infection stage (A), early infection stage (B), and late infection stage (C). Midrib longitudinal section: typical branching of C. theobromae hyphae at right angles (D) (xy = xylem, Ot = C. theobromae hyphae, br = hyphae typical branching). Scale bars = 50 µm (A-C); 30 µm (D)

14 PELITA PERKEBUNAN, Volume 33, Number 1, April 2017 Edition Secondary metabolites as potential phytoalexins, secretion sites and proposed resistance markers to VSD in cocoa stage (Table 1). There was then an increase and 2,3 dihidrobenzofuran could be considered in production of I-limonene (a terpenoid as indicators of a triggered defense signaling compound) and p-ethylguaiacol (a phenolic mechanism, and are produced by host plants compound) in both clones at the early stage induced by C. theobromae, mainly in late of C. theobromae infection, and again, there infection stage. I-limonene, p-ethylguaiacol, was no significant difference between the and 2,3 dihydrobenzofuran were synthesized VSD-resistant and VSD-susceptible clones during C. theobromae infection stages through (Table 1). However, at the late infection stage, different metabolite pathways. I-limonene production of I-limonene, p-ethylguaiacol, and is a monoterpenoid that is synthesized 2,3 dihidrobenzofuran (terpenoids), were through methyl erythritol phosphate (MEP) significantly higher in resistant clones than in the terpenoid backbone metabolism. in susceptible clones (Table 1). Limonene has been reported as a mono- Early infection defined as the first time terpenoid that exhibits an antifungal action C. theobormae hyphae detected in the xylem that is similar to the commercial fungicide, vessel of infected leaf, but VSD symptoms carbendazim. Limonene and carbendazim not yet appear. Early infection occurred in have been tested against Rhizoctonia solani, week 14th (TSH 858) and week 18 th (Sca 6) Fusarium oxysporum, Penicillium digitatum following placement of cacao seedlings under and Aspergilus niger (Marei et al., 2012). VSD-infected cacao plants. Control was Fungi produce a cellulase enzyme that can cacao seedlings of the same age, placed degrade cell walls during pathogenesis (Milling outside area of infected cacao plants. & Richardson, 1995), and it was reported Late infection is defined as when both that limonene has a strong inhibitory effect, C. theobormae and VSD symptom have been similar to that of commercial fungicides, on detected. Late infection in TSH 858 and Sca 6 cellulase activity (Marei et al., 2012). occurred at 15th and 19th weeks respectively, Monoterpenoid is a type of metabolite after cacao seedlings placed under VSD product synthesized in large quantities through infected cacao plants. Control was cacao a signal transduction pathway, as a result of seedlings of the same age placed outside area activity (KEGG, 2015). Jasmonic of infected cacao plants. acid is a signal molecule that induces a signal In the early stage of C. theobromae transduction pathway, which leads to the produc- infection there was increasing production tion of phytoalexins as part of the resistance of I-limonene and p-ethylguaiacol in both mechanism of Glycine max, in response to VSD-resistant and VSD-susceptible clones, Rhizoctonia solani infection (Aliferis et al., with no significant difference between the 2014). two types of clone at this stage (Table 1). Phenolic compound p-ethylguaiacol is This might be due to similar basic responses synthesized through phenylpropanoid path- of both VSD-resistant and VSD-susceptible ways. Secretion of the compound from cells to the infection, and similar rates of plants is induced by wounds and is involved phytoalexin formation (Kuc, 1992). Increased in the communication and defense systems production of I-limonene and p-ethylguaiacol, during Agrobacterium and Rhizobium infection as well as 2,3 dihidrobenzofuran, occured (Bhattacharya et al., 2010). Accumulation of more significantly in the VSD-resistant clones phenolic compounds associated with a hyper- at the late infection stage (Table 1). It is sensitive response by a plant to pathogen suggested that I-limonene, p-ethylguaiacol infection has been found in coffee plant

PELITA PERKEBUNAN, Volume 33, Number 1, April 2017 Edition 15 Santoso et al. Late infections TSH 858 Susceptible clone Control Late infections Sca 6 Sca % from total chromatogram % ion from Resistant Resistant clone , where x = chromatogram peak area of x Control 00 0.70 b 0.70 b 1.62 a 1.80 a 1.37 a 1.94 a 0.00 c 1.99 a 0.01 16.66 a 15.55 a 8.99 ab 5.68 b 0.06 1.23 b 2.34 a 2.43 a 2.01 ab Value 0.1550.231 1.440.502 a 1.06 a 1.52 1.070.005 a a 1.370.009 a 0.92 2.23 2.170.124 a a a 0.00 2.160.022 c a 2.25 1.53 2.28 0.700.857 a a a a 4.25 5.06 1.030.199 ab ab a 1.40 3.04 0.91 2.06 a a a a 3.98 2.02 b b 2.10 a 2.94 1.04 1.310.103 a b a 7.60 7.00 a a 1.97 a 3.73 1.10 2.810.323 a a a 4.96 ab 2.730.417 a 0.99 4.82 2.480.144 a a a 6.38 2.500.042 a a 4.99 2.61 3.980.909 a a a 6.97 3.260.756 a a 1.60 1.08 1.71 5.21 a a a a 2.26 1.25 4.99 ab a a 1.08 3.85 1.90 a a a 2.20 ab 1.27 4.86 a a 1.64 1.08 1.29 a a b 1.61 a 0.88 a 1.36 a P P C. theobromaeC. . Early Infections TSH 858 Susceptible clone Control stage of infection by of infection stage = 5%; *Data were transformed by Early a Infections Sca 6 Sca % from total chromatogram % ion from Resistant Resistant clone Control ----- Value 0.0470.426 1.370.227 b 2.000.032 a 0.83 1.030.548 a b 20.37 a 1.080.001 a 3.13 2.39 1.120.722 a a 20.20 a a 0.73 1.820.852 b a 15.71 2.00 1.88 0.71 5.24 ab a a a ab 5.69 12.70 1.54 2.140.687 a ab b a 0.93 4.93 2.200.596 a a a 0.89 b 2.49 5.080.006 a a 2.35 4.29 1.630.000 a a a 5.60 0.00 a 5.34 2.380.107 b a a 1.79 1.65 2.090.394 a a a 6.31 5.60 2.50 2.460.020 a a a a 5.46 2.20 1.770.627 a a a 2.61 2.78 2.84 5.140.130 ab a ab a 1.91 1.13 0.71 5.790.544 a b a a 1.89 2.79 2.86 b 3.17 a0.060 a a 2.51 0.71 4.92 2.16 a a a b 2.86 2.46 ab 1.35 ab 3.71 b a 5.34 1.63 1.32 2.24 a a a b 2.70 1.19 a b 2.04 1.98 a a 1.77 2.21 ab a 1.62 a 2.18 a P P

Susceptible clone TSH 858 e day before e day cacao placed seedlings under cacao infected trees by VSD in pre-infection, the early stage of infection, and the late the of infection, and early stage in pre-infection, the r are not significantly different, based on Tukey test Resistant clone Sca 6 Scaclone % from total chromatogram % ion from Value P metabolite, pre RT retention= (minutes); = infections time on 3.6084.0824.275acetate* Methyl 4.802 Diacetyl5.342 2-Methylfuran*12.543acid Ethylic 13.066 Acetol I-limonene13.658 Corylon 0.60814.144 p-Methoxyphenol14.863Propyl- 2–N- Oxetan 15.508 3.06 2-Methoxy-4-methylphenol15.712 0.480 -dihydrobenzofuran 2,3 15.664 0.863 0.435 p-ethylguaiacol16.128 2,5 0.544Dimethoxytoluene 0.634 1.1016.492acetophenone 2-Methoxy 8.68 0.89317.344 3.39 2.68 0.932 - - 2,6-Dimethoxyphenol 0.19317.925 2.99 6.25 Eugenol19.352 0.979 3.83 Toluene, 3,4,5-trimethoxy 0.9119.889 0.881 1.34 1.60 4-Allyl-2,6-dimethoxyphenol -20.888 - 0.719 8.35 Phytol 2.91 4.97 0.493 0.088 Palmiticacid 3.14 6.80 3.37 - 4.60 3.90 0.89 2.12 1.37 - 1.10 4.97 0.727 - 3.34 4.36 0.644 1.98 0.89 4.06 0.638 - 2.69 1.15 4.37 2.41 0.70 RT Metabolites Note: Figures in the same line followed by the same lette Table 1.peak area chromatogram profiles Cacao metabolite on based

16 PELITA PERKEBUNAN, Volume 33, Number 1, April 2017 Edition Secondary metabolites as potential phytoalexins, secretion sites and proposed resistance markers to VSD in cocoa defense mechanism against Colletotrichum (Figures 3B & 3D). Phenolic compounds were kahawae (Loureiro et al., 2012). also detected in palisade tissue (Figure 3A). 2,3-dihydrobenzofuran is a heterocyclic Glandular trichomes containing phenolic and compound that is synthesized through degra- terpenoid compounds were distributed across dation of certain aromatic compounds (KEGG, both cacao leaf surfaces, although mainly on 2015). Asperfuran is an example of an anti- the leaf midrib (Figures 3C & 3E). Phenolic fungal 2,3 dihydrobenzofuran produced by compounds also appeared in numerous a strain of Aspergillus oryzae. Asperfuran idioblast cells in the cortex of the midrib inhibits chitin synthase produced by (Figures 4A, 4B, & 4C). Coprinus cinereus (Pfefferle et al., 1990). Similar accumulation sites were found Chitin is a major component of fungal cell in the leaves of VSD-resistant and VSD- walls, which play a vital role in hyphal tip susceptible clones during the infection stages growth and fungal morphogenesis. Chitin of O. theobromae. Histochemical staining is synthesized by chitin synthase and it is showed that phenolic and terpenoid content well known that the latter is inhibited by com- of the phloem in VSD-resistant clones was mercial fungicides (Kong et al., 2012). The higher than that of susceptible clones. This 2,3 dihydrobenzofuran compound has also was indicated by increasing intensity of dark been reported to be produced by Bauhenia brown color of the phloem at the time of purpurea and shows antifungal activity infection (Figure 5, Table 4). against Candida albicans (Abyaneh & Rai, The secretion of terpenoid and phenolic 2013). compounds during C. theobromae infection Role of terpenoid and phenolic compounds involves phloem tissues. The role of phloem in cacao resistance mechanism to pathogens in the secretion of phenolic compounds has have been reported as phytoalexins in respect been reported in the case of Picea abies of Verticillium dahlia infection. Production infected by Ophiostoma polonicum (Brignolas of triterpenoid arjunolic acid and the phenolic et al., 2007). It is understandable that phenolic compounds 3,4 dihydroxyacetophenone and and terpenoid contents in the phloem of 4-hydroxyaceto-phenone, significantly increases VSD-resistant clones were higher than in in cacao infected by Verticillium dahlia susceptible clones. This might be related to (Resende et al., 1996). In addition to these the secretion of the compounds during the terpenoid and phenolic phytoalexins, elemental infection stage. Cacao leaves have glandular sulphur is produced as an antifungal substance, and non-glandular trichomes (Nakayama et al., in cacao during Verticillium dahlia infection 1996). In some plants, glandular trichomes (Cooper & Williams, 2004; Resende et al., 1996). have a role as physical and chemical barriers to spores (Martin & Clover, 2007; Chattopadyay et al., 2011). However, there is lack of study Metabolite Accumulation Sites findings regarding the metabolite content of Metabolite accumulation sites were found cacao glandular trichomes. This research in healthy leaves of both VSD-resistant and showed that glandular trichomes of cacao, VSD-susceptible clones at pre-infection stage. containing phenolic and terpenoid com- Both clones had leaf laminae consisting of pounds, were distributed on both surfaces single-layered epidermis tissue with idioblast of cacao leaves, mainly on the leaf midribs. cells containing phenolic and terpenoid However, the trichomes were generally more compounds in the upper and lower epidermises abundant on the abaxial than on the adaxial

PELITA PERKEBUNAN, Volume 33, Number 1, April 2017 Edition 17 Santoso et al. leaf surface. Cacao glandular trichomes are resistant (Sca 6) and VSD-susceptible clones of the multiseriate capitate type: composed (TSH 858) did not differ statistically, sug- of a basal cell, two-cell uniseriates and eight gesting that the glandular trichome is not cells making up the secretory head. The dis- closely related to cacao resistance mechanisms tribution of glandular trichomes on both VSD- to VSD.

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 idioblast epidermis (D) and glandular trichomes (E). Glandular trichomes in water as control (F). (pl = phenolic compounds, tr = terpenoids). Scale bar: 50 mm (A), 20 mm (B - F)

Tabel 2. Secondary metabolite production sites during O. theobromae infection, based on histochemical staining Pre infection Early infection Late infection Cell/tissue Sca 6 TSH 858 Sca 6 TSH 858 Sca 6 TSH 858 PTPTPTPTPTPT 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.

18 PELITA PERKEBUNAN, Volume 33, Number 1, April 2017 Edition Secondary metabolites as potential phytoalexins, secretion sites and proposed resistance markers to VSD in cocoa

Figure 4. Histochemical staining of phenolic compounds (A-F) and terpenoids (G-L) in cacao leaf midrib of Sca 6 (A-C and G-I) and TSH 858 (D-F and J-L) showing positive results in phloem during pre-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 mm (A-L)

PELITA PERKEBUNAN, Volume 33, Number 1, April 2017 Edition 19 Santoso et al.

Epidermis as a Barrier clones. We suspect that pathogens have more opportunities to penetrate susceptible The leaf epidermis tissue of VSD-resistant than resistant plants through the former clones was significantly thicker than that of wider stomatal openings. susceptible clones, for both leaf surfaces (Table 3). However, size and density of stomata, A previous study explained that hyphae as well as the distribution of glandular trichomes, penetrated young leaves by growing directly were not significantly different between through cuticle layers into mesophyll tissues VSD-resistant and VSD-susceptible clones. (Prior, 1979). This study found that cacao Interestingly, stomatal pores on the VSD- leaves have a very thin cuticle layer, even susceptible clones were found to be wider on the leaves of resistant clones. Our result than those of resistant clones. offers the possible explanation that physical and biochemical resistance mechanisms to Pathogenesis caused by infectious agents VSD infection may involve leaf epidermis begins when the pathogen contacts host plants. tissue characteristics. The leaf epidermal tissue, Certain anatomical characteristics of leaves can on upper and lower surfaces, of resistant act as physical barriers to pathogens penetrating clones was significantly thicker than that of a host plant. Most pathogens gain access to susceptible clones on both surfaces. In addi- the plant interior through natural openings such tion, our results also confirmed that phenolic as stomata (Agrios, 1988). and terpenoid compounds accumulated on The VSD-resistant and VSD-susceptible both surfaces of the leaf epidermis. Phenolic clones were not significantly different in compounds that accumulated in the idioblast terms of stomatal density, length and width. of epidermis tissue may be involved in biological The study suggested that these stomatal interaction between cacao and pathogens. characteristics were not directly related to cacao Epidermis tissue plays a role in the penetration resistance mechanisms to VSD. This finding by pathogens of the host plant through cell differs from a previous study that reported a wall degradation (Agrios, 1988). Therefore, we positive association between stomatal density propose that epidermis characteristics and the and cacao resistance to VSD (Susilo & Anita- compounds accumulated in the epidermis, be Sari, 2009). However, our research found considered as anatomical and biochemical that the stomatal pores of susceptible markers of cacao resistance to VSD. clones were wider than those of resistant

Table 3. Anatomical characters of the leaves of Theobroma cacao clones Sca 6 and TSH 858 Cocoa clone Anatomical characteristics P Value Sca 6 TSH 858

Upper epidermis thickness, µm 0.000 22.616 a 16.953 b Lower epidermis thickness, µm 0.000 10.448 a 8.679 b Palisade tissue thickness, µm 0.000 22.335 b 27.018 a Density of adaxial glandular trichomes, mm-2 0.271 1.897 a 1.743 a Density of abaxial glandular trichomes, mm-2 0.278 5.282 a 4.974 a Length of glandular trichomes head, µm 0.314 28.705 a 32.688 a Width of glandular trichomes head, µm 0.308 26.700 a 29.057 a Stomatal density, mm-2 0.184 1138.5 a 1076.9 a Stomatal index 0.007 14.842 a 13.380 b Length of stomata, µm 0.063 12.410 a 12.730 a Width of stomata, µm 0.083 13.764 a 14.120 a Length 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 a = 5%.

20 PELITA PERKEBUNAN, Volume 33, Number 1, April 2017 Edition Secondary metabolites as potential phytoalexins, secretion sites and proposed resistance markers to VSD in cocoa

CONCLUSIONS tion with Ophiostoma polonicum. Forest Pathology, 25, 253–265. I-limonene, p-ethylguaiacol and 2,3 dihi- Chattopadhyay, S.; K.A. Ali; S.G. Dos; N.K. Das; drobenzofuran may act as potential phyto- R.K. Agarwal; T.K. Bandopadhyay; alexins produced by the host after infected A. Sarkar & Bajpai (2011). Association by pathogen mainly on the late infection of of leaf micro-morphological characters C. theobromae. Terpenoid and phenol with powdery mildew resistance in compounds were accumulated on idioblast field-grown mulberry (Morus spp.) of upper and bottom epidermis of cacao leaves. germplasm. AoB / PLANTS, 1–11. Epidermis thickness of resistant clone was Chaves, F.B. & T.J. Gianfagna (2007). Necrotrophic significantly greater than that of susceptible phase of Moniliophtora perniciosa clone on both surfaces. Leaf epidermis tissue causes salicylic acid accumulation in and the accumulated compounds in epidermis infected stem of cacao. Physiological idioblast are proposed as a physical and bio- Molecular Plant Pathology, 69, 104-108. chemical markers of cocoa resistances against Cooper, R.M. & J.S. Williams (2004). Elemental VSD disease. sulphur as an induced antifungal substance in plant defense. Journal of Experimental Botany, 55, 1947–1953. ACKNOWLEDGEMENT Dennis, J.J.C. (1991). Epidemiology and Control of Vascular Streak Dieback of Cacao The authors thank to Director of ICCRI in Papua New Guinea. MSc thesis. for supporting funding this research through La Trobe University, Australia. Insinas Research Grant 2015 and also to Ministry Guest, D. & P.J. Keane (2007). Vascular streak of Research, Technology and Higher Education dieback: A new encounter disease of of the Republic of Indonesia. cacao in Papua New Guinea and South- east Asia caused by the obligate Basidi- REFERENCES omycete Ceratobasidium theobromae. American Phytopathology Society, 97, Abyaneh, M.R. & M. Rai (2013). Antifungal 1654–1657. Metabolites from Plants. Springer- Verlag Berlin Heidelberg, New York. Halimah, D. & S. Sukamto (2007). Vascular streak dieback intensity on some clones of Agrios G.N. (1988). Plant Pathology. 3rd Ed. Indonesian coffee and cacao collection. Academic Press Inc, New York. Pelita Perkebunan, 23, 118–128. Aliferis, K.A.; D. Faubert & S. Jabaji (2014). Harborne, J.B. (1987). Metode Fitokimia A metabolic profiling strategy for the Penuntun Cara Modern Menganalisis dissection of plant defense against Tumbuhan. (Translated: K. Padmawinata; fungal pathogens. Plos One. 9, 1–13. I. Soediro; & S. Niksolihin). Institut Bhattacharya, A.; P. Sood & V. Citovsky (2010). Teknologi Bandung Press, Bandung. The roles of plant phenolic in defense ICCO (2014). Quarterly Bulletin of Cacao and communication during Agrobac- Statistics (Cacao year 2013/2014). terium and Rhizobium infection. International Cocoa Organization. Molecular Plant Pathology, 11, 705–19. London. Brignolas, F.; F. Lieutier; D. Sauvard; A. Yart; Johansen, D.A. (1940). Plant Microtechnique. A. Drouet & A. Claudot (2007). McGraw-Hill. New York. Changes in soluble phenol content of Norway spruce (Picea abies) phloem Junianto, Y.D. & Sri-Sukamto (1986). Vascular in response to wounding an inocula- streak dieback pada tanaman kakao di

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in planta detection of the pathogen Susilo, A.W. & Annita-Sari (2009). Respon and a new taxonomy. Fungal Biology, ketahanan beberapa hibrida kakao 116, 11–23. (Theobroma cacao L.) terhadap serangan penyakit pembuluh kayu. Sass, J.E. (1951). Botanical Microtechnique. Pelita Perkebunan, 27, 77–87. 2nd Ed. The Iowa State College Press, Iowa. **0**

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