Heritability of Phenols in the Resistance of Theobroma Cacao Against Phytophthora Megakarya, the Causal Agent of Black Pod Disease

J. Phytopathology 155, 519–525 (2007) doi: 10.1111/j.1439-0434.2007.01268.x 2007 The Authors Journal compilation 2007 Blackwell Verlag, Berlin

Department of Plant Biology, Faculty of Science, University of Yaounde´ 1, Yaounde´, Cameroon

Heritability of Phenols in the Resistance of Theobroma cacao against Phytophthora megakarya, the Causal Agent of Black Pod Disease

P. F.Djocgoue 1,21,2, T.Boudjeko 1,31,3, H. D.Mbouobda 1, D. J.Nankeu 1, I.El Hadrami4 and N. D.Omokolo 1 AuthorsÕ addresses: 1Laboratory of Plant Physiology, Department of Biological Sciences, Higher Teacher’s Training College, PO Box 47, Yaounde, Cameroon; 2Department of Plant Biology, Faculty of Science, P.O. Box 812, Yaounde, Cameroon; 3Department of Biochemistry, Faculty of Science, P.O. Box 812, Yaounde, Cameroon; 4Laboratoire de Biotechnologies, Protection et Valorisation des Ressources Ve´ge´tales, Equipe Biotechnologies, Ecophysiologie et Valorisation des Plantes, De´partement de Biologie, Faculte´des Sciences-Semlalia, BP 2390, 40 000 Marrakech, Morocco (correspondence to P. F. Djocgoue. E-mail: [email protected] or [email protected]) Received October 26, 2006; accepted February 26, 2007

Keywords: Theobroma cacao, Phytophthora megakarya, resistance, heritability, necrosis, phenols

Abstract ica (Wood and Lass, 2001). Cocoa is an important The black pod disease caused by Phytophthora megak- crop in many tropical countries, including Brazil, arya is responsible for 80% of the cocoa production loss Ghana, Ivory Coast, Nigeria and Cameroon. Global in Cameroon. To assess the resistance of cocoa plants annual earnings from exports of cocoa average against this pathogen, necrotic lesions, phenolic content US$2.9 billion (Gray, 2000). This plant is a source of and qualitative alteration of phenolics were conducted income for Cameroonian farmers. However, black pod in ICS84 and ICS95 clones (two Trinitario introduced rot caused by Phytophtora megakarya is the most seri- from Trinidad) and their hybrids (families F30 and F25) ous disease affecting cocoa production in Cameroon. derived from reciprocal cross breeding between these Beans from pods attacked by the disease are not suit- two parental clones after inoculation. The existence of able for processing and must be discarded. Losses can strong hybrid vigour has been shown. Ninety percentage reach 100% of annual production if no control meas- of the hybrid’s genotypes manifested a positive heterosis ures are taken (Despreáux et al., 1988). The methods effect for the development of lesion size. This suggests available for controlling cocoa black pod rot are fungi- the existence of hybrid vigour with a genetic additive cide application, use of resistant cultivars and other effect. F3086, F2509, F2552 and F2586 hybrids were appropriate cultural practices. An increase in the characterized by localized lesions. Those hybrids geno- effectiveness of control can be expected when these types can be considered as elite clones. In relation to methods are combined. However, chemical control is analysis of total phenolics and lesion size, no maternal expensive, commercially non-viable and environmen- effect was detected in the transmission of these charac- tally harmful. Resistance to black pod has become a ters. A significant and negative correlation (r ¼ )0.683) major breeding target. (P < 0.01) has been observed between necrosis evolu- Besides, a strong correlation exists between the pro- tion and phenolics accumulation. The values of the her- duction and the use of hybrids. In fact, the main pro- itability of lesion size and the total phenolic content in ducing countries are those who use a high percentage offsprings did not permit to show the maternal effect. of hybrids in their farms (Indonesia 76%, Ivory Coast Qualitative analyses of phenolics showed high flavo- 69.2%, Malaysia 69%, Ghana 63.2%, Cameroon nones content in cocoa leaves. Qualitative analyses of 25.1% and Equador 17.4%. In Cameroon, most of the phenolics in ICS84, ICS95 clones and their hybrids cultivated cocoa trees were originally derived from old showed a modification of the phenolics profiles, notably varieties introduced by German colonialists, and from concerning apigenin and luteolin derivatives due to the second generation seeds obtained from newer hybrid inoculation. These compounds, along with others that cultivars. Hybrid selection was based on heterosis were not identified, could have a role in the reaction and observed in crossing genetically distinct genotypes. mechanism of defence of cocoa against P. megakarya. Local and introduced clones available in clonal banks generally were used as hybrid progenitors. Due to their Introduction yield capacity, environmental adaptation and vigour, Theobroma cacao (cacao) is a tropical tree native to these hybrids have been exploited on a large the rain forest understory of South and Central Amer- scale. These hybrids varieties have also shown large

www.blackwell-synergy.com 520 Djocgoue et al. phenotypic variation for many traits and are not read- ern Cameroon) were used to create two progenies. In ily accepted by farmers (Ndoumbe-Nkeng et al., 2001). the previous study, ICS clones (Introduced Trinitario) Cocoa breeders continue to face the problem of high appeared to be more promising parents than SNK heterogeneity between individuals derived from one clones (Local Trititario; Djocgoue et al., 2006). Cros- cross and heterogenous transmission of genetic traits sing using hand-pollination techniques (Cilas, 1991) to the progeny. The Ôhybrid vigourÕ is manifested by was conducted at the Nkoemvone Research Station of increased size, growth rate and/or other parameters IRAD in March and April 2001. The two families resulting from the increase in heterozygosity in the F1 obtained were: F30: $ ICS84 · # ICS95 and F25: $ generation following crosses between inbred lines (Gal- ICS95 · # ICS84. lais, 1990). Nyasse (1997); Djocgoue (1998) and Nyasse et al. (2002) showed that there is a correlation Leaf inoculation and analysis between the resistance of cocoa to P. megakarya and The leaf test is an artificial inoculation method that the size of the necrotic lesion following artificial inocu- can be used to assess the resistance of genotypes. lation to screen pods and leaves for disease develop- Whole detached leaves from one or two-month-old ment. plants were washed thoroughly with tap water and Many authors showed the involvement of phenolics sterilized with 70% ethanol for 30 s. The experimen- in the plant defence mechanism against parasitic tal design consisted of three replicate plants with six attacks (El Hassni et al., 2004; Tan et al., 2004; Omok- leaves per plant. The inner surface of leaves were sca- olo and Boudjeko, 2005). In cacao tree, Djocgoue rified along the midrib and inoculated by placing a (1998) and Omokolo et al. (2002) observed an accumu- mycelial disc (4 mm) of Phytophthora megakarya lation of phenolics during the development of necrosis obtained from a 7-day-old PDA culture medium and within the mesocarp of the cortex in cocoa’s pod infec- incubated at 25–26C in the dark, in a humid cham- ted by P. megakarya. These authors noticed that the ber. Control leaves were inoculated with sterile agar most tolerant cocoa tree’s clones to the black pod dis- disc using the same methods. The isolate of ease have pods with the richer phenolics content. How- P. megakarya used was collected from a naturally ever, neither work has yet been performed on the infested pod from the Nkolbisson station. Necrotic nature of the accumulated phenolics in the resistance lesions appeared 2 days after inoculation and the size mechanism. Generally, phenolics accumulated at differ- of these lesions was measured daily for 6 days. To ent levels in infected tissues in response to pathogen determine the effect of sex of the parents on the invasion. However, numerous works have shown that transmission of resistance, heritability (h2) was estima- the involvement of phenolics in the plant’s mechanism ted according to Falconer (1974). Heritability was defense could be of qualitative nature. For example, it obtained by slope of the regression line between par- has been shown that the resistance of apple (Malus ents and their progenies using size of lesion and domestica)toVentura inaequalis is related to the high phenolics data. content of catechin and the proanthocyanidin present in leaves (Treutter and Feucht, 1990). Daayf et al. Phenolics analysis (1997) reported the accumulation of methylester of For the analysis of phenolic compounds, samples were P-coumaric acid in leaves of cucumber (Cucumis sati- taken 6 days after inoculation from healthy tissue at vus) infected by Sphaerotheca fuliginea. In the date approximately 2 cm outside of the lesion. Extraction palm/Fusarium oxysporum f. sp. albedinis pathosystem, and quantitative measurement of phenolics were per- there is a higher accumulation of inducible hydroxy- formed as described by Omokolo and Boudjeko (2005). cinnamic acid derivatives in resistant cultivars (El Total phenolic compounds were extracted twice using Hadrami et al., 1997). 0.1 N HCl. One gram of fresh tissue was ground in The objectives of the present study were to: (i) 3 ml HCl. After 30 min incubation at room tempera- evaluate the development of lesions in response to ture, the ground material was centrifuged at 6000 g for P. megakarya on leaves of two parental genotypes cat- 30 min. The supernatant was decanted and the precipi- egorized as less susceptible (ICS84) and moderately tate re-suspended in 3 ml 0.1 HCl and incubated at susceptible (ICS95) to black pods disease and the room temperature for 15 min followed by another cen- hybrids F30 and F25 obtained after reciprocal cross- trifugation. The supernatant was collected and mixed breeding, (ii) analyse the qualitative and quantitative with the first to constitute the phenolic extract. The phenolic compounds in healthy, wounded and inocu- concentration of phenolic compounds was determined lated leaves of these individuals, and (iii) assess the in the supernatant spectrophotometrically at 725 nm, heritability of resistance to P. megakarya using this according to Marigo (1973), using the Folin–Ciocalteu data. reagent. Phenolic contents were expressed in milligram equivalent of chlorogenic acid/gram of fresh weight. Materials and Methods Qualitative analysis of phenolics was carried out as des- Cocoa plant material cribed by El Hassni et al. (2004). Briefly, frozen tissues Two cocoa clones (ICS84 and ICS95) available at the from different treatments were extracted three times Institute of Agricultural Research for Development with 80% aq. MeOH at 4C under continuous stirring. (IRAD) at the Nkoemvone Research Station (South- The homogenate was centrifuged at 7000 · g for 3 min Phenols in T. cacao/P. megakarya interaction 521 and the supernatant was stored at )20C until required Results for analysis by HPLC using a Waters 600E HPLC Evolution of the lesion (Waters, Paris, France) equipped with a Waters 990 On leaves wounded and inoculated with an agar disc photodiode Array Detector (Waters, Paris, France) and containing P. megakarya mycelium, the development Millipore Software (Waters, Paris, France) for data of the necrosis appears 2–3 days after inoculation in analysis. An efficient gradient of acetonitrille-o- phos- both clones and their hybrids. Generally, in the F30 phoric acidified bidistilled water (pH 2.6) was used with family, the size of the necrosis varies between 1.45 cm an Interchrom C18, 5 lm reversed phase column. (Second day) and 8.87 cm (sixth day). At day 2, the Phenolics were identified on the basis of their retention development of lesion was less important in the F3019, time and their spectra in comparison with standards F3032 and F3086 hybrids (see Table 1). On the con- (P-coumaric acid, caffeic acid, synapic acid, catechine trary at the sixth day, individuals F3032, F3086, and epicatechine, luteoline-7- glucoside and apigenine F3094 and F3011 had the smallest necrotic lesion size. from Sigma-Aldrich, Saint Quentin Fallavier, France). In F25 family, the necrosis length varies between When necessary, standards were used in co-injection to 0.5 cm at day 2 and 8.00 cm at day 6. Two days after confirm the identity of certain compounds. Content of inoculation, the size of the lesion was less important in hydroxycinnamic acids derivatives, apigenine deriva- F2552, F2586 and F2509 hybrids (Table 1). Six days tives and luteoline derivatives were estimated from the after inoculation, the same clones presented the small- area of peaks having the same absorption spectrum. est necrotic lesion size.

Statistical analyses Variation of the phenolics content Data presented are the means ± SE of at least three In healthy leaves, the total phenolic content was independent experiments. anova and the Student-New- higher in F2586 (2.57 mg/g of Fw), F2552 (2.10 mg/g man–Keuls test were used to compare the susceptibility of Fw) and F3045 (2.05 mg/g of Fw) individuals levels of better progenies resulting from different cros- (Table 2). When leaves were wounded or inoculated ses, to assess hybrid vigour (Begun and Gabriel, 1981) total phenolic content increased and varied from 16% and to compare phenolics content. Hierarchical classi- to 55% in the F30 family and from 14% to 56% in fication of parents and their progenies was obtained by the F25 family (Table 2). At 90% degree of homo- using principal component analysis (PCA). anova and geneity, direct hierarchical classification of soluble PCA were performed using SAS-system (Anonymous, total phenol content in healthy, wounded and inocu- 1997). lated leaves differentiated into three groups for the

Table 1 Average lesion size (cm) on the Average lesion size (cm) midrib of Theobroma cacao leaves Genotypes Day 2 Day 3 Day 4 Day 5 Day 6 of hybrids derived from F30 and F25 families Parents ICS95 2.00 ± 0.11abc 5.57 ± 0.25de 6.57 ± 0.53ef 8.30 ± 0.67d 9.20 ± 0.54d ICS84 2.05 ± 0.28abc 2.92 ± 0.35a 4.75 ± 0.99abc 6.75 ± 0.70bc 8.42 ± 0.29cd F30 F3018 2.4 ± 0.21cd 4.75 ± 0.86bcd 6.40 ± 0.93def 7.07 ± 0.17bc 7.87 ± 0.25bc F3019 1.68 ± 0.30ab 4.05 ± 0.42abc 6.12 ± 0.39def 7.62 ± 0.47cd 8.87 ± 0.25d F3022 2.25 ± 0.19bcd 3.15 ± 0.20a 5.60 ± 0.64cde 6.35 ± 0.50abc 7.00 ± 0.21ab F3032 1.45 ± 0.53a 3.75 ± 0.47ab 4.32 ± 0.47ab 5.12 ± 1.16a 6.57 ± 0.80a F3045 4.82 ± 0.49g 5.92 ± 0.89e 6.60 ± 1.21f 7.12 ± 0.86bc 7.75 ± 0.55bc F3048 3.85 ± 0.45e 5.45 ± 0.54de 6.97 ± 0.49f 6.97 ± 0.49bc 7.72 ± 0.62bc F3052 2,.30 ± 0.21bcd 5.00 ± 0.35cde 5.62 ± 0.45cde 6.10 ± 0.27ab 7.20 ± 0.40ab F3086 1.52 ± 0.49a 3.00 ± 0.21a 4.00 ± 0.38a 5.20 ± 0.41a 5.12 ± 1.80a F3094 3.05 ± 0.34e 3.85 ± 0.45ab 5.30 ± 0.54bcd 6.45 ± 0.54abc 6.55 ± 0.41a F3011 2.70 ± 0.43de 3.10 ± 0.63a 4.47 ± 0.45abc 6.15 ± 0.58ab 6.77 ± 0.33a Parents ICS95 2.00 ± 0.11abc 5.57 ± 0.25de 6.57 ± 0.53ef 8.30 ± 0.67d 9.20 ± 0.54d ICS84 2.05 ± 0.28abc 2.92 ± 0.35a 4.75 ± 0.99abc 6.75 ± 0.70bc 8.42 ± 0.29cd F25 F2508 3.16 ± 0.30d 3.93 ± 0.25b 5.13 ± 0.25cd 6.60 ± 0.65b 7.96 ± 0.05c F2509 0.50 ± 0.08a 2.46 ± 0.32a 3.60 ± 0.36ab 4.33 ± 0.32a 5.73 ± 0.25a F2526 4.86 ± 0.20e 5.86 ± 0.75c 6.80 ± 1.15e 7.53 ± 1.45bc 8.00 ± 1.00c F2548 1.87 ± 0.9c 2.27 ± 0.34a 4.82 ± 0.41bcd 6.12 ± 0.59b 6.70 ± 0.35b F2551 1.75 ± 0.50c 4.07 ± 0.43b 5.92 ± 0.89d e 7.12 ± 0.62bc 7.72 ± 0.48c F2552 0.77 ± 0.09ab 2.32 ± 0.69a 4.30 ± 0.42abc 4.85 ± 0.58a 5.65 ± 0.12a F2561 2.00 ± 0.40c 4.50 ± 0.40b 4.90 ± 0.27bcd 6.25 ± 0.28b 6.40 ± 0.45ab F2563 1.80 ± 0.08c 5.42 ± 0.61c 6.45 ± 0.52e 7.15 ± 0.12bc 7.80 ± 0.40c F2584 4.35 ± 0.50e 5.37 ± 0.42c 5.70 ± 0.40cde 6.45 ± 0.54b 6.85 ± 0.12b F2586 1.08 ± 0.02b 2.63 ± 0.45a 3.26 ± 0.81a 5.03 ± 0.15a 5.73 ± 0.87a

*Values with the same letter in the same column and in the same family are not signiﬁcantly (P < 0.05) diﬀerent. 522 Djocgoue et al.

Table 2 Scale 100 95 90 85 80 75 Total soluble phenolic content (lg/mg Fw) in healthy, wounded and Phytophthora megakarya inoculated leaves of ICS95 and ICS84 par- (a) ental clones, F30 and F25 hybrid families of Theobroma cacao F3014 F3094 Phenolic content (lg/mg Fw) F3011 F3019 Genotypes Healthy Wounded Inoculated F3052 F3045 Parents ICS84 ICS95 1.11 ± 0.07b 1.42 ± 0.04bc 1.80 ± 0.05c ICS95 ICS84 1.20 ± 0.09bc 1.27 ± 0.08b 2.17 ± 0.06def F3086 F30 F3022 F3018 1.50 ± 0.05d 1.75 ± 0.11de 2.27 ± 0.06ef F3032 F3019 1.50 ± 0.05d 1.76 ± 0.07de 1.97 ± 0.08cd F3048 F3022 2.03 ± 0.04f 2.50 ± 0.05g 2.83 ± 0.05g F3032 1.76 ± 0.03e 1.89 ± 0.06e 3.44 ± 0.19h Scale 100 95 90 85 80 75 F3045 2.05 ± 0.10e 2.20 ± 0.05f 2.33 ± 0.06f F3048 0.746 ± 0.01a 0.81 ± 0.04a 0.916 ± 0.04a (b) F3052 1.46 ± 0.06d 1.70 ± 0.05de 2.23 ± 0.05def F2561 F3086 1.08 ± 0.07b 1.27 ± 0.05b 1.40 ± 0.01b ICS84 F3094 1.54 ± 0.12d 1.91 ± 0.05e 2.34 ± 0.09 f ICS95 F3011 1.35 ± 0.02cd 1.58 ± 0.01cd 2.03 ± 0.12cde F2540 Parents F2551 ICS95 1.11 ± 0.07a 1.42 ± 0.04ab 1.80 ± 0.05ab F2500 ICS84 1.20 ± 0.09ab 1.27 ± 0.08a 2.17 ± 0.06b F25 F2563 F2508 1.76 ± 0.06d 1.68 ± 0.06bcd 2.11 ± 0.08ab F2526 F2509 1.49 ± 0.14c 2.20 ± 0.05f 3.17 ± 0.14de F2584 F2526 1.40 ± 0.05bc 1.56 ± 0.01bc 1.70 ± 0.05a F2509 F2548 1.50 ± 0.05c 1.85 ± 0.02de 2.06 ± 0.06ab F2552 F2551 1.64 ± 0.09cd 2.09 ± 0.05ef 1.93 ± 0.26ab F2586 F2552 2.10 ± 0.03e 2.50 ± 0.05g 3.01 ± 0.05d F2561 1.03 ± 0.03a 1.23 ± 0.07a 2.19 ± 0.08b Fig. 1 Direct hierarchical classification obtained with soluble total F2563 1.53 ± 0.05cd 1.80 ± 0.08cd 1.92 ± 0.14ab phenols of leaves of ICS84, ICS95 clones and hybrids from F30 (a) F2584 1.60 ± 0.05cd 1.87 ± 0.20de 2.56 ± 0.09c and F25 (b) families of Theobroma cacao structuring each of hybrid F2586 2.57 ± 0.11f 2.76 ± 0.07h 3.53 ± 0.20e family into three groups *Values with the same letter in the same column and in the same family are not significantly (P < 0.05) different. High performance liquid chromatography (HPLC) Qualitative analysis of phenolics in healthy, wounded F30 family (Fig. 1a) and two groups for the F25 and inoculated leaves from parental clones ICS84 and family (Fig. 1b). The first group of the F30 family ICS95 and from two hybrids (F3032 and F2586), consisted of none individuals with average phenolics showed the diversity of the compounds present content in inoculated leaves. The second group con- (Figs 3–5). They were largely represented by hydroxy- sisted of F3022 and F3032 characterized by high cinnamic acid derivatives and flavonoids. Apigenin phenolics content in inoculated leaves. The third was the major compound and represented approxi- group consisted of F3048, showing a very low pheno- mately 50% of the soluble phenolics in cocoa leaves lics content in inoculated leaves (Fig. 1a). In the F25 irrespective of whether the leaves were healthy, woun- family, the first group consisted of the two parental ded or inoculated (Fig. 5). Some compounds like the F ICS84 and ICS95 clones and seven hybrids. The sec- compounds (F4, F5, F6; Figs 3a,b and 4) and com- ond group consisted of the F2509, F2552 and F2586 pounds L (L2, L3) were stimulated after wounding or hybrids characterized by higher phenolic contents in inoculation. The L compounds were apparently luteo- inoculated leaves. lin derivatives and were present in the ICS84 and ICS95 clone, and in the F3032 hybrid (Figs 3–5). The Heritability and correlation apigenin derivatives content increased with stress and The values of the heritability (h2) have been deter- the increase was more pronounced in inoculated leaves mined according to the size of lesion and phenolics of ICS84 clone and F3032 hybrid (Fig. 5). content. Concerning the character size of lesion, values obtained are 0.68 and 0.69, respectively, for F30 Discussion ($ICS84 · # ICS95) and F25 ($ICS95 · # ICS84) Our experiment allows us to analyse the heritability of crosses (Fig. 2a,b). For the accumulation of pheno- resistance to P. megakarya by the evaluation of the lics, the values are 0.70 and 0.79, respectively, for necrotic area and phenolic compounds in healthy, F30 and F25 families (Fig. 2c,d). In our trials, a sig- wounded and inoculated leaves of ICS84 and ICS95 nificant negative correlation (r ¼ )0.683 for F25 fam- clones and hybrids resulting from the reciprocal cros- ily and r ¼ )0,490 for F30 family) at P < 0.01 is sing $ICS84 · # ICS95. This study shows that wound noticed between the necrosis length and the phenolics inoculation of 1 or 2-month-old leaves of cocoa- content. derived from the genotypes ICS84 and ICS95, Phenols in T. cacao/P. megakarya interaction 523

2.5 (c)

9 (a) 8 2 7 6 5 1.5 4 y = 0.6783x + 1.3884 3 2 R = 0.9662 1 2 1 y = 0.7013x + 0.7752 Means of progenies (cm) 0 R 2 = 0.9394 0246810 0.5 Means of parents (cm) Means of progenies (mg/g pf) 0 0 0.5 1 1.5 2 2.5 Means of parents (mg/g pf)

8 (b) 7 3 (d) 6 2.5

5 2 4 1.5 3 Fig. 2 Evaluation of heritabilities 1 y = 0.7867x + 0.8503 2 2 (h ) using the regression slope bet- y = 0.6889x + 0.9384 R2 = 0.9247 Means of progenies (cm) 0.5 ween parental clones and their 1 R 2 = 0.9749 progenies for lesion size (a, F30; 0 Means of progenies (mg/g pf) 0 b, F25) and phenolic content 0246810 0 0.5 1 1.5 2 2.5 (c, F30; d, F25) Means of parents (cm) Means of parents (mg/g pf)

02040Similar results were obtained by Djocgoue et al. (2006) 280 nm × 1 (a) when leaves of the same clones were inoculated with P. megakaya. Some hybrids like F3086, F2509, F2552 F6 were characterized by small lesions. The hybrids pro- 4 duced from the two clones were more tolerant to F5 F2 L2 P. megakarya than the best parent ICS84. Also, no F4 significant difference has been observed between the heritability of the lesion size for the two crosses 1 (h2 ¼ 0.68 for F30 and h2 ¼ 0.65 for F25). This should be due to the absence of maternal effect of the heritability of resistance of cocoa to P. megakarya when the 02040lesion size on the main vein was evaluated. In the healthy plants, the tolerant clone ICS84 and 280 nm × 1 (b) 90% of the hybrid’s genotypes have more phenolics F6 than the clone ICS95. Also, an increase of soluble phe- F2 nolic content in stress conditions was observed. This increase is more intense in infection condition. These 4 L2 findings are consistent with results reported by Musseti F3 3 L1 5 L3 et al. (2000) and Siranidou et al. (2002) who showed F1 an increase of phenolic content in conditions of infec- 2 tion in potato’s tissues and wheat’s tissues respectively. Some progenies (F3032, F2509, F2586, F2552) showed an important increase in phenolics with small and Fig. 3 HPLC chromatogram at 280 nm of phenolic compounds in restricted lesions in inoculated conditions. However, Theobroma cacao leaves of ICS84; healthy (a) and inoculated (b) with P. megakarya, showing: (1) the dominance of apeginin (F1, the F3086 hybrid was particular; this hybrid has small F2, F, F4, F5, F6); (2) an increase of luteolin derivatives Ôcom- lesions and low phenol content. This is in contrast to pounds L1 and L3Õ in infected leaves what is found in progenies from the $ SNK413 · # SNK10 cross where hydrids, which produce small lesion and high concentration of phenolics were indi- developed necrosis of the main vein. Generally, the viduals where the female parent was the resistant clone necrotic lesion is reduced in hybrid’s genotypes com- (Boudjeko et al., 2006). The manifestation of hybrid pared to that of their parent. 90% of the hybrid’s gen- vigour and the values of the heritability (h2 ¼ 0.70 for otypes manifested a positive heterosis effect for the F30 and h2 ¼ 0.78 for F25) obtained for phenolic con- development of lesion size. This suggests the existence tents show that this character was inherited by the two of hybrid vigour with a genetic additive effect. The size progenies and indicate by this fact a good combination of necrotic lesions was larger in ICS95 than in ICS84. aptitude of parental clones belonging to the group of 524 Djocgoue et al.

0 20 40 the Trinitario resulting from Trinidad (ICS; Djocgoue 280 nm × 1 (a) et al., 2006). Generally, the accumulation of phenolics following infection by a pathogen involves the neosynthesis of F5 specific phenol compounds (Conceiçao et al., 2006). F2 Qualitative analysis of phenolics in leaves by HPLC showed a higher accumulation of some luteolin deriva- 1 3 5 F1 F3 tives and apigenin derivatives (flavonones) and some hydrocycinnamic acid derivatives. Recent research has L1 2 shown that treatment of Hypericum perforatum cells with jasmonic acid before inoculation with Colletotri- chum gloeosporioides led to selective accumulation of 02040 flavonones (Conceiçao et al., 2006). 280 nm × 1 (b) In conclusion, this study shows that certain individuals of the two progenies (F3032, F2509, F2552 and F2586) derived from the cross ICS84 · ICS95 (two F6 4 Trinitario of Trinidad) showed an important increase F5 F2 in phenolics with small and restricted lesions when F4 inoculated with P. megakarya. Furthermore, the qual- L1 itative analyses of phenolics in ICS84, ICS95 clones and their hybrids showed a modification of the phenolics profile, notably concerning apigenin and luteolin derivatives due to the inoculation. In a similar experi- Fig. 4 HPLC chromatogram at 280 nm of phenolic compounds in Theobroma cacao leaves of ICS95; healthy (a) and inoculated ment on SNK10 and SNK413 (two local Trinitario) (b) with P. megakarya, showing: (1) the dominance of apeginin (F1, and their offspring, we have shown the accumulation F2, F, F4, F5, F6); (2) an increase of compound 4, an hydroxycin- of luteolin only in the less susceptible clone SNK413 namic acid derivatives and the progeny whose mother was the clone SNK413

Healthy Wounded Inoculated Healthy Wounded Inoculated 2 2 (a) (c) b

1.6 b 1.6

1.2 1.2 a 0.8 a 0.8 a a b b a a a a a a a 0.4 a a 0.4 a Hydroxycinnamic acids derivatives Hydroxycinnamic acids derivatives g/mg fresh weight) of luteolin. apigenin and g/mg fresh weight) of luteolin. apigenin and m m ( ( 0 0 Luteolin Apigenin HC Luteolin Apigenin HC Phenols Phenols

Healthy Wounded Inoculated Healthy Wounded Inoculated 2 2 (b) (d)

1.6 1.6

c b 1.2 1.2 b Fig. 5 Quantitative (lg/mg fresh a a weight) of luteolin, apigenin and 0.8 a 0.8 Hydroxycinnamic acids derivatives in leaves of ICS84 (a), ICS95 (b) b a a a clones and F3032 (c) and F2586 0.4 0.4 a a (d) hybrids inoculated with a a a

Hydroxycinnamic acids derivatives Phytophthora megakarya. Values and Hydroxycinnamic acids derivatives g/mg fresh weight) of luteoline. apigenine m

g/mg fresh weight) of luteolin. apigenin and with the same letter in the same (

m 0 0 ( clone or the same hybrid and for Luteolin Apigenin HC Luteolin Apigenin HC the same compound are not signi- Phenols Phenols ﬁcantly (P < 0.05) diﬀerent Phenols in T. cacao/P. megakarya interaction 525

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