Abstract: Morpho-physiological characteristics of 12 genetically diverse cocoa genotypes with various levels of resistance were examined to determine which features were associated with resistance. The 12 cocoa genotypes were evaluated by leaf discs and detached pod inoculations for resistance to P. palmivora and P. megakarya. Pod husk thickness and epicuticular waxes on abaxial and adaxial surfaces of leaf and on cocoa pod surfaces were significantly and negatively correlated with resistance to black pod disease. Percentage moisture content was positively associated with resistance to black pod disease. Epicuticular wax on pod husk was significantly correlated with penetration resistance but however was not significant with post-penetration resistance suggesting its role in restricting penetration of the fungus. Principal Component Analysis (PCA) shows that the first two components contribute to explain 97.02% (PC1 = 87.87%, PC2 = 9.29%) of the total variation of morpho-physiological traits in cocoa. Amount of cuticular wax on pod surface, percentage moisture content of pod husk and leaf were the major contributors of total variation of morpho-physiological factors evaluated. Multiple linear regression analysis indicated that morpho-physiological traits explained 99.2, 91.2 and 81.3% of the variation in pod lesion number, pod lesion size and leaf disc score, respectively. This suggests that amount of wax on pod and abaxial surface of leaf, thickness of exocarp at the ridge and percentage moisture content could be used as marker traits to select for resistance to black pod disease.
INTRODUCTION
Black pod is an important fungal disease that reduces yield and quality of cocoa beans (Nyasse et al., 2007). In Ghana, during the rainy season, black pod is the most limiting disease for cocoa production (Akrofi et al., 2003) and frequent fungicide sprays are commonly used to prevent crop losses. Planting resistant varieties is considered to be the most effective and cost efficient management option for the disease, particularly for small scale farmers who make up the greatest proportion of cocoa growers in developing countries (Adomako, 2006; Nyasse et al., 2002; Iwaro et al., 2005).
Progress in breeding for cocoa varieties which are better adapted to less susceptibility to black pod disease is still restricted for several reasons. Cocoa breeders are always skeptical not only because of the morpho-physiological complexity of the matter but mainly due to limited data on both the variation among available cocoa collections and the genetics of key characters involved (Van der Vossen, 1997). Despite resistance of the cocoa pod being known to constitute one of the major defenses of fighting against the black pod disease, little is known regarding the morpho-physiological mechanisms upon which this resistance is based (Van der Vossen, 1997).
Disease resistance is governed by a number of morphological and physiological features of host plant. These traits confer disease resistance by checking the entry of pathogen into the host tissues (Singh, 2005). In a study of the modes of resistance of cocoa to Phytophthora species infection, Iwaro et al. (1997) concluded that resistance operates at two distinct stages of infection. They are the penetration stage, which restricts the entry and establishment of the pathogen, thus reducing the frequency of lesions and the post-penetration stage, which reduces the rate of spread of the pathogen and hence the rate of lesion expansion. He obtained a strong correlation between stomatal frequency and lesion frequency and size on pods. He indicated that the mechanism of resistance at the penetration stage of infection could be attributed in part to morphological characteristics. Cuticle thickness was identified as important factors related to resistance to scab infection caused by Sphaceloma batatas in Sweet potato (Bajit and Gapasin, 1987). Similarly, resistance of winged bean to Phoma sorghina was found to be related to surface wax, cuticle thickness and stomatal frequency (Kumar et al., 1985). Besides the role of epicuticular waxes in preventing plant desiccation in water-limiting environments (Riederer and Schreiber, 2001), surface waxes may protect plants from pathogens (Jenks et al., 1994; Carver et al., 1996). Patton et al. (1980) looked at stomatal influences on infection and suggested that wax occlusion of stomata may be a reason for failed infections after artificial inoculations of leaves. Okey et al. (1996) obtained a strong positive correlation between bark moisture content and development of canker in cocoa. Nyadanu et al. (2011) reported a strong relationship between pod husk thickness, moisture content and resistance to Phytophthora palmivora.
Some researchers have suggested that morphological characteristics of plants related to disease resistance could be employed in screening for resistance (Wink, 1988). This approach, apart from providing an understanding of the mechanism of resistance, may lead to the development of easier and more cost-effective methods of assessment of resistance, which could make selection and breeding programmes more effective.
The objective of this study therefore was to evaluate the relationship between resistance to Phytophthora species (measured as lesion number and size on pods, leaf disc scores and natural field infection) and the morphological characteristics (pod husk thickness, % moisture content, surface wax load) in cocoa pod and leaf.
MATERIALS AND METHODS
The experimental material consisted of a diverse array of 12 cocoa genotypes (Pa7/808, Na33, T60/887, T63/971, Imc76, Pa 150, Sca9, Imc67, Imc53, Sca6, T85/799 and T79/501) maintained at the germplasm plot of Cocoa Research Institute of Ghana (CRIG). The experiments were conducted at CRIG during 2009/2010 and 2010/2011 seasons.
Inoculum preparation: The isolates of P. palmivora and P. megakarya were grown on carrot agar medium and from a ten-day-old culture, a zoospore suspension was obtained by inundating each culture plate (9 cm diameter) with 10 mL sterile distilled water (chilled to 10°C), refrigerated for 25 min (5°C) and incubated in the dark at 25°C for 30 min. The zoospore concentration of the suspension was determined using a haemocytometer and adjusted to 200,000 mL-1.
Leaf disc test: Leaf disc preparation and inoculation as described by Nyasse et al. (1995) and Tahi et al. (2006) was carried out. The flushes of leaves were tagged for the cocoa genotypes. The ages of the leaves for each treatment were established by following the growth of young flushes from bud break in the field. Leaves of good physiological condition (young lignified leaves) without insect attacks and of similar age and exposure to sunlight were collected. Sixteen leaf discs of 1.5 cm diameter from each genotype were made with a cork borer and replicated four times. Leaf discs were placed with their abaxial surface upwards on wetted plastic foam in five trays of 70 cm long, 60 cm wide and 15 cm high. Discs of the cocoa genotypes were randomly arranged in groups of 12 within each tray, giving 16x12 = 192 discs per tray. The discs were inoculated and incubated at room temperature (25°C) in plastic trays and covered with another plastic tray in the laboratory to prevent direct sunlight until observations were made. Symptoms were scored 6 days after inoculation using a 0 to 5 point scale depending on the size of necrosis (0 = absence of symptoms, 1 = very small necrotic spots, 2 = larger number and size of necrotic spots, 3 = coalescence of brown spots into medium-sized, 4 = large uniform brown lesions and 5 = very large brown lesions, often expanding outside the area covered by the inoculum droplet) as described by Nyasse et al. (1995). The experiment was carried out twice.
Detached pod test: Detached pod test at penetration and post-penetration stages of infection as described by Iwaro et al. (1997) was carried out. The flowers of the cocoa genotypes were selfed (using hand pollination) so that their exact ages could be determined at the time of harvest.
Inoculated pods were, arranged in a randomized complete block design and incubated at room temperature in moist plastic chamber boxes. Three replicates consisting of five pods each were assayed. After incubation for four days, the number of infection sites per inoculum site was counted. On the 7th day of incubation, sizes of the established lesions were traced on a transparent paper. The lesion sizes were determined from brown paper cutouts trimmed to the size of each lesion and were measured with a leaf area meter.
Field observations: Natural pod infections in the field were evaluated in 2009 and 2010 on individual crosses of the 6x4 factorial and 6x6 diallel mating designs. Pods infected by Phytophthora (Bp), Rodent damaged pods (R) and Healthy ripe pods (H) and Other Damages (OD) were counted each month after each harvesting round. The percentage (%) of rotten pods (Bp) was estimated in relation to the total number of pods produced by the cross:
Morphological and physiological characteristics: Pod husk thickness (exocarp/endocarp), surface wax load and moisture content of both pod and leaf were assessed for the cocoa genotypes.
Thickness of pod husk: Pods were cut transversely at the equator to expose the thickness of the husk using a very sharp knife. The thickness of the husk was measured using a transparent ruler. Each pod was measured at seven different points on the ridge and furrow. Measurements were taken for the thickness of exocarp and endocarp on the ridge and furrow of pods. Five pods were assessed per each genotype to obtain a mean measurement. The experiment was conducted two times.
Moisture content of pod husk and leaf: The percentage moisture content of the pod husk was determined following the method of Susheelamma et al. (1990). Five pods for each genotype per replication were collected from the field and tissues were obtained from the pod husk at the equator of the pod using a cork borer (18 mm diameter). For the leaf samples, five leaves for each genotype per replication were collected from the field and discs were obtained from the leaf using a cork borer (18 mm diameter). The fresh weight (M1) of the tissue was determined using an electronic balance (Mettler Toledo, Model PB3001-L, made in Switzerland). The tissue was then dried in an oven at 80°C for 72 h and the dry weight (Md) recorded. The percentage Moisture content (Mc) was calculated using the formula:
Pod and leaf surface wax load: Surface wax was extracted by dipping the distal end of pods up to the equator into chloroform for 30S. The extract was transferred into a weighed flask (W1). The chloroform was evaporated using Rotary evaporator (Ratavapor BÜCHI, EL 131, made in Switzerland). The flask with the sample was placed in desiccators in a cool dry place for 24 h. The flask with the sample was weighed (W2). The extracted part of the pod (which turns brown) was cut into four pieces and after removing the inner tissue, their outlines were traced on brown paper. This was used to determine the pod area (A) cm-2 using a leaf area meter. Three pods were assessed per progeny per replication. Wax was extracted from both abaxial and adaxial surfaces of leaf by washing each surface at a time with chloroform. The area of the leaf was also determined using leaf area meter. Five leaves were assessed per progeny per replication.
The weight of the wax load on pod/leaf surface was calculated using the formula:
where, W2 is weight of sample+ flask, W1 is weight of empty flask, A is Surface area of pod or leaf.
Statistical analysis: Data were subjected to Analysis of Variance (ANOVA) using GenStat® 11th version (GenStat, 2008). Significance of differences among the genotypes for each trait was tested by F-test. When the ANOVA showed significant genotypic differences, the significance of differences between the genotypic means was judged by Least Significant Difference (LSD) at p≤0.05. Simple correlation and multiple linear regression analysis was performed to understand the relationship between the morpho-physiological traits and resistance to black pod disease in leaf and pod caused by P. palmivora and P. megakarya. Principal components and cluster analysis was also carried out to identify the most important morpho-physiological factors and diversity of cocoa genotypes in morpho-physiological factors.
RESULTS
Relative susceptibility of cocoa genotypes to Phytophthora species: Reaction of the 12 cocoa genotypes used in this study to Phytophthora palmivora and P. megakarya is presented in Table 1. Significant differences (p<0.001) in mean severity values were detected among the genotypes in the inoculation tests (Pod Lesion Number (PLN), Pod Lesion Size (PLS), Leaf Disc Score (LDS)). The PLN on pods among the genotypes varied from 2.93 for Sca6 to 15.23 for Imc67. Significantly smaller PLS were recorded on Sca6, T60/887 and Pa 150 than for the other genotypes. PLS on Imc76 and Sca9 were moderate; but significantly larger sizes were produced on the rest of the genotypes. Disease severity scores of LDS varied from 1.57 for Sca6 to 4.24 for T63/971. The lesion number, lesion sizes and leaf disc scores increased when P. megakarya was used as the inoculum.
Variation of quantity of cocoa epicuticular wax on leaf and pod: Total leaf and pod cuticular waxes were extracted from the cocoa genotypes. There were significant variations in the amount of wax on the surfaces of leaf and pods of cocoa. Table 2 presents the amount of cuticular wax on abaxial and adaxial surfaces of leaf and on the pod of cocoa. Amount of wax on the abaxial surface of leaf of the cocoa genotypes varied largely between 7.97±0.39 and 38.25±0.23 with a Cefficient of Vriation (CV) of 6.70. On the adaxial surface, the amount of wax varied between 10.02±0.54 and 33.59±0.91 with a Cefficient of Vriation (CV) of 11.9. The amount of cuticular wax on the surface of pod of the cocoa genotypes varied largely between 12.54±1.39 and 59.84±2.15 with a coefficient of variation of 8.50. Significantly higher cuticular waxes were produced on the abaxial and adaxial surfaces of leaf and on pod of T60/887, Pa150, Sca6 and Pa7/808 than on the leaf and pod of the other genotypes. Epicuticular waxes were significantly fewer on the leaf and pod of Sca9, Na33, T63/971 and Imc 67. Comparatively, there were higher amount of cuticular wax on the surface of pod than on the surface of leaf. Also, the amount of epicuticular wax on the abaxial surface of leaf was relatively higher than on the adaxial surface.
| Table 1: | The parental mean values for pod lesion number, pod lesion size and leaf disc scores of the 12 cocoa varieties used as parents in the 6x4 factorial and 6x6 diallel mating designs |
| PLN: Pod lesion number, PLS: Pod lesion size, LDS: Leaf disc score, Pp: Phytophthora palmivora, Pm: Phytophthora megakarya, S.e.: Standard error. For each column, figures followed by the same letter(s) are not significantly different at 95% | |
| Table 2: | Amount of epicuticular wax load on leaf and pod surfaces of cocoa genotypes |
| For each column, figures followed by the same letter(s) are not significantly different at 95% | |
| Table 3: | Pod husk thickness of the cocoa genotypes at the ridge |
| Genotypes with the same letters in columns are not significantly different | |
Thickness of the cocoa pod husk and the moisture content of leaf and pod: There were significant differences (p<0.001) among the cocoa genotypes in both exocarp and endocarp and total thickness of pod husk along the ridge and furrow, respectively (Table 3 and 4). There were very little variation among the cocoa genotypes in thickness of exocarp and endocarp along the ridges and furrows.
| Table 4: | Pod husk thickness of the cocoa genotypes at the furrow |
| Genotypes with the same letters in columns are not significantly different | |
The cocoa genotypes Sca6, Pa150, Imc76, T60/887 and Pa7/808 had a significantly thicker exocarp along the furrow and were not significantly different as compared with the other genotypes, while Na33 and Sca9 had the thinnest. The cocoa genotypes Sca6, Pa150, Imc76, T60/887 and Pa7/808 possessed relatively thicker endocarp along the furrow than the other genotypes. The total thickness (exocarp+endocarp) of the pod husk along the furrow was significantly thicker for Pa150 and Sca6 than the other genotypes.
The cocoa genotypes Sca6, Pa150 and T60/887 had thicker exocarp along the ridge. Na33 and Imc67 had the thinnest. The genotypes Sca6, Pa150, Imc76, T60/887 and Pa7/808 possessed thicker endocarp along the ridge than the other genotypes. The total thickness (exocarp+endocarp) of the pod husk along the ridge was significantly thicker in Pa150 and T79/501 than the other genotypes. In general, the thickness of the pod husk diminished slightly along the furrows as compared with the ridge but followed almost the same pattern as in the ridges.
The thickness of the exocarp was not greater than the thickness of the endocarp in all the genotypes. Some cocoa genotypes had thicker endocarp than exocarp. For example, Pa150, T60/887, Imc76 and Sca6 had thicker endocarp than exocarp along the furrow.
The moisture content of the cocoa pod husk varied significantly (p<0.001) among the cocoa genotypes in both leaf and pod. Sca6, Pa150, T60/887 and T85/799 had the lowest moisture content while high values were recorded for Na33, T63/971, Sca9 and Imc53 in both leaf and pod under similar environmental conditions. The amount of moisture reduced in leaf and pod infected with P. megakarya suggesting that the fungus utilizes moisture in its development (Table 5).
Association between morpho-physiological factors and black pod disease resistance: The correlation coefficients of wax load on the abaxial surface of leaf, wax load on the adaxial surface of leaf, wax load on the surface of cocoa pod, thickness of exocarp at ridge, thickness of endocarp at ridge, total thickness of pod husk at ridge, thickness of endocarp at furrow, total thickness of pod husk at furrow and percentage moisture in leaf and pod were significant and negative (p<0.05) with pod lesion number (r = -0.75 to -0.98) (Table 6).
| Table 5: | Percentage moisture content in leaf and pod husk of cocoa genotypes |
| Genotypes with the same letters in columns are not significantly different | |
| Table 6: | Correlations of morpho-physiological factors with components of resistance to P. palmivora and P. megakarya |
| X1: Leaf abaxial wax, X2: Leaf adaxial wax, X3: Pod wax, X4: Exocarp thickness at ridge, X5: Endocarp thickness at ridge, X6: Total thickness of pod husk at ridge, X7: Exocarp thickness at furrow, X8: Endocarp thickness at furrow, X9: Total thickness of pod husk at furrow, X10: Leaf %moisture, X11: Pod husk % moisture, MFI+: Mean field infection of factorial crosses, MFI*: Mean field infection of diallel crosses. Multiple linear regression; lesion numbers on pod caused by P. palmivora (PLNPp) = -315**+1.46X1**-4.09X2**+1.36X3-2258X4-2139X5**+2225X6*-1199X7+1125X8+1147X9+4.82X10*-2.68X11** (R2 = 96.1%, R2 (adj.) = 78.5%). Multiple linear regression; lesion numbers on pod caused by P. megakarya (PLNPm)= -0.21**+0.59X1**-2.29X2**+0.86X3**-1509X4-1434X5*+1490X6**-717X7-670X8+683X9+3.21X10**-1.69X11* (R2 = 99.2%, R2 (adj.)= 97.9%). Multiple linear regression; lesion sizes on pod caused by P. palmivora (PLSPp) = -2918*+19.8X1**-45.9X2*+13.1X3*-202X4** +191X5*+199X6-117X7-110X8*+112X9+43.8X10**-24.8X11** (R2 = 94.5%, R2 (adj.) = 70.0%). Multiple linear regression; lesion sizes on pod caused by P. megakarya (PLSPm)= -168*+12.4X1**-25.9X2*+5.86X3**-935X4-872X5*+916.7X6-6652X7-6306X8+6416X9+27.6X10**-15.7X11** (R2= 91.2%, R2 (adj.) = 75.7%). Multiple linear regression; leaf disc scores caused by P. palmivora (LDSPp) = -17.4**-0.05X1*-0.05X2*+0.07X3**-96.9X4*-91.4X5**+95.6X6-27.7X7-26.1X8+26.1X9+0.24X10**-0.09X11* (R2 =92.2%, R2 (adj.) = 78.7%). Multiple linear regression; leaf disc scores caused by P. megakarya (LDSPm) = 41.5*-0.45X1*+0.93X2**+0.28X3***+401X4*+383X5*-395X6+199X7+184X8-189X9-0.55X10**+0.34X11** (R2= 81.3%, R2 (adj.) = 48.5%). *,**,***= Correlation and regression coefficients significant at p = 0.05, p<0.05 and p<0.001, respectively | |
Correlation coefficients of wax load on the abaxial surface of leaf, thickness of exocarp at ridge, thickness of endocarp at ridge, thickness of endocarp at furrow and percentage moisture in pod husk were significant and negative (p<0.05) with pod lesion size (r = -0.75 to -0.87).
| Table 7: | Correlation among the morpho-physiological characters |
| *= significant at p = 0.05, ** = significant at p<0.05, ***= significant at p<0.001. Lab: Leaf abaxial wax, Lad: Leaf adaxial wax, Pwax: Pod surface wax, Lmoist: % moisture content in leaf, Pmoist: % moisture content in pod, Rendo: Pod husk endocarp thickness at the ridge, Rexo: Pod husk exocarp thickness at the ridge, Rtotal: Pod husk total thickness at the ridge, Fendo: Pod husk endocarp thickness at the furrow, Fexo: Pod husk exocarp thickness at the furrow, Ttotal: Pod husk total thickness at the furrow. X: Morpho-physiological character | |
Correlation coefficients of wax load on the abaxial surface of leaf, thickness of exocarp at ridge, thickness of endocarp at ridge, total thickness of pod husk at ridge and percentage moisture content in leaf were significant and negative (p<0.05) with leaf disc score (r = -0.77 to -0.93) (Table 6).
Correlation coefficients of amount of wax on abaxial and adaxial surfaces of leaf, amount of wax on surface of pod, total thickness of pod husk at the ridge, total thickness of pod husk at the furrow, percentage moisture content in leaf and pod were significant and negative (p<0.05) with mean field infection of crosses in 6x4 factorial and 6x6 diallel designs (data not shown) (Table 6). The association was however positive for percentage moisture content in pod husk and leaf with resistance to Phytophthora species.
Multiple linear regression analysis indicated that morpho-physiological traits explained 99.2, 91.2 and 81.3% of the variation in PLN, PLS and LDS caused by P. megakarya, respectively (Table 6).
Character association of morpho-physiological factors: Morpho-physiological characters were significantly correlated among themselves. However, correlation coefficients of wax load on abaxial and adaxial surface of leaf, wax load on pod surface, exocarp thickness at ridge, endocarp thickness at ridge and total thickness at ridge with thickness of exocarp at furrow were positive but not significant (p>0.05) (Table 7).
Principal components analysis of morpho-physiological characters: Principal Component Analysis (PCA) shows that the first two components contribute to explain 97.02% (PC1 = 87.87%, PC2 = 9.29%) of the total variation of morpho-physiological traits in cocoa. Amount of cuticular wax on pod surface and percentage moisture content of pod husk were the major contributors of total variation of morpho-physiological factors in PC1. In PC2, amount of cuticular wax on pod surface and percentage moisture in leaf and pod husk were the major contributors to total variation (Table 8). Graphic representation of the principal components analysis (Fig. 1) shows the 12 cocoa genotypes separated according to their morpho-physiological characteristics.
| Table 8: | Principal component analysis of morpho-physiological traits of 12 cocoa genotypes |
| Fig. 1: | A two-dimensional graph of the principal component analysis on 12 cocoa genotypes and 11 morpho-physiological factors of leaf and pod of cocoa as variables |
Genotypes Imc53, Imc76 and T63/971 are clearly classified as different from the rest. The PC1 which discriminates genotypes according to amount of wax on pod surface and percentage moisture content in pod, classify T60/887, Sca6 and Pa150 as different because of higher amount of wax on the surface of pod of these genotypes. Likewise, PC2 allows separation of genotypes T63/971 and Na33 from the others because these genotypes have higher percentage moisture in their leaf and pod husk. The acute angles among amount of wax on leaf abaxial surface, leaf adaxial surface and pod surface suggest a close relationship among them. Also, the graph shows that percentage leaf moisture content and percentage pod moisture content are closely related (Fig. 1).
Cluster analysis (Fig. 2 and 3) based on PC1 and PC2 and on the original data, respectively, shows the variability among the 12 cocoa genotypes based on the morpho-physiological traits. Two major clusters were evident with their subgroups distinctively separating the black pod resistant and black pod susceptible genotypes.
| Fig. 2: | Dendrogram of 12 cocoa genotypes based on principal components 1 and 2 of the morpho-physiological traits |
| Fig. 3: | Dendrogram of the 12 cocoa genotypes based on the original data of the morpho-physiological factors in cocoa |
Pa 150/887 and T60/887 were sub-clustered and Na33 and T63/971 were also sub-clustered.
DISCUSSION
The study shows considerable genetic variability among cocoa genotypes for PLN, PLS and LDS caused by P. palmivora and P. megakarya. The differential response of cocoa genotypes further suggested that resistance to black pod disease was under genetic control and should therefore be liable to genetic improvement. Ample genetic variability for black pod resistance has also been reported in cocoa by Iwaro et al. (1997), Nyadanu et al. (2009), Nyasse et al. (2002) and Tahi et al. (2006).
There are many obstacles that fungal pathogen must overcome before they are able to initiate disease in plants. First, the pathogen must locate and adhere to susceptible tissue and then initiate infection. The first barriers encountered are generally cuticle and cell wall.
In this study, significant variations were observed among cocoa genotypes for the amount of cuticular wax on both abaxial and adaxial surfaces of leaf and on the surface of cocoa pods. Resistance to P. palmivora and P. megakarya measured as lesion numbers on pods, lesion sizes on pods and leaf disc scores increased with an increase in the amount of wax on the surface of the cocoa genotypes. The results suggest that fungal penetration into intercellular spaces of pod and leaf could be favoured in genotypes or pod or leaf sides with lower quantities of surface waxes, while higher wax quantities observed in other genotypes could reduce rates and number of fungi invading the mesophyll or pod husk. This agrees with the findings of Zinsou et al. (2006) who reported surface waxes as a mechanism of resistance against Xanthomonas blight in cassava.
The significant variation among cocoa genotypes for thickness of pod husk and percentage moisture content provides opportunity to select desirable cocoa genotypes for these traits. This agrees with the results of Iwaro et al. (1997) and Nyadanu et al. (2011) who reported significant differences in cocoa genotypes for the thickness of pod husk. Cocoa genotypes observed to be resistant to black pod disease, tended to have thicker pod husk than susceptible genotypes. Therefore, pod husk thickness is probably an important resistant factor that needs to be incorporated into breeding lines for resistance to P. palmivora and P. megakarya.
The reduction of moisture content in leaf and pod after infection with P. megakarya suggests that the pathogen utilizes moisture for its growth. Higher moisture content of pod husk therefore favours the growth of the fungus. Cocoa genotypes with lower percentage moisture content tended to be more resistant than genotypes with higher percentage moisture content of pod husk and leaf. The cocoa genotypes with thicker pod husks were observed to have thicker endocarp than genotypes with thinner pod husks, which may explain why cocoa genotypes with thicker pod husk had lower moisture content. These results support those of Okey et al. (1996), Bertrand et al. (1976) and Tippet and Hill (1983) who showed a positive correlation between bark moisture content and level of canker susceptibility in cocoa. Clones which had high moisture content were found to be more susceptible while those with low moisture content were less susceptible to P. palmivora stem infection.
Significantly negative and strong correlation was observed between most of the morphological characters and resistance to black pod disease except thickness of exocarp at the furrow. The results suggest that, as morphological factors increase, infection caused by Phytophthora species in cocoa leaf and pod decreases. However, as percentage moisture content in leaf and pod increases, infection caused by Phytophthora species increases. The findings from this study suggests that cuticular wax on leaf and pod, thickness of pod husk endocarp, exocarp and total thickness and percentage moisture content of leaf and pod are contributing factors to resistance of black pod disease in cocoa.
The significant and strong relationship between cuticular wax on leaf and pod with resistance to Phytopthora species agrees with the results of Sena Gomes et al. (1995) who observed higher concentration of epicuticular wax on cocoa pods of the Phytophthora-tolerant clones PA 150 and SCA 6, in relation to the more susceptible clone SIC 2. Resistance to Botrytis cinerea increased with an increase in the amount of wax on the surface of berries (Gabler et al., 2003). The results however, disagrees with those of Iwaro et al. (1997) who observed insignificant and poor negative correlation between cuticular wax on cocoa pod surface and lesion frequencies caused by Phytophthora palmivora. More studies are needed to understand the role of epicuticular wax in resistance of cocoa against Phytophthora species. Analysis of chemical contents of waxes from cocoa leaf and pod surfaces and effects of waxes on environmental factors are being carried out.
The relationship between thickness of pod husk and resistance to black pod disease reported in this study agrees with earlier work on relationship between pod husk thickness and resistance to Phytophthora palmivora in cocoa (Nyadanu et al., 2011). Cuticle thickness was identified as important factors related to resistance to scab infection caused by Sphaceloma batatas in Sweet potato (Bajit and Gapasin, 1987). Similarly, resistance of winged bean to Phoma sorghina was found to be related to cuticle thickness (Kumar et al., 1985). Sarig et al. (1998) found that skin thickness was correlated with resistance in berries but suggested that fungal invasion was related more to cell density in the skin than to its absolute thickness. The positive relationship observed between percentage moisture content of leaf and pod and resistance to black pod disease agrees with earlier work reported by Nyadanu et al. (2011) who reported positive relationship between percentage moisture content of cocoa pod and pod lesion number and size caused by P. palmivora in some cocoa clones. Okey et al. (1996) also reported positive relationship between stem bark moisture of cocoa and susceptibility to canker.
The morphological traits were significantly correlated among themselves. The positive relationship between cuticular wax and pod husk thickness suggests that the two traits can be simultaneously improved without any compensatory negative effects. However, the negative relationship between these two traits and percentage moisture content in leaf and pod suggest that they should be improved independently.
The PCA showed that cuticular wax load on pod and percentage moisture content in pod and leaf were the major contributors of variation in the morphological traits. The cocoa genotypes T60/887, Sca6, Pa150, Pa7/808 and T85/799 observed to have high amount of epicuticular wax in leaf and pod and less moisture contents could be good sources of materials to improve morpho-physiological traits in cocoa. The acute angle among amount of wax on the abaxial and adaxial surfaces of leaf and pod surface suggest that the surfaces of leaf could be used to predict surfaces of pod. This finding could offer an explanation to why lesion numbers and sizes on leaf surface correlated with lesion numbers and sizes on pod surface as reported by Nyasse et al. (1995, 2002), Iwaro et al. (1997) and Nyadanu et al. (2009).
The clustering of resistant genotypes differently from the susceptible genotypes indicates a genetic relationship among resistant genotypes in relation to the morpho-physiological traits of cocoa leaf and pod.
CONCLUSIONS
The findings indicate that epicuticular wax, pod husk thickness and percentage moisture content were associated with black pod disease resistance. Multiple linear regression analysis indicated that morpho-physiological traits explained 99.2, 91.2 and 81.3% of the variation in pod lesion number, pod lesion size and leaf disc score, respectively and suggest that amount of wax on pod and abaxial surface of leaf, thickness of exocarp at the ridge and percentage moisture content could be used as marker traits to select for resistance to black pod disease. The role of chemical content of wax in resistance to black pod disease needs to be studied.
ACKNOWLEDGMENTS
Financial support provided by Ghana Cocoa Growing Research Association (GCGRA), UK, is highly appreciated. Technical support provided by Messrs Emmanuel Ewe and Ernest Akortia, Madam Mercy Ofori and Rafiatu Kotei, all of CRIG is highly acknowledged.