MALAYSIAN
COCOA JOURNAL ------
Editor Dr. Rosmin Kasran
Vice Editor Dr. Lee Choon Hui
Secretary Dr. Tan Chia Lock
Editorial Committee Dr. Alias Awang Dr. Douglas Furtek Dr. Ramle Kasin Harnie Harun Hii Ching Lik Suzannah Sharif
Published by
MALAYSIAN COCOA BOARD 5-7th Floor, Wisma SEDCO Lorong Plaza Wawasan, Off Coastal Highway 88999 Kota Kinabalu, Sabah, Malaysia
©All rights reserved. No part of this publication may be reproduced in any form or by any means without permission in writing from Malaysian Cocoa Board
CONTENTS
ENTOMOLOGY
ANATOMICAL STUDIES OF THE SCLEROTIC LAYER OF COCOA POD DEVELOPMENT IN COCOA 1 Alias, A., Azhar, I., Hadley, P. and Hatcher, P.E.
DISTRIBUTION OF COCOA POD BORER EGGS AT DIFFERENT LEVELS OF SHADE IN SMALL-SIZE PLANTATION 9 Saripah, B., Hassan, S. T. S. and Sajap, A. S.
CONTROL OF COCOA POD BORER USING INSECTICIDES AND COCOA BLACK ANTS 14 Saripah, B.
SUSTAINING COCOA BLACK ANTS, Dolichoderus thoracicus (Smith) USING ARTIFICIAL NEST IN THE COCOA ECOSYSTEM 23 Saripah, B.
BIOTECHNOLOGY
ISOLATION OF DNA AND RNA FROM COCOA POD BORER, CONOPOMORPHA CRAMERELLA (SNELLEN) AND CLONING OF ITS GENES 31 Goh, L.P.W., Chia, Y.C., Tan, C.L.
IDENTIFICATION OF GENETIC MARKERS FOR POD BORER RESISTANCE BY SEQUENCING 14 COCOA GENOMES 40 Roslina, M.S., Rosmin, K., Lea, J., Navies, M., Nor Aisyah, Sumayyah, A.K., Zafirah, Z., Fahmie, W. and Larry, C.
PRODUCT DEVELOPMENT
COMPARISON OF ANTIOXIDANT PROPERTIES OF COCOA PODS AND SHELLS 49 Azila Abdul Karim, Azrina Azlan, Amin Ismail, Puziah Hashim, Nur Azilah Abdullah
SHORT COMMUNICATIONS
PRO AND CONS IN APPLYING CANOPY REPLACEMENT METHOD FOR VASCULAR STREAK DIEBACK INFECTED MATURE COCOA 57 Rozita O., Nik Aziz, N.M. and Azhar, I.
REPLACE YOUR TOPS WHEN YOU’RE INFECTED! 60 Rozita O., Nik Aziz, N.M. and Azhar, I.
Malaysian Cocoa Journal, 8/2014
ANATOMICAL STUDIES OF THE SCLEROTIC LAYER OF COCOA POD DEVELOPMENT IN COCOA
Alias, A1., Azhar, I1., Hadley, P2. and Hatcher, P.E2. 1Malaysian Cocoa Board, Lock Beg 211, 88999 Kota Kinabalu, Sabah, Malaysia 2School of Biological Sciences, University of Reading, RG6 6AS, UK
Malaysian Cocoa J. 8: 1 – 8 (2014) ABSTRACTS - Plants use several types of external and internal barriers as their first defence mechanisms against herbivorous insects by interfering with their feeding, oviposition or even their use of plants as shelter, which includes hardness of the sclerotic layer. Therefore, knowledge of the sclerotic layer hardness and the timing of development can provide useful information for the development of an appropriate methodology for screening clones resistant to cocoa pod borer. Results showed that the hardness of the pod differed between clones. The sclerotic layer was apparent as early as two months after pollination, but was soft. No lignified cells were observed and cells only had a primary cell wall in the young pods. The pods reached the maximum hardness between four and five months, depending on clone. In some clones, the hardness of sclerotic layer gradually declines as pods ripen. A possible explanation of the decreasing hardness is that mature pods lose their firmness with the beginning of the degradation process of cell walls, which is a common phenomenon in other drupe fruits during the ripening process.
Keywords : Sclerotic layer, cocoa, Pod development, Cocoa pod borer resistance, Anatomy
INTRODUCTION sclerotic layer thickness between genotypes, but no subsequent study has been conducted. Whether there Cocoa pod borer (CPB), Conopomorpha cramarella is genotypic variation in the timing of sclerotic layer (Snellen) [Lepidoptera: Gracillariidae] is a pest of development is yet to be elucidated. The objective of cocoa in Malaysia for more than three decades since the study was to investigate the anatomical changes it was discovered in 1980. This pest is not only a of the sclerotic layer at various developmental stages threat in Malaysia, but also in other South East Asia in clones. A histological study of the pod walls was countries such as Indonesia, Papua New Guinea, conducted to ascertain the internal characteristics of India and Philippines. Of all possible control the pod. The information obtained should provide a measures, chemical control is considered to be the better understanding of pod response to CPB attack most reliable method for controlling CPB. The search and why certain clones are more tolerant than the for resistant clones to CPB in Malaysia has been others. undertaken for more than 15 years with limited success, partly because of the lack of understanding on its resistance mechanism. MATERIALS AND METHOD
Evidence available so far indicates that a The pod samples of six selected clones, SIC 1, ICS 1, hard sclerotic layer provides an effective barrier to ICS 16, EET 308, EET 353 and CC 10, were selected the completion of CPB larval development in pods, for this study. Five pod wall samples were taken at although the development of these layers is not well the primary furrow of each cocoa pod as these areas understood. Studies have shown that a thick sclerotic are regarded as being preferred by CPB for layer in the pod wall is a barrier to the completion of oviposition, giving 15 samples for each clone. The the larval development in cocoa pods (Day, 1985; pod walls approximately 6 mm long x 6 mm wide Azhar and Lim, 1987), but these layers develop were cut down to the endocarp layer and fixed in towards the end of pod maturation. The pods may be 75% alcohol for 48 hrs. The pod wall samples were attacked by CPB before the sclerotic layer is properly then transferred from the fixative into an alcohol developed. More importantly, knowledge of the series for dehydration. This was followed by internal characteristics of the pod is far from preparation of paper moulds to hold the pod wall complete. So far, a preliminary microscopic study by tissues. Fresh molten paraplast was poured off into Adomako and Fordham (1985) revealed variation in paper moulds at one-fourth of the paper moulds
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depth. The pod wall tissues were placed in paper became lignified as pod development continued and moulds once the paraplast was about to harden. Fresh sclereid cells began to develop a secondary cell wall. paraplast was added to cover the whole pod wall The area of non-lignified cells, which have been tissue and fill up the moulds. Hot mounted needles referred to as channels (Susilo, 2005) began to were used to adjust the position of the pod wall tissue decrease and subsequently disappeared as the pod in the paper moulds when necessary. Air was gently matured. Two types of sclereid cell were found in the blown across the surface of the paraplast until it sclerotic layer, brachysclereid (often known as stone formed a skin of solid wax. The paper moulds were cells) and filiformsclereid. Both cells were observed then, removed from the hot-plate and plunged into a in a sequential arrangement (Plate 2). beaker containing iced water and left for 20 min. The embedded tissues were cut into 12 µm thick sections Thickness of the sclerotic layer using a microtome. The cut sections were then placed There was a significant difference in the thickness of onto glass slides for staining with Safranin O and the sclerotic layer between clones (F(5, 48)=54.51, counter stained with Alcian blue. P<0.001) and also between pod age (F(3, 48)=200.43, P<0.001). The duration taken for the sclerotic layer to The slides were examined with a Zeiss- reach maximum thickness also differed between Axioskop Plan2 light microscope (Zeiss, clones (Figure 1). Clone EET 308 reached the Oberkochen, Germany) fitted with an ‘AxioCam’ maximum thickness three months after fruit set camera linked to a computer. Images of the sclerotic compared to clones CC 10, EET 353, ICS1, ICS 16 layer were processed by AxioVision Release 3.1 and SIC 1, which attained the maximum thickness image recording software (Zeiss, Oberkochen, one month later. In some cases, the thickness of the Germany). Three good images of each pod wall sclerotic layer decreased significantly after four sample were selected, giving a total of 45 digitized months, suggesting that the pods were in the process images of the sclerotic layer for each pod age and of ripening. This was clearly demonstrated in clones clone. A standard size of 1.3 mm x 1.03 mm at CC 10 and ICS 1. A significant interaction was also magnification of 100X was used for each image. The noted between clones and pod ages (F(15, 48)=4.82, thickness of the sclerotic layer were measured using P<0.001), suggesting that the thickness of the the ImageJ software programme (Rasband, 1977) sclerotic layer during each sampling period varies (Plugins>Analyze>Grid tools) using tools within and between clones. Analyze>Measure. Each image was measured 10 times at 100 µm intervals between measurements.
Statistical analysis The effects of clone and pod age on the hardness and thickness of the sclerotic layer were tested by two- way analysis of variance (SAS Institute, 1988). Multiple comparisons using a least significant difference (LSD) were performed to examine the differences between clones and pod ages at 5% levels. Prior to analysis, the data were tested for normality, and if necessary, the data were either square root or log transformed to normalize the variance.
RESULTS
Development of sclerotic layer The development of the sclerotic layer in the six selected cocoa clones from 2 – 5 months old is shown in Plate 1. The sclerotic layer was apparent as early as two months after pollination. However, at this stage, the cells in the sclerotic layer were not yet lignified and only had a primary cell wall. The cells
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CC 10
EET 308
EET 353 fruit set 3 months after fruit set 4 months after fruit set 5 months after fruit set
Plate 1. Transverse sections of the development of the sclerotic layer of the six selected clones at four different pod ages at a magnification of 100X with scale bar of 20 µm
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ICS 1
ICS 16
SIC 1
2 months after fruit set 3 months after fruit set 4 months after fruit set 5 months after fruit set Plate 1. Transverse sections of the development of the sclerotic layer of the six selected clones at four different pod ages at a magnification of 100X with scale bar of 20 µm (continued)
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Filiformsclereid Brachysclereid cells cells
Plate 2. Transverse sections of the filiformsclereid and brachysclereid cells in the sclerotic layer of a three-month- old cocoa pod at a magnification of 200X with scale bar of 200 µm
1.07
0.6 2 month 3 month 4 month 5 month
m)
m pod pod age
0.4 LSD
0.2 Thickness ( Thickness
0.0 CC10 EET 308 EET 353 ICS 1 ICS 16 SIC 1 Clone
Figure 1. Mean thickness of the sclerotic layer at four different pod ages of the six selected clones (Vertical bars show ± the standard error of mean. Each clone had three replicates per pod age. Means of untransformed data are presented)
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DISCUSSION Although lignin is known as one of the plant defences to many herbivorous insects (Panda and Although the sclerotic layer can develop as early as Khush, 1995), it does not necessarily affect their two months after fruit set, it does not provide a growth performance. The growth performances of significant barrier to CPB attack because it is geometrid larvae of Aethalura punctulata (Dennis relatively soft. This was confirmed by the and Schiffermuller) and Trichopteryx carpinata histological study, where no lignified cells were (Borkhausen) did not differ when fed on transgenic observed in the sclerotic layer of a two-month-old lines of leaves and control leaves, even though the cocoa pods after staining with Safranin O. A brilliant former had a high lignin content (Tiimonen et al., red stained by Safranin O also indicates the presence 2005). A similar observation was made by Williams of lignin in the cell (Wilson et al., 1991). The cells et al. (1998), who concluded that lignin did not begin to lignify in pods aged three months onwards. appear to be associated with resistance to fall The deposition of lignin in the cells is more likely armyworm (Lepidoptera: Noctuidae) and associated with the development of secondary cell Southwestern corn borer (Lepidoptera: Crambidae). walls (Domingo et al., 1994; Lukjanova and Mandre, 2006) and it is strongly related to plant growth, where The finding of this study can be used in the immature plants have lower lignin content than management of CPB. If female CPBs deposited eggs mature plants. High lignin content made the stems of on a two-month-old cocoa pod, the first larval instars tall fescue harder as they matured (Chen et al., 2002). may suffer high mortality because the pods at this This was confirmed by Lucas et al. (1995) in 14 plant stage are still subjected to cherelle wilt. On the other species. In cocoa, even though the lignin content of hand, the first larval instars may inflict high mortality the pod was not examined, it could be assumed that if CPB eggs are laid on the pods older than four the mature pods had higher lignin content, and thus months because of the hardening and thickening of which may act as barrier of defence against CPB the sclerotic layer. The survival of CPB larvae could larvae. This assumption is supported as mature pods be greater if female CPB deposited eggs on a three- had a harder sclerotic layer than immature pods month-old cocoa pod and the larva completes its life (Alias, 2011) although in some cocoa genotypes, the cycle before the sclerotic layer became harder and hardness of the sclerotic layer tends to decrease thicker. However, Azhar and Long (1996) reported gradually as the pods reach maturity although this that a high number of CPB eggs was found between may not be significant. A possible explanation of the the pods aged three and four weeks before ripening. decreasing thickness and hardness is that the mature It can be argued that the survivorship of CPB larvae pods had lost their firmness with the beginning of the on pods of such an age will decline due to hardening degradation process of cell walls. A loss in firmness and thickening of the sclerotic layer. Moreover, some is a common phenomenon in other drupe fruit during of the pods will be entering the ripening phases, and the ripening process because of increasing activity of the pods could be harvested before CPB larvae cell-wall degrading enzymes which lead to the manage to complete their life cycle. This suggests chemical and structural alterations in cell walls that there is a critical window between larval (Dilley, 1970). Recent studies in avocado and pear penetration through the sclerotic layer and the have confirmed that the fruit cell walls lose their development of the lignified sclerotic layer structure during ripening (Ben-Arie et al., 1979). increasing pod maturity, and that this is approximately between pod ages of 3 - 4 months after The staining intensity of the sections not pollination. Therefore, chemical spraying should be only varied between clones, but also between pod targeted to this specific pod age to ensure their ages, suggesting that staining intensity is not a good effective control. indicator of cell lignin content. Variation in staining intensity is likely due to differences in the cutting ACKNOWLEDGEMENTS sections thickness although the microtome was set to cut at 12 µm. This would affect the absorption rate of The authors would like to thank the Director General Safranin O and Alcian blue during staining. A of the Malaysian Cocoa Board for permission for similar observation was made by Wilson et al. publish this paper. A special thank to Mr. Kelvin (1991), who noted that staining intensity was not a Lamin, Deputy Director General of the Malaysian sensitive indicator at high temperature. Cocoa Board for his advice and constructive comments; Mr. Albert Ling, biometrician, for his
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assistance in analyzing the data; and the staff and Journal of Agricultural and Food Chemistry technicians at the Cocoa Research and Development 50: 5568-5565. Centre of the Malaysian Cocoa Board, Perak and Sabah for their contribution to this project. The Day, R. K. (1985). Control of the cocoa pod borer authors are indebted to the Ministry of Agriculture (Conopomorpha cramerella). Ph.D. Thesis. Nature and Food Quality of the Netherlands for The University of London. 299 pp. funding the project. Dilley, D. R. (1970). Enzymes. In The Biochemistry of Fruits and their Products (Ed. Hulme, A. REFERENCES C.) pp. 179-207. Academic Press, London.
Adomako, B. and Fordham, R. (1985). Cocoa pod Domingo, C., Gomez, M. D., Canas, L., Hernandez- wall anatomy in relation to pod borer attack. Yago, J., Conejero, V. and Vera, P. (1994). Poster presented at “Cocoa Research – the A nove1 extracellular matrix protein from U.K. Involvement”. University of tomato associated with lignified secondary Nottingham, Sutton Bonnington. 10-12 cell walls. The Plant Cell 6: 1035-1047. April 1985. Haya, R., Navies, M., Kelvin, L., Albert, L., Alias, A. Azhar, I. (1990). Studies on sclerotic layer hardness and Nuraziawati, M.Y. (2007). Screening for of cocoa pods. MARDI Research Journal cocoa pod borer resistance based 18: 63-69. morphological descriptors. Presentation at the 2007 Conference on Plantation Azhar, I. and Lim, D. H. K. (1987). An investigation Commodities. Putra World Trade Centre, on the use of host plant resistance and crop Kuala Lumpur, Malaysia. 3-4 July 2007. manipulation in the management of cocoa pod borer. In Management of the Cocoa Pod Lucas, P. W., Darvell, B. W., Lee, P. K. D., Yuen, T. Borer (Eds. Ooi, P. A. C., Chan, L. G., D. B. and Choong, M. F. (1995). The Khoo, K. C., Teoh, C. H., Jusoh, M. M., Ho, toughness of plant cell walls. Philosophical C. T. and Lim, G. S.) pp. 83-101. Malaysian Transactions of the Royal Society of London Plant Protection Society. Kuala Lumpur. B – Biological Sciences 348:363-372.
Azhar, I. and Long, G. E. (1996). Effect of cocoa Lukjanova, A. and Mandre, M. (2006). Anatomical and pod age on egg distribution and egg features and localization of lignin in needles parasitism of the cocoa pod borer in of Scots pine (Pinus sylvestris L.) on dunes Malaysia. Entomologia Experimentalis et in South-West Estonia. Proceedings of the Applicata 81: 81-89. Estonian Academy Sciences Biology Ecology 55: 173-184 Bekele, F. (1996). A preliminary study of pod husk hardness of accessions in the International Panda, N. and Khush, G. S. (1995). Host Plant Cocoa Genebank, Trinidad. Cocoa Research Resistance to Insects. CAB International Unit Report (Unpublished report). 9 pp. Publication, United Kingdom. 431 pp.
Ben-Arie, R., Kislev, N. and Frenkel, C. (1979). Rasband, W. S. (1997). ImageJ. U. S. National Ultrastructural changes in the cell walls of Institutes of Health, Bethesda, ripening apple and pear fruit. Plant Maryland, USA, http://rsb.info.nih.gov/ij/, Physiology 64: 197-202. 1997-2007.
Chen, L., Auh, C., Chen, F., Cheng, X., Aljoe, H., SAS Institute. (1988). SAS/STAT User’s guide. Dixon, R. A. and Wang, Z. (2002). Lignin Version 6. Fourth edition. SAS Institute Inc., deposition and associated changes in Cary, North Carolina, USA. 1686 pp. anatomy enzyme activity, gene expression, and ruminal degradability in stems of tall Susilo, A. W. (2005). Relationship between the fescue at different developmental stages. characteristics of sclerotic layer of cocoa
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pods and their resistance to cocoa pod borer. transgenic silver birch (Betula pendula Presentation at the Malaysian International Roth)? Planta 222: 699-708. Cocoa Conference, Kuala Lumpur, Malaysia. 17-19 July 2005. Williams, W. P., Davis, F. M., Buckley, P. M., Hedin, P. A., Baker, G. T. and Luthe, D. S. Teh, C. L., Pang, J. P. Y. and Ho, C. T. (2006). (1998). Factors associated with resistance Variation of the response of clonal cocoa to to fall armyworm (Lepidoptera: Noctuidae) attack by cocoa pod borer (Conopomorpha and southwestern corn borer (Lepidoptera: cramerella (Lepidoptera: Gracillaridae)) in Crambidae) in corn at different vegetative Sabah. Crop Protection 25: 712–717. stages. Journal of Economic Entomology 91: 1471-1480. Tiimonen, H., Aronen, T., Laakso, T., Saranpaa, P., Chiang, V., Ylioja, T., Roininen, H. and Wilson, J. R., Deinum, B. and Engels, F. M. (1991). Haggman, H. (2005). Does lignin Temperature effects on anatomy and modification affect feeding preference or digestibility of leaf and stem of tropical and growth performance of insect herbivore in temperate forage species. Netherlands Journal of Agricultural Science 39: 31-48.
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DISTRIBUTION OF COCOA POD BORER EGGS AT DIFFERENT LEVELS OF SHADE IN SMALL-SIZE PLANTATION
Saripah, B.1, Hassan2, S. T. S. and Sajap2, A. S. 1Malaysian Cocoa Board, 5-7th floor, Wisma SEDCO, Lorong Plaza Wawasan, Off-Coastal Highway, Locked Bag 211, 88999 Kota Kinabalu, Sabah, Malaysia 2Universiti Putra Malaysia, 43300 Serdang, Selangor Email: [email protected]
Malaysian Cocoa J. 8: 9 - 13 (2014) ABSTRACT - Cocoa pod borer, Conopomorpha cramerella Snellen (Lepidoptera: Gracillariidae) (CPB) is the most serious pest of cocoa and the infestation caused severe losses of cocoa crop hence production in Malaysia although several control approaches had been implemented. Proper management tactics and control approaches were very crucial in reducing this pest’s infestation. However, management tactics especially pruning of cocoa canopy and thinning of available shade trees in mature cocoa plantation gained less attention by cocoa growers. Thus, this study was conducted to examine effects of different levels of available shade trees especially Gliricidia macculata and coconut in small-size plantation (1.8ha). The block divided into different shade categories, which were Light, Medium and Heavy Shade Level; and parameter studied was the CPB eggs distribution. The results showed that different levels of shade in small-size plantation did not affect the distribution of CPB eggs and there was no significant difference in different shade levels. This could be due to the flying capability of CPB, that can fly within short distance, thus was capable to oviposit their eggs at adjacent trees regardless to the different shade level available in small cocoa plantation.
Keywords: Cocoa pod borer, Shade, Management tactics, Pest infestation
INTRODUCTION attention by cocoa growers. It is common to see entire cocoa block being completely covered by Cocoa trees require shade at all stages of growth in heavy shade especially the Gliricidia spp, due to their the field and providing shade is necessary to ensure negligence in thinning the shade tree. survival of young cocoa trees. Immature cocoa tree requires amount of full sunlight and light requirement Cocoa is subjected to attacks by a wide increases as the crop reaches maturity (Azhar et al. range of insects’ pests wherever it is grown. Cocoa 2009). Cocoa seedlings require 25% of full sunlight Pod Borer (CPB) is the most serious insect pest in and establishment of shade should commence South East Asia, particularly in the sprawling Malay immediately after land clearing. Planting of young Archipelago including Malaysia, Philippines and cocoa seedlings in 100% full sunlight should be Indonesia (Shapiro et al., 2008). CPB is also present avoided if existing and established shade is already in Papua New Guinea, Samoa, Northern Territory of available. If the shade is too heavy, cocoa will not Australia, Irian Jaya, India, China and Taiwan. Since grow and yield well. The most common shade tree in its first discovery in the Tawau, Sabah in late 1980, cocoa plantation is semi-permanent or permanent CPB continue threatened the cocoa industry in shade trees such as Gliricidia maculata or fruit trees, Malaysia. CPB infestation causes heavy crop losses temporary shade as coconut fronds, plastic bags or all over Malaysia if the pest is not kept under control. short-term crops such as maize or tapioca. During Infestation of CPB provides a persistent problem to maturity stages, number of shade trees such as growers due to increasing production cost, and hence banana or Gliricidia must be reduced. Shaping and reduced yield (Azhar et al., 2000). thinning of Gliricidia is done by removing the undesirable branched, while banana should be In order to estimate recent population of removed after three years. Removal of shade trees CPB and later on will helps in the management will increase cocoa yield because light intensity is decision, sampling of CPB egg is very important one of the main factor in limiting yield and affect the (Azhar and Long, 1991). Egg is the most practical population of insects in cocoa plantation. However, stage to use in population estimation since egg counts the importance of thinning shade trees gains little will provide a more recent estimate of moth activity.
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Understanding the distribution and abundance of capable to deposit their eggs in less shaded areas CPB egg is important in estimating the population based on their flight ability. density and distribution parameters later on. Three basic types of population patterns in a geographic Relationship between effects of shade and area are random, clumped or uniform and most insect pests infestation especially CPB is still debatable, yet populations are spatially aggregated or clumped, but there is no specific research on the effect of shade on the degree of aggregation often varies among the CPB infestation. Thus, this research was carried populations and among species (Root and out to establish the relationship of different shade Cappuccino, 1992). Depending on the spatial scale, levels with CPB population and infestation in small most insects were aggregated due to the patchy cocoa plantation. The study must affirm on the distribution of food sources, temperature, humidity, relationship between the effects of shade levels on predators, ovipositional sites, relationship between CPB infestation. Lack of knowledge on the individuals, and their varied environment (Campbell importance of thinning their existing shade trees and Hagstrum, 2002). might have explained CPB existence and population build-up in their cocoa plantations. Flight activity may also provide information on variation in insect population density within a given environment, and to the relationship of the MATERIALS AND METHODS insect with alternate host distribution and phenology (Nansen et al., 2002). Spatial variation in different The study was conducted at 1.8-hectare block within habitats may help to determine the relative 10 months of data collection. The block were divided importance of the natural habitats and the relationship into three plots, which were Light (Plot B), Medium with flight activity. Most field research on insect (Plot A) and Heavy (Plot C) in accordance with pests focuses on their damage and population different shade levels. Light Plot with average yearly dynamics in fields, while their spatial distribution light penetration taken at 9.00 am was 1,906 to 2,235 pattern and spatial migration patterns generally given Lux, where as the temperature range from 28.4°C to less attention. Locomotors activity of insects usually 29.6°C. Medium Plot with average yearly light received little attention by researchers. The flight penetration was 1,356 to 1,871 Lux, with the ability of winged adult arthropods may lead to temperature range from 28.0°C to 28.7°C. Heavy Plot dispersal, hence decreasing of aggregation. Insects with average 673 to 821 Lux and the temperature and their immobile stages result in high aggregations, range from 26.9°C to 27.9°C. Fifty (50) trees were in comparison with winged insects which can fly. selected for egg sampling in each plot. Each plot was then divided into four sub-plots with two trees as Some insects have great capability to fly for guard rows, to ensure sample trees were represent the long distance, thus migration process is easier within whole plot. Approximately 12 or 13 trees were small-scale area. The capacity for flight especially in selected at random in each sub-plot. Visual counts off females will increases the potential rate of spread and CPB eggs on the pod surface especially on cocoa the attractant to lights that results in egg deposition furrows were done where the cocoa pod is the sample (Wallner et al., 1995). Environmental factors such as unit. The data were arranged in matrix with different temperature, moisture, light and wind may play an plots and various shade levels. Data were pooled by important role in the dispersal of insects. Increase in monthly basis in Excel® program. By using SAS® temperature might affect any stage of the life cycle UNIVARIATE procedure analysis, eggs distributions and therefore limit distribution and abundance were normalized using the square-root (SQRT) through its effects on survival, reproduction and transformation on egg numbers per pod per month. development (Karuppaiah and Sujayanad, 2012). Subsequently, the transformed data were analyzed However, in contradict an adult CPB (Figure 1) was using the ANOVA (SAS® system for Windows® usually stated as a weak flyer, due to their delicate V8), and means from each block were compared body, behavioral and morphological characteristics. using Least Square Difference (LSD) multiple In this study, relationship between different shade comparison test. Taylor’s power law Index was levels available in the same block and CPB egg chosen to measure the degree of aggregation. distribution was yet to be questionable. Small-scale block with uneven shade levels may provide resting and breeding sites in heavy shaded areas but still
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RESULTS AND DISCUSSIONS recorded VMR less than 1 in October, January and March (Medium Plot); July, September and Mean of CPB eggs for each plot were shown in Table November (Light Plot); November and March at 1. There were no significant different in mean Heavy Plot. The value was resulted from uniform number of CPB eggs in all plots (p<0.05). Findings distribution on those particular months in each plot. showed that the mean of CPB eggs was almost similar regardless to the shade level in each plots. The distributions of CPB eggs on cocoa This result may be influenced by the location of each pods were evaluated using Taylor’s power law (Table plot, which was adjacent to each other. Even with 3). Dispersion of CPB eggs in 20 sampling occasions different shade levels of these plots, probably the showed the b values were larger than 1 in all plots. CPB moth was capable to fly within the short range Regression formula of CPB eggs dispersion at from one plot to another. Medium Plot was Y= (2.014 ± 0.362) + (3.551± 0.273X). While at Light Plot, the regression formula To evaluate the distribution pattern of CPB of CPB eggs distribution was Y= (1.827 ± 0.422) + eggs within observation plot, several different (1.439 ± 0.146). Regression formula of CPB eggs techniques can be applied. The simplest index of distribution at Heavy Plot was Y= (0.901 ± 0.305) + dispersion and the most fundamental method in (1.165 ± 0.154). The slope values of Taylor’s power evaluating the dispersion is using the variance to law at Medium Plot (b = 3.551) were significantly mean ratio (VMR). Distribution analysis using VMR greater than 1. While for Light Plot, the slope value in almost all observations shows significantly higher was 1.439 and 1.165 at Heavy Plot. Overall, b values statistics than 1 (s2/ x >1), involving six of 10 months at all plots was larger than 1, thus the CPB eggs of observations at Medium Plot, 7 from 10 months dispersion was aggregated or clumped with strong (Light Plot) and 8 from 10 months at Heavy Plot positive correlation. Distribution of CPB eggs was (Table 2). The results show aggregated distribution almost similar among study plots. Taylor’s power of the CPB eggs (Figure 2) among sampled trees law coefficients provided the best fit (R2) at Medium regardless of shade levels. Observations also Plot.
Table 1. Mean number of CPB eggs at Light (Plot B), Medium (Plot A) and Heavy (Plot C)
Plot No. of sampling Mean ± SD
Medium 20 0.240a ± 0.22
Light 20 0.285a ± 0.271 Heavy 20 0.301a ± 0.311
Means followed by same letter are not significantly different at p ≥ 0.05.
Figure 1 Adult cocoa pod borer moth Figure 2 Aggregated distribution of the CPB eggs
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Table 2. Variance to mean ratio of CPB eggs at Light (Plot B), Medium (Plot A) and Heavy (Plot C)
Variance to Mean Ratio Month A B C July 1.264 0.979 2.261 August 1.110 1.469 1.738 September 0.972 0.987 1.759 October 0.000 6.701 2.050 November 1.263 0.000 0.000 December 1.402 1.406 1.412 January 0.961 1.264 1.456 February 4.063 3.044 1.731 March 0.980 1.770 0.000 April 3.198 2.048 1.631
Table 3. Taylor’s power law distribution parameters at Light (B), Medium (A) and Heavy (C) Plots
Taylor's Power Law
Plot R square r log a SE b Se t P F
A 0.955 0.977 2.014 0.362 3.551 0.273 5.56 0.005 169.76 B 0.9243 0.961 1.827 0.422 1.439 0.146 4.33 0.0025 97.63
C 0.8772 0.936 0.901 0.305 1.165 0.154 2.96 0.0182 57.16
DISCUSSIONS In order to protect their eggs from any environmental factors such as wind or rain, and from natural In this small-scale plantation with different levels of enemies, CPB usually deposit their eggs on the cocoa shade were available, aggregated distribution of CPB pod furrow, and this will lead to the aggregated eggs was recorded using variance to mean ratio distribution. (VMR) and Taylor’s power law (TPL), where both indices show high values of index of aggregation. Cocoa canopy itself plays a role as a shade, Aggregation of CPB eggs in most of the observation and may influence the aggregation pattern of CPB denoted that eggs having similar distribution trend in eggs. CPB usually prefers cocoa trees with closed all plots although with varied light penetration and canopy, and this provides sufficient shade by the shade levels, which confirmed that most insect cocoa canopy itself. CPB usually resting underside populations are spatially aggregated or clumped. the horizontal cocoa branches or jorquette in Causes of CPB eggs to aggregate in this study might daylight, thus close canopy may provide a suitable be due to availability of food source, cocoa canopy, resting sites and ovipositional place. This may flight potential ability and small size plots that were contribute CPB to less depending on the available adjacent to one another. Another reason that may shade trees. In addition, most of shade trees available contribute to aggregated pattern of CPB eggs on pod in this block was tall coconut trees, thus coconut trees surface in all plots was the survival of the CPB eggs.
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itself have less functional as a shade to cocoa trees, REFERENCES even in medium or heavy shade level plots. Azhar, I., annd Long, G. E. (1991). The ecology of Although CPB was known as a weak flyer, cocoa pod borer in Malaysia: Dispersion it is believe that CPB was capable to fly within short pattern and sampling plan for eggs on pods. distances. With the 2m x 3m x 3m cocoa planting Journal of Plant Protection in the Tropics, 8: system, this system still, allow CPB to fly from one 103-110. tree to another. Furthermore, mature cocoa trees tend Azhar, I., Alias, A., and Meriam, M. Y. (2000). to have sparse canopy, thus joining canopy between Research on the management of cocoa pod trees in a same rows was easily occurs. CPB can fly borer in Malaysia. In Bong, C. L., Lee, C. H., between trees, and capable to fly between study plots. & Shari F.S (Eds.), Proceedings INCOPED Ability of CPB to fly within short distance and small- 3rd International Seminar on Cocoa Pests and size plantation may contribute to no significant Diseases (pp 105-113). Kota Kinabalu, KK: different on CPB eggs between plots. With tall Malaysian Cocoa Board (MCB) and coconut trees that approximately 25 to 30 meters and International Permanent Working Group for low number of Gliricidia sp trees, it provides more Cocoa Pests and Diseases (INCOPED). open environment at this block. Open environment Azhar, I., Kelvin, L., Denamany, G., Saripah, B., and with tall coconut trees as in all study plots may not Nuraziawati, M. Y. (Eds). (2009). Cocoa affecting CPB flying activities. Thus, this field may Planting Manual. Sabah: Malaysian Cocoa provide almost similar environment for CPB to lay Board. their eggs, even in different shade levels. Karuppaiah, V. and Sujayanad, K. (2012). Impact of climate change on population dynamics of insect pests [Electronic version]. World CONCLUSIONS Journal of Agricultural Sciences, 8(3): 240- 246. The aggregation index in all plots may reflect the Nansen, C., Meikle, W. G. and Korie, S. (2002). behavioral interaction between insect and the Spatial analysis of Prostephanus truncates environment and the heterogeneity of habitat. (Bostrichidae: Coleoptera) flight activity near However, the small-size block, which is only 1.8ha maize stores and in different forest types in with range less than 40 meters from the first row of southern Benin, West Africa. Annals of the cocoa to the last row of cocoa may produces Entomological Society of America 95(1): 66- homogeneity of nature, thus lead to almost similar 74. trend of dispersal, regardless to the different shade Root, R. B., and Cappucino, N. (1992). Patterns in level. Overall, distribution pattern of CPB eggs in population change and the organization of the block with different levels of shade were aggregated, insect community associated with goldenrod. and shows almost similar trends between small size Ecological Monographs 62: 393-420. plots. Different shade levels in small-size plantation Shapiro, L. H., Scheffer, S. J., Maisin, N., Lambert, not affected the CPB egg dispersion pattern in this 10 S., Purung, H., Sulistyowati, E., Vega, F. E., months observation. Gende, P., Laup, S., Rosmana, A., Djam, S. and Hebbar, P. (2008). Conopomorpha ACKNOWLEDGEMENTS cramerella (Lepidoptera: Gracillariidae) in the Malay Archipelago: Genetic signature of a Authors would like to thank the Director-General of bottleneck population? Annual Entomology Malaysian Cocoa Board, Dr. Lee Choon Hui for Society of America 101(5): 930-938. permission to publish this paper. We also thank Wallner, W. E., Humble, L.M., Levin, R. E., former Director-General of Malaysian Cocoa Board, Baranchikov, Y. N. and Carde, R. T. (1995). Dato’ Dr. Azhar Ismail, Deputy Director General, Response of adult lymantriid moths to Mr. Kelvin Lamin, Mr. Roslan Sa’adi and staffs of illumination devices in the Russian Far East. Entomology Unit at CRDC Hilir Perak for their Journal of Economical Entomology, 88: 337- technical assistance. 342.
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CONTROL OF COCOA POD BORER USING INSECTICIDES AND COCOA BLACK ANTS
Saripah, B. Malaysian Cocoa Board, 5-7th floor, Wisma SEDCO, Lorong Plaza Wawasan, Off-Coastal Highway, Locked Bag 211, 88999 Kota Kinabalu, Sabah, Malaysia Email: [email protected]
Malaysian Cocoa J. 8: 14 - 22 (2014) ABSTRACT Assessments on the infestation of Cocoa pod borer (CPB) using four treatments; application of monthly insecticides spraying, bimonthly insecticides, augmentation of Cocoa black ant (CBA) and no management approach (control) was observed within a period of 24 months. Mature pods from all treatments were evaluated for good and loss pods, CPB’s infestation categories and Average Damage Severity Index (ADSI) values. Based on loss pods due to CPB’s infestation, the results showed that CPB infestation was most serious in bimonthly insecticide treatment that surrounded by abandoned cocoa plantation with only 8 out of 24 months recorded more than 50% of good pods. A monthly insecticide treatment that surrounded with well-managed cocoa plantation recorded 13 months of more than 50% of good pods. The most promising results were obtained in the Cocoa black ant treatment with 17 out of 24 months with more than 50% of good pods. Prior to ANOVA analyses, ADSI value of bimonthly insecticide treatment was significantly different at p≥0. 05 (3.224a) with monthly insecticide treatment (2.730b), CBA (2.697b) and control (2.696b). Heavy infestation category of CPB infestation was observed in bimonthly insecticide treatment. At the monthly insecticide, control and CBA treatment trends of infestation category were recorded throughout the sampling occasions. There was a significant difference between treatments for CPB infestation, where bimonthly insecticide treatment recorded highest CPB infestation (1.752a), followed by control (1.623b), monthly insecticide (1.504c) and CBA treatment (1.50c). Throughout the study period, an area with augmentation of CBA showed the most promising results; with high number of good pods, low ADSI values and light infestation category compared with both areas with insecticide application.
Keywords: Cocoa, Theobroma cacao, Cocoa pod borer, Conopomorpha cramerella, Insecticides, Cocoa black ants, Dolichoderus thoracicus
INTRODUCTION various insect pests belonging to the orders of Lepidoptera, Coleoptera, Diptera and Hemiptera. Various control measures have been used to reduce Growers used both chemical and non-chemical the infestation of Cocoa Pod Borer, Conopomorpha approaches in controlling CPB, and the management cramerella Snellen (Lepidoptera: Gracillariidae) approach of cocoa insect pest in Malaysia is mainly since its early outbreaks in 1986. The control based on chemical application (Azhar, 1992). Most measures include management practices, chemical of cocoa growers are keen to implement insecticides control, semio-chemical, mechanical control and spraying due to their quick action and availability in biological control. Each control measure should be the market. Spraying is usually conducted using easily used by the farmer. Farmers usually prefer manual knapsack sprayers due to their suitability technique that is cheap, easy to handle, with long where cocoa trees are not too tall, with small acreage lasting results, and give high returns. The adoption of and limited resources. Pyrethroids are the most control measure by farmers will also depend on their popular group of insecticides used against CPB perception of the cost, not necessarily in monetary (Azhar et al., 2000). The bimonthly spraying interval terms but in time and effort. The costs of the pest is usually adopted, although two or three spraying control to farmers will depend on the type of control program (during the two ascending peaks cropping technique used (Dent, 1991). periods and other optional spraying during the trough period) of 5-6 sprayings at a 10-day intervals were Pyrethroids are among the most commonly recommended per year (Sidhu et al., 1987). The use insecticides worldwide, which accounting for application of insecticides should be made according more than 30% of global use (Shukla et al. 2002). to the level of infestation rather than on the fixed Synthetic pyrethroid is widely used for a control of schedule basis. However, smallholders usually apply
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prophylactic spraying based on fixed bimonthly treatments; block surrounded by abandoning cocoa calendar basis regardless of the current level of CPB plantations, and block surrounded by well-managed infestation; hence the cost will increase. Although cocoa plantations. Two different spraying calendars control measure had been applied, CPB still were implemented which are insecticide spraying at continued to inflict higher yield loss especially in bimonthly and at monthly basis. The objective of this heavily infested areas. If the infestation is serious, the study is to determine the effects of insecticide crop loss to farmers can be substantial, although they spraying at two different environments and two are following the recommended calendars by different spraying calendars, and CBA in the cocoa chemical companies. The infestation cause field. The effectiveness of both control approaches unacceptable level of damage, and it may also be was determined by using several parameters such as influenced by the feeding and oviposition preferences good and loss pods, pod damage category and for pods. Insects may look for the suitability of their Average Damage Severity Index (ADSI) values. food value and the fruit texture to deposit their eggs, and it is possible that CPB shows similar responses. Insecticides may not be able to cover entire cocoa MATERIALS AND METHOD pod surface, thus give CPB a chance for egg deposition. This later on will reduce the effectiveness The study was conducted at the Cocoa Research and of chemical control. Development Center (CRDC), Malaysian Cocoa Board Hilir Perak, Malaysia. The experimental The use of biological control in the designs were RCBD at four blocks with three management of insects has become an alternative replicates. There are three treatments (Table 1), method in controlling CPB. One of the sources of bimonthly insecticide spraying (T1), monthly biological control is using ants. In Malaysia, only a insecticide spraying (T2), control (T3) and few ant species were observed to have potential in augmentation of CBA (T4). The bimonthly controlling cocoa pests, especially the Cocoa black insecticide area was adjacent to an idle and ants (CBA), Dolichoderus thoracicus Smith abandoned cocoa blocks in the back, left and right (Hymenoptera: Formicidae). CBA is an endemic sides. Meanwhile, selected areas for monthly species and does not spread naturally into many insecticide and control treatments was adjacent to cocoa areas. However, the use of CBA is feasible blocks with common management practices. CBA where cocoa is inter-planted under coconut and may block was selected at isolated places, and at a thrive well due to the availability of nesting sites on distance from blocks with insecticide application. the coconut crown. The population of the CBA can Two spraying calendars were implemented; first was be enhanced by augmentation of artificial ant nests using the dosage recommended by the chemical made from a plastic bag and stuffed with dry cocoa company and second by twelve spraying occasions leaves. Saripah and Azhar (2007) reported that cocoa per year. Spraying was conducted every two weeks at pods that had been covered by CBA may hinder CPB T1 and every month at T2, and targeted to the cocoa from laying the eggs on the pod surface. Meanwhile, pods 7-9 cm and above. For T4, empty ant nests were Azhar (1992) reported that cocoa pods continued to hung at the ant farm source, and after 5-6 weeks, have a CPB infestation in the presence or absence of colonized artificial ant nests were transferred to the CBA. However, the proportion of infestation was study blocks. Augmentation of colonized artificial ant higher on pods without attending ants than those with nests was carried out four times, with 150 nests were attending ants. The study also indicated that pods constructed every 6 months. The nests were placed without attending CBA also recorded higher randomly inside the block and hung at the jorquette moderate and heavier damage than those with of cocoa trees. All ripe cocoa pods were harvested attending CBA. every two weeks to determine the number of good and loss pods, pod damage category (Figure 1) and Comparison of the effectiveness of CBA and ADSI values. For the pod damage, there are five insecticide in controlling CPB infestation must be main categories, as listed in Table 2. based on the field data. This biological approach was selected due to cocoa-coconut ecosystems in the For ADSI assessments, mature cocoa pods selected block, which may give potential to CBA to will be split and categorize into the degree of thrive well in this cropping system. Two types of infestation technique referring to Azhar (1995). The cocoa environment were selected for insecticide ADSI were calculated using the formula:
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[(0xn1) + (1xn2) + (2xn3) + (3xn4) + (4xn5)] /N, where n1 : number of pods in category 0 (Healthy) n2 : number of pods in category 1 (Slight) n3 : number of pods in category 2 (Light) n4 : number of pods in category 3 (Medium) n5 : number of pods in category 4, (Heavy) N : total number of pods examined
Collected data were arranged by monthly V8). Data for all parameters were collected for a basis, according to different treatments and pooled in period of 24 months from the first spraying occasions Excel® program. Data were then analyzed using SAS and augmentation of artificial nests. statistical software (a SAS® system for Windows®
Table 1. Treatments and justification of application Treatments Justification of application T1 Insecticides spraying every two weeks (Bimonthly insecticides) Decis (Class 3) Deltamethrin with 13 ml/10 liters water, 0.27 liter/hectare Active ingredients: 1.4% w/w, Inert ingredients : 98.6 % w/w T2 Insecticides spraying every four weeks (Monthly insecticides) Decis (Class 3) Deltamethrin with 13 ml/10 liters water, 0.27 liter/hectare Active ingredients: 1.4% w/w, Inert ingredients : 98.6 % w/w T3 (Control) No control approach were implemented T4 (Cocoa black ants) Four times of nests augmentations using 600 colonized artificial ant nests
Table 2 Categories of CPB infestation Score Category Description
0 Healthy Healthy (no larva penetrated the sclerotic layer (SCL) and all beans inextricable) 1 Slight Slight damage (Larvae penetrated the SCL with sign of infestation inside the pod, such as the frass and cell growth on the inner endodermis, but all beans are inextricable) 2 Light Light damage (<20% of the beans are inextricable) 3 Medium Moderate damage (21-50% of the beans are inextricable) 4 Heavy Heavy damage (>50% of the beans are inextricable)
Figure 1. Assessments for pod damage category. Damage from left was healthy, slight, light, medium and heavy infested category
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RESULTS months recorded more than 50% of good pods. However, only 3 months recorded more than 70% of Assessments on the number of good pods generally good pods throughout the data collection. In the determine the seriousness of the infestation with control area, 9 months obtained more than 50% of respect to the treatment. The number of good pods in good pods, and 3 months recorded more than 70% of all treatment shown in Figure 2.The percentage of good pods. All of the months with less than 30% of good pods recorded more than 50% of overall total CPB infested pods were recorded in the first three pods was obtained in 8 out of 24 months of data months of data collection. Generally in almost all collection at bimonthly insecticide treatments, and treatments, the infestation of CPB was low when the only one month recorded more than 70% of good number of sampled pods was high, and vice-versa the pods. At monthly insecticide treatments, 13 and 7 infestation was high when the number of sampled months recorded more than 50% and 70% of good pods was low. pods. Meanwhile, at CBA treatment, 17 out of 24
Figure 2. Percentage of good and total pods at T1 (bimonthly insecticide), T2 (monthly insecticide), T3 (control) and T4 (Cocoa black ants)
The pod damage category of each treatment than 20% of the beans was inextricable; medium (21 was shown in Figure 3. A heavy category was a to 50%), healthy (all beans inextricable with no sign reflection of the seriousness of CPB infestation with of CPB) and slight (all beans inextricable with little more than 50% of infested pods, and was dominated sign of infestation). In summary, the infestation at throughout sampling occasions at bimonthly bimonthly insecticide block was insecticide treatment. This was followed by light heavy>light>medium> healthy>slight. Pod damage category, which described light infestation with less category at monthly insecticide was fluctuated, with
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heavy category dominated at 2-7 months and 16 to 24 treatments, light category dominated at the first four months of data collection. Light category was months of data collections and from 13 to 17 months. dominated from 7 to 18 months of data collections. The other months were dominated by heavy damage Fluctuation trends also occurred at control treatments category. The slight was the least recorded category between heavy and light category; as well as healthy in all treatments throughout 24 months of data and medium category. Meanwhile, at CBA collections.
Figure 3. Pod damage category at T1, T2, T3 and T4. Damage were divided into five categories; healthy, slight, light, medium and heavy
Statistical analysis was determined using peak crop seasons, the highest ADSI was recorded in ANOVA procedures as described in Table 3. The bimonthly insecticide treatment, followed by control, results show that ADSI values of bimonthly monthly insecticide and CBA. During low crop insecticide treatment (3.222a) were the highest and seasons, CBA recorded lowest ADSI values (2.905) significantly different (p ≥ 0.05) from the other three although this was not significantly different from treatments. Monthly insecticide, CBA and control other three treatments. treatments showed no significant differences between each other. From 24 months of data collection, 13 Table 5 shows the CPB infestation months were described as peak-crop months, and 9 categories throughout two years of data collections. months as low-crop months with respect to the There was a significant difference between both monthly pod number. Therefore, ADSI during peak insecticide-spraying treatments. Bimonthly and low crop seasons was analyzed separately as insecticide recorded higher CPB infestation than shown in Table 4. The results showed that ADSI monthly insecticide treatment. There was also a during low crop was higher than that during peak significant difference between the bimonthly crop seasons regardless of different treatments. insecticide with CBA treatments and control area. No Although different treatments were implemented, significant difference was recorded between CBA there was no significant difference for ADSI in every and monthly insecticide treatments. treatment, either during low or peak crop seasons. In
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Table 3. ADSI values at bimonthly insecticide, monthly Insecticide, Control and CBA Blocks
Treatment ADSI Value N T1 (Bimonthly) 3.224a 24 T2 (Monthly) 2.730b 24 T4 (CBA) 2.697b 24 T3 (Control) 2.696b 24 Means followed by same letter are not significantly at 5% level.
Table 4. ADSI values during peak and low crop seasons.
Peak crop season Low crop season Treatment N ADSI Treatment N ADSI T1 13 3.075a T1 9 3.875a T3 13 2.636a T3 9 2.989a T2 13 2.563a T2 9 2.906a T4 13 2.492a T4 9 2.905a
Means followed by same letter are not significantly at 5% level.
Table 5. CPB infestation category at bimonthly insecticide, monthly insecticide, control and CBA treatments
Treatment N Mean T1 (Bimonthly) 24 1.752a T3 (Control) 24 1.623b T2 (Monthly) 24 1.500c T4 (CBA) 24 1.504c Means followed by same letter are not significantly at 5% level.
DISCUSSIONS to abandoned cocoa areas may reduce the success rate of insecticides, although with bimonthly Throughout two years of observation, infestation of insecticide spraying calendars. No management CPB in all treatments can be considered as serious, was carried out at surrounding area, leaving a with the respect of a number of good pods, ADSI suitable place for CPB to breed and to escape from values and infestation category. The infestation was insecticides spraying. This is because CPB can fly high especially during low crop season, when the in short range, thus they may shift to other number of available pods was low. Low pod locations where no insecticide applied. The CPB number gave chance to the same pod to be attacked may flee from abandoned areas to these blocks several times by CPB, although insecticide during the spraying gap, which is within 14 days. spraying were conducted at bimonthly or on a Therefore, before application of insecticides, it is monthly basis. This was in agreement with the important to have information on the surrounding findings obtained from Way and Khoo (1989). areas adjacent to the cocoa plantation. They noted that despite repeated insecticide treatments following recommended calendars by Ineffectiveness of insecticide spraying the manufacturers, infestation caused by CPB may also be due to inefficient application continues to cause an unacceptable level of techniques, timing and pest’s feeding activities. damage, even with regular application of Mortality or sublethal effects of pesticides occur insecticides. through three different modes of action; which are direct contact with the insecticide, residual uptake It was surprising when the CPB by contacting residues on another surface, or food infestation was the highest in bimonthly chain uptake (Croft, 1990). Direct spraying of insecticides areas. The infestations were serious, insecticides may be effective, however when the and maybe due to their surrounding areas. The insecticide residues have dissipated, it was believed location of the selected block itself, which adjacent they will be less harmful to the insect pests. Some
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Malaysian Cocoa Journal, 8/2014 insecticides were believed to cause a dramatic This will cause the population to be made up reduction in flight response of Hymenoperan wasps mostly resistant individuals. A resistant individual as reported by Stapel et al. (2000). However, there may reproduce faster, or survived better during is a sharp decline in flight response within day 4 to free-spraying calendars. In order to reduce day 18 and no effects after day 20. Similar resistance risk, application of different pesticides, response may occur when CPB was able to fly and especially different class and different mode of deposit their eggs on the pod surface after action were very important. This method might not insecticide residues have dissipated with time. be the best solution, but it may allow pest population at a lower level until other management In some cases, insecticides may affect strategies can be developed to reduce the behavior as well as physiology at levels that do not infestation later on (Bellinger, 1996). Lowest rate lead to direct mortality. Insects may decrease their and avoid repeat applications of the same chemical ability to produce more offspring due to changes in class must be promoted to the cocoa growers. The behavioral modifications such as mate location and use of only single insecticide throughout the study oviposition (Haynes, 1988). Therefore, to reduce period, may contribute to low success rate of CPB successful risk of oviposition behavior, targeted control. In addition, side effect of the usage of area must cover the entire pod surface with the some pesticides results in unfortunate addition of any spray adhesive or stickers to evade consequences to the non-target organisms (Fishel, quick dissipation of insecticides from the pod 2011). Some pesticides are harmful to pollinators surface. Furthermore, there is a hindrance using such as wasps and honey bees, using direct losses insecticide application, due to an insidious of the insect populations and indirect losses of crop characteristic of CPB larva which totally inside the yield because of the lack of adequate pollination. pods. Matured larvae will tunnel out from the cocoa pods, emerge as pre-pupa and immediately From the findings obtained in this study, develop cocoon for their pupation. Previous biological control may be considered as an research by Hill (1983) demonstrated that chemical alternative pest management strategy for dealing control against the larvae of the pink bollworm, with insect pests. Augmentations of CBA artificial Pectinophora gossypiella on cotton was usually nests may become as one of the control approaches ineffective because they feed within the fruiting in managing CPB infestation due to their bodies where they are protected. The spraying may effectiveness that almost similar with insecticide not be targeted directly at the larvae, and there application. Giesberger (1983) previously reported might be a time lag between the application and the that efforts was expanded to establish CBA in effect (Wood et al. 1992). If the spraying did not cocoa farms In Indonesia, and this aroused higher reach the target, which can be the adult CPB, their interest after the CBA was proven to be a eggs can be susceptible too. Therefore, any control potentially effective biological control agent approach using direct contact must be targeted against mirid infestation. Wood and Chung (1989) either to the egg or adult stages of CPB. reported that the establishment and maintenance of CBA remains as an attractive approach in Failure of insecticides to reach targets, controlling CPB infestation, although broad- especially for cocoa pods in dense canopy may also spectrum chemicals became widely used in the lesser success rate of insecticides control. Dense world. High population of CBA on the cocoa pod canopy, especially in insecticides areas may shelter and increasing level of abundance in the field can CPB especially during daylight. Hence insecticides be indicative criteria for successful introduction of sprayed need to be targeted directly to pods, as well CBA (Way and Khoo, 1989). as the canopy. Spraying on the dense canopy may affect the adult that usually resting on the jorquette A well-managed cocoa-coconut or cocoa canopy itself. Unfortunately, spraying of environment as in this study was suitable for the insecticides throughout the study was only targeted development of strong CBA population due to the to the cocoa pods with 7-9 cm in length and above. tendency of nest making in coconut crowns. Augmentation of artificial ant nests stuffed with Prolonged pesticide usage may lead to dry cocoa leaves will help in the population built- insecticides resistant. Pesticide resistance is the up. However, nests must be replaced several times ability of an insect to survive a pesticide treatment a year. Simultaneous augmentation of colonized applied at a rate that other individuals in the pest artificial ant nests and introduction of empty ant population cannot survive (Goodel et al., 2001). nests to replace deteriorated and broken nests must This is an inherited characteristic, and the more be carried out regularly (Saripah and Azhar, 2007). insecticide applied, the more quickly susceptible It was suggested that the nests must be replaced 3-4 individuals were removed from the population. times per year and the augmentation of cocoa
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Malaysian Cocoa Journal, 8/2014 mealy bug chips must be carried out more often. REFERENCES This is to ensure sufficient food source for CBA, especially on the cocoa pod surface. CBA will Azhar, I. (1992). Progress and development of always attend to cocoa mealy bugs on the pod cocoa pod borer research in MARDI. surface, protecting the pods continually, thus MARDI Occasional Paper, 5: 1-6. preventing adult CPB from laying their eggs. For the enhancement of the CBA population in the Azhar, I. (1995). An overview on the management field, the cocoa area must be free from any of key insect pests of cocoa with major pesticide application. This was due to some emphasis on the Cocoa Pod Borer, insecticides can indirectly affect some Conopomorpha cramerella. The Planter 71 Hymenopteran species, and may cause mortality (835): 469-480. and reduce the population later on (Stapel et al, 2000). Azhar, I., Alias, A., and Meriam, M. Y. (2000). Research on the management of cocoa pod borer in Malaysia. In Bong, C. L., Lee, C. CONCLUSIONS H., & Shari F.S (Eds.), Proceedings INCOPED 3rd International Seminar on The study reveals that CPB damage was less Cocoa Pests and Diseases (pp 105-113). serious and significantly lower in the treatment Kota Kinabalu, KK: Malaysian Cocoa sprayed in the monthly insecticides area and Board (MCB) and International Permanent implementation of CBA, as compared to control Working Group for Cocoa Pests and and bimonthly insecticides. The results indicating Diseases (INCOPED). that even the spraying was conducted at bimonthly basis, the success rate was low when adjacent areas Bellinger, R. G. (1996). Pest resistance to were an abandoned cocoa plantation. CPB from pesticides. Retrieved 1 September 2012 neighboring area may fly to treat block, and reduce from the successful control of this pest. The bimonthly http://ipm.ncsu.edu/safety/factsheets/resista insecticide treatment showed higher ADSI values n.pdf compared with monthly insecticides, CBA and control treatments. The ADSI value was the lowest Croft, B. A. (1990). Arthropod biological control for CBA treatment during low crop season. Low agents and pesticides. Wiley and Sons: New infestation might occur due to the low number of York. Pp 703. available pods, thus CBA may thrive well and can cover a greater number of pods. Throughout the Dent, D. (1991). Insect Pest Management. observations, CBA was able to reduce CPB Wallingford, UK: C.A.B International. infestation, in term of good pods and ADSI values. It can be concluded that the use of CBA shows Fishel, F. M. (2011). Pesticides effects on nontarget great potential as an alternative approach in organisms. University of Florida IFAS controlling CPB, with almost similar results by Extension. http://edis.ifas.ufl.edu/pi122. insecticides. However, the CBA must be Retrieved 3 September 2012. distributed evenly on every trees and can cover almost all pods for better protection against CPB. Giesberger, D. (1983). Biological control of the Hence, augmentation of CBA should be Helopeltis pest in Java. In Toxopeus, H., & encouraged for an alternative of CPB control. Wessel, P. C. (Eds), Archieves of Cocoa research Vol. 2. Cocoa Research in Indonesia (pp. 91-180). Wageningen: ACKNOWLEDGEMENTS American Cocoa Research Institute and IOCC. The author would like to thank the Director- General of Malaysian Cocoa Board, Dr. Lee Choon Haynes, K. F. (1988). Sublethal effects of Hui for permission to publish this paper. We also neurotoxic insecticides on insect behavior. thank former Director-General of Malaysian Cocoa Annual Review of Entomology 33: 149-168. Board, Dato’ Dr. Azhar Ismail, Deputy Director General, Mr. Kelvin Lamin, Mr. Roslan Sa’adi and Hill, D. S. (1983). Agricultural insect pests of the staffs of Entomology Unit at CRDC Hilir Perak for tropics and their control. Second Edition. their technical assistance. Cambridge: Cambridge University Press.
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Sidhu, M.S., Sim, C.H., and Johney, K.V. (1987). Practical aspects of chemical spraying for cocoa pod borer management in Sabah. In Ooi, P. A. C., Luz, G. C., Khoo, K. C., Teoh, C. H., Md. Jusoh, M., Ho, C. T & Lim, G. S (Eds.), Management of the Cocoa Pod Borer (pp. 19-41). Kuala Lumpur, KL: Malaysian Plant Protection Society.
Stapel, J. O., Cortesero, A. M., and Lewis, W. J. (2000). Disruptive sublethal effects of insecticides on biological control: Altered foraging ability and life span of a parasitoid after feeding on extrafloral nectar of cotton treated with systemic insecticides. Biological Control 17: 243-249.
Way, M. J., and Khoo, K. C. (1989). Relationship between Helopeltis theobromae damage and ants with special reference to Malaysian cocoa smallholdings. Journal of Plant Protection in the tropics 6: 1-11.
Wood, B. J., Chung, G.F., Sim, S.C. and Chee, C.F. (1992). Trials on control of the cocoa pod borer Conopomorpha cramerella (Snellen) in Sabah by regular complete harvesting. Tropical Pest Management, 38(3): 271-278.
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SUSTAINING COCOA BLACK ANTS, Dolichoderus thoracicus (SMITH) USING ARTIFICIAL NEST IN THE COCOA ECOSYSTEM
Saripah, B. Malaysian Cocoa Board, 5-7th floor, Wisma SEDCO, Lorong Plaza Wawasan, Off-Coastal Highway, Locked Bag 211, 88999 Kota Kinabalu, Sabah, Malaysia Email: [email protected]
Malaysian Cocoa J. 8: 23-30 (2014) ABSTRACT - Cocoa Black Ants (CBA), Dolichoderus thoracicus was introduced as a biological control agent against Cocoa Pod Borer (CPB), Conopomorpha cramerella at Cocoa Research and Development Center (CRDC) Hilir Perak, Malaysian Cocoa Board. Introduction of artificial ant nests stuffed with dried cocoa leaves was undertaken to enhance the CBA population at four blocks in cocoa-coconut ecosystems. Each block was divided into four plots, Plot A (one nest per trees alternately, Plot B (every tree), Plot C (two nest per tree) and Plot D as control plot. Eight augmentations of artificial ant nests were carried out throughout the study at approximately three months interval. Cocoa mealy bugs (CM), Cataenococcus hispidus was also introduced by putting 2” x 3” CM chips especially on the cocoa cherelles within the trees or next to the cocoa flowers as food source for ants. The result of the trials showed that Plot C harbored strongest trails, which consisted heavy, medium and longest ground trails throughout the studies period, followed by Plot A and B. It is suggests that the density of artificial nest influence the ground trails and CBA activities on the cocoa tree.
Keywords: Cocoa, Theobroma cacao, Cocoa Black Ants, Dolichoderus thoracicus, Cocoa mealybug, Cataenococcus hispidus, Artificial ant nest
INTRODUCTION usually had relatively lower infestations by the CPB, Helopeltis and rodent pests (Azhar, 1992). Various order of insects can be found in the cocoa The presence of CBA particularly in large numbers ecosystem and the ants (Hymenoptera: Formicidae) is effective in controlling CPB infestation (Saripah are among the dominant species of insects. Forty and Azhar, 2007), but they are not distributed three species of ants were collected from the homogenously on the pods in the blocks (Azhar, plantations in Malaysia (Azhar, 1985), but a few 1995). Thus, to ensure CBA population are evenly species were dominant and play a role in limiting distributed within the block, good shelter for pest infestations. Oecophylla smaragdina, protection and food source of CBA have to be Dolichoderus thoracicus, Anoplolepis longipes and provided. Interspecies competition with other ants Crematogaster spp. were observed to have great must be removed or eliminated (Saripah and Azhar, potential in controlling cocoa pests. Among this, 2003). only Cocoa Black Ants (CBA), Dolichoderus thoracicus Smith (Hymenoptera: Formicidae) was CBA is mutually associated with the effective in controlling the losses caused by mirid, Cocoa Mealybug (CM), Cataenococcus hispidus Helopeltis theivora (Azhar, 1989), one of the major Morrison (Homoptera: Pseudococcidae). High pest in the 1980s and 1990s. Giesberger (1983) population of CBA through its mutual interactions reported the efficacy of CBA as a biological with the CM can limits the infestation of CPB. control agent for controlling mirid infestation in However, the presence of CM which provides Indonesia for more than 40 years in large scale honeydew as a food for CBA might influence its areas. effectiveness against CPB. CM sucks the sap from cocoa pods and young shoots and excretes CBA mostly found in the coastal belt of honeydew while CBA provides protection from Perak, Selangor and Johor and have a great predators or parasites, and the colonies maintain potential as a biological control agent against the hygiene (Azhar, 2000). Other factors that influence major cocoa pest in South East Asia which is the population of CBA in cocoa-coconut Cocoa Pod Borer (CBA), Conopomorpha ecosystems are optimal shade level, canopy cramerella Smith (Lepidoptera: Gracillariidae). coverage, available foraging or migration alleys for Cocoa planted under coconut usually harbors high local spread and availability of nesting sites. The ant populations and ensures the potential of CBA nests of CBA mainly formed in the coconut crown as a bio-indicator of healthy agro-ecosystems. High due to the large amount of debris found on the populations of CBA in cocoa-coconut ecosystems crown. Temporary or transient nests can be found
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Malaysian Cocoa Journal, 8/2014 in cocoa canopy with two or three leaves attached The presences of ground trails which together to perform as a nest. However, these indicated the CBA activities were recorded for the transient nests were unstable; therefore introduction period of 30 months at monthly interval. CBA and augmentation of CBA into new cocoa areas are activities on the cocoa trees were examined at fifty required. Artificial ant nests are made from plastic trees per plot and the number of CBA per foot, bags stuffed with dry cocoa leaves and hung on the number of CBA moving in and out from the cocoa trees attended by CBA. By increasing the artificial nests, CBA population and number of numbers of artificial nests, the populations of CBA transient nests were recorded fortnightly. The were enhanced (Saripah and Azhar, 2007). Ground strength of CBA colony on each plot was trails are usually found in existing heavily monitored by counting the number of CBA moving populated area of CBA, especially from cocoa trees in and out, approximately 30cm from artificial to the coconut palms because their workers are nests. Data on CBA per 30 cm were recorded by foraging for food at the coconut crowns. Therefore, counting the number of CBA in the main trail the study was conducted to evaluate the ground crossing an arbitrary line at the basal part of cocoa activities of CBA after the introduction artificial tree, approximately a foot above the ground, over a ant nests in cocoa-coconut ecosystem. period of five minutes (5-min count). The trail pattern was divided into three categories, which are light, moderate and heavy ground trails. Light trails MATERIALS AND METHODS were recorded when the number of individual CBA was approximately 200 to 500 within a foot, 500 to The study was conducted at Entomology Unit, 2,000 for moderate and more than 2,000 Cocoa Research and Development Center (CRDC), individuals for heavy ground trails. Similarly, the Malaysian Cocoa Board Hilir Perak, Malaysia dynamics of the CBA foraging pattern were (Longitude E.100 M, 52’ 0’, Latitude N3 53’ 42). monitored by recording the contraction and Four blocks of mature cocoa trees (block’s size) expansion on the ground trails every two weeks. were selected and replicated in four different plots The number and strength of the trails, as well as with coconut and Gliricidia as shade trees. Empty distance (shortest and longest) trails were also ant nests were hung at Ant Farm Source that had noted. The dynamics of CBA foraging pattern was high population of CBA. The nests were tied at the visualized by plotting the trail pattern, then jorquette and left to be colonized by CBA under transformed into illustrated figures to see the natural conditions. After five to six weeks, the ground activities of CBA which usually influenced nests were transferred to all experimental blocks. by the number of nests augmented in each plot. The Twenty-five colonized artificial ant nests were data recorded in all experiments were arranged and hung in Plot A, 50 nests in Plot B and 100 nests at pooled in Excel® program and analyzed using plot C at every nests augmentation schedules. SAS® (SAS® system for Windows® V8). Augmentations of artificial ant nests were carried out approximately every three months to replace deteriorated, broken or un-colonized artificial ant RESULTS nests caused by pruning, heavy rain and other environmental factors. Cocoa mealybug (CM) was Sixty-four series of observations on the CBA introduced in all blocks at two or three days before ground trails were recorded and five sampling first augmentation of colonized artificial ant nests observations were discussed in this paper; before using CM chips. CM chips were obtained from the augmentation of artificial nests, two weeks after ripen cocoa pod epicarp that had CM on the cocoa first introduction, after six month, after a year and pod surface. The epicarps of cocoa pod with CM after 16 months of CBA introduction. Existing were cut into approximately 2cm x 3 cm chips. trails prior to the introduction of colonized artificial Subsequently, the chips were placed on the cocoa ant nests were observed to examine CBA activities pods that were less than three months old or next to under natural condition. Observation on the ground the cocoa flowers cushion. CM chips were trails at Blocks 10B, 13B, 18B and 23B were introduced after every harvesting occasion using illustrated as shown in Figures 1 to 4. second or third instars and adult CM. The study was replicate in four different blocks.
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Plot B
INDICATOR : Cocoa tree Active nests Empty nests Coconut tree Gliricidia
Heavy trails Medium trails Light trails
Figure 1. Ground trails of CBA at Block 10B before augmentation, after 2 weeks, after 6 months, 12 months and 16 months
Before the introduction of artificial ant artificial ant nest was introduced. CBA might nests, several heavy ground trails were found at shifted from artificial ant nests to Plot D since Block 10B, which shows that the population of ground trails were observed from cocoa trees to CBA was strong in almost at all parts of the block. coconut trees. CBA from Plots A, B and C might Similar pattern of population was found at Block forage for food at the coconut crown at Plot D, due 18A, and strongest trails were recorded at plots B to the absence of chips introduced in plot and D. Light populations of CBA were recorded throughout the study period. After several months, with only few ground trails found in Blocks 13B most of the nests were occupied by CBA, therefore and 23B. A month after the first augmentation of the lesser number of ground trails were observed in nests, strong heavy ground trails were observed at almost all blocks. High number of active nests, Blocks 10B and 18B almost in all plots, except in especially in Plots B and C showed the population Plot B at Block 10B. Heavy trails were observed in of CBA increased with time, and the number of Plots C and D at Block 10B and all plots at Block nests augmentation. 18B. Interestingly, heavy ground trails were also observed at Plot D especially in the first two months after nests augmentation, although no
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Figure 2. Ground trails of CBA at Block 13B before augmentation, after 2 weeks, after 6 months, 12 months and 16 months
Figure 3. Ground trails of CBA at Block 18B before augmentation, after 2 weeks, after 6 months, 12 months and 16 months
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Figure 4. Ground trails of CBA at Block 23B before augmentation, after 2 weeks, after 6 months, 12 months and 16 months
Observation on the heavy, medium and Plot C. This suggests that low number of light ground trails were carried out at Plots A, B, C augmented nests denote the strength of the trails. and D at all blocks. Heavy ground trails were found Plot D, the control plot harbored the lowest number higher at Plot C, (Table 1) followed by Plots A, B of trails regardless to the trails strength. Longest and at the control plot (D). Similar observations ground trails found in Plot C, followed by Plots D, were recorded for medium ground trails indicated A and B which suggest that number of artificial that number of augmented artificial ant nests might nests will influenced the distance of ground trails. influenced by the number of ground trails. More The distance will increase with the number of nests artificial nests were hung at the block, more heavy hung at particular plot. Longest trails were found at and medium ground trails were observed in Plot D, proportionately to Plots A and B. Plot C respective plot. Different result were found for harbored shortest ground trails, followed by Plots light ground trails, which Plot A with one nest per D, B and A. tree harbored the most trails, proportionately to
Table 1. Ground trails activities at Plots A, B, C and D
Plot Ground Trails Activities (Mean) Heavy Medium Light Longest Shortest A 6.010a 2.055a 5.251a 6.725a 1.375a B 3.930a 1.501a 3.100a 6.200a 1.050a C 7.052a 2.207a 2.805a 8.700a 0.550a D 2.859a 1.355a 1.210a 8.025a 0.800a Means with the same letter at the same vertical rows are not significantly different at P ≥ 0.05
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Observations on ground trails throughout at all plots. For medium ground trails, it was found the sampling period are shown in Table 2. Heavy that CBA were actively foraging after six months and light ground trails were highest at sampling 2, introduction. Longest ground trails were highest which was two weeks after first augmentation date. after two weeks of CBA augmentation, The CBA were actively roaming and foraging for unfortunately getting decreased in the third, fourth food outside the nests after two weeks of the and fifth observations. Similar trends were introduction. This might be due to insufficient food recorded for shortest ground trails, with the shortest source provided by CM for these two weeks. Lack trails in the fifth observation. The results showed of food source will encourage CBA to forage for that trail numbers and trail strength decrease over food outside the nests especially on the coconut period of time. crowns. Therefore, ground trails easily can be seen
Table 2. Ground trails activities at different sampling occasions; 1 (before augmentation), 2 (two weeks after first nests augmentation), 3 (after six months), 4 (after 12 months) and 5 (after 16 months) Sampling Ground Trails Activities (Mean) Heavy Medium Light Longest Shortest 1 7.438a 0.000a 0.875a 12.594ab 2.063a 2 9.750a 2.751a 5.063a 15.375a 1.500ab 3 6.500a 3.252a 4.938a 5.469ab 0.719ab 4 0.688a 1.938a 3.000a 1.875ab 0.250ab 5 0.375a 0.938a 1.563a 1.750ab 0.188ab Means with the same letter at the same vertical rows are not significantly different at P ≥ 0.05
The number of CBA per foot varied Relationship between CBA populations, numbers between plot, with highest number recorded at Plot of CBA moving in and out from artificial nests C, followed by Plots B, A and D (Table 3). Plot C were observed in all plots throughout the studies harbored highest number CBA that moved from (Figure 5). The fluctuating trend of the ants artificial nests. The number of CBA moving in at movement at Plot C, reflected diverse activities Plots A and B were insignificant between the plots, recorded at this plot compared with other plots. and the number of CBA moving out did not Passive activities can be seen at Plot D and might significantly different between Plots A, B and C. be attribute by low number of individual CBA recorded at this plot.
Table 3. Activities of CBA; Number of CBA per foot, number of CBA moving in and moving out from the artificial ant nests in five minutes, CBA population and number of transient nests CBA activities Plot Number of Number of Number of CBA Number of CBA CBA moving CBA moving population transient nests per foot in out A 13.618c 49.575b 57.802a 1.930c 2.877c B 14.389b 53.512b 63.644a 2.029b 2.945b C 16.496a 16.918a 84.926a 2.139a 3.099a D 5.295d 14.110c 0.081b 0.894d 1.216d * Means with the same letter at the same vertical rows are not significantly different at P ≥ 0.05
Figure 5. Relationship between CBA population, CBA moving in and moving out from artificial nests counted for 5 minutes at Plots A, B, C and D
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Similar results were recorded for number months of artificial nests augmentation. However, of transient nests, which are the temporary nests the number of nests were in a range of 10 to 15 that usually built up in the cocoa canopy. Transient throughout the sampling period, similar results nests in the cocoa canopy are common features and found at Plot B. Plot D harbored lowest transient the numbers were recorded highest at Plot C nests with usually less than 10 nests been observed (Figure 6). The number of transient nests at Plot C in every sampling occasion. was more than 15 nests especially in the first three
Figure 6. Number of transient nests recorded at Plot A, B, C and D
DISCUSSION augmentation assisted in population built-up, although some of the nests harbored low number of Introduction of colonized artificial ant nests can CBA. Replacing the nests from tree with low CBA increase the number of CBA per trees especially in population to high CBA population contributed in areas with low CBA population. This findings was increasing the population. The studies found that supported by Saripah and Azhar’s (2007) studies. introduction of two nests per individual trees will Continuous augmentation of colonized artificial successful in strengthening the population of CBA nests influenced the population hence, ensures at cocoa-coconut ecosystem and it was supported successful control of CPB especially in heavily by Azhar et al. (2000) studies. infested areas. The sustainability of CBA population throughout the study period was Under natural condition, the ground trails influenced by their feasibility, availability of the can be seen among the cocoa trees and coconut nesting sites and more migrating alleys from fallen trees. The sustainability of CBA population seems coconut fronds in cocoa-coconut ecosystem. to be influenced by the availability of the nesting However, introduction an artificial ant nests sites and migrating alleys from fallen coconut resulting in decreasing of ground trails in all plots. fronds. However, it is very interesting to note that Two main factors contributed to this observation, the numbers of ground trails decreased when the i.e. the availability of nesting sites and sufficient artificial ant nests were introduced. This may food source provided by CM. Introducing CM suggest that the ants will occupied and colonized chips at regular basis on the cocoa pods will nesting sites when available. In the first sampling increased the CBA population. Sufficient occasions, heavy trails which leading to the honeydew source influenced CBA to shift their coconut crowns were observed in Blocks 10B and foraging activities from coconut crown to the CM 18A. Foraging for foods especially on the coconut at the cocoa pods, leading to a decrease in the crowns may lead to the strength of the trails found ground trails. on the ground. CBA may also built up nests with dry cocoa leaves on the ground. After two weeks of It also known that artificial ant nests is nests augmentation, heavy ground trails were very useful in enhancing the population of CBA in visible at Blocks 10B and 18A. Availability of cocoa-coconut ecosystems (Azhar, 1989). nesting sites may also contribute to the decrease in Therefore, colonized artificial ant nests increase the ground trails especially on the third, fourth and number of CBA per trees especially in low existing fifth observations. CBA were quickly colonized the CBA block (Saripah and Azhar, 2007), nests and subsequently influence passive activities subsequently enhance CBA population. On the on the ground. CBA were only need to move from other hand, augmentations of empty artificial ant the artificial nests to the cocoa pods at the same nest in the area which have high density of ants trees for a food source. Therefore, it limits their will sustained its population. Numbers of nests activities on the ground. Nik Rahayu (2001) augmented in the study also play a role in limiting reported that CBA were found colonized artificial the activities outside the nests. Continuous ant nests as early from the first week the nest been
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Malaysian Cocoa Journal, 8/2014 augmented. The results might be similar with this Conopomorpha cramerella. The Planter. 71 study, especially on the decreasing number of (835): 469-480. ground trails. Azhar, I (2000). Cocoa Black Ant (CBA) as a bioindicator of the cocoa and coconut CONCLUSION agroecosystem health measured indirectly through the level of Cocoa Pod Borer The number of artificial ant nests affect CBA infestation. Proceedings of 13th International activity, and continuous augmentation of the Cocoa Research Conference. 9-14 October artificial nests and CM will sustain the CBA 2000. Kota Kinabalu: Cocoa Producers population. Availability of nests for shelter and Alliance. Pp 437-446. protection influence a more uniform and aggregated activities at the cocoa trees, especially Azhar, I., Nur Samhanim A.J. and Hasan, S.T.S. on the cocoa pods. Longevity of the CBA on the (2000). Variations in colony strength and cocoa pods rather than on the ground indirectly local foraging pattern of the Cocoa Black deters CPB from laying their eggs at the pod Ant, Dolichoderus thoracicus, in cocoa- surface, thus reduce the infestation later on. coconut ecosystem. Proceedings of INCOPED 3rd International Seminar. 16-17 October 2000. Kota Kinabalu: Malaysian ACKNOWLEDGEMENTS Cocoa Board, Pp 33-43.
I thank Dr. Lee Choon Hui, Director General of Giesberger, D. (1983). Biological control of the Malaysian Cocoa Board; Dato’ Dr. Azhar Ismail, Helopeltis pest in Java. In Archieves of Cocoa former Director General; Mr. Kelvin Lamin, research Vol. 2. Cocoa Research in Indonesia former Deputy Director General; Mr. Haya Ramba, (Toxopeus, H. and Wessel, P.C., ed). Director of Upstream Technology Division, Mr. Wageningen: American Cocoa Research Roslan Saa’di on the illustrated figures, and staffs Institute and IOCC. of Entomology Unit, Cocoa Research and Development Centre (CRDC) Hilir Perak, Nik Rahayu, N.M. (2001). Penentuan kekuatan Malaysian Cocoa Board for the data collections. koloni semut hitam koko, Dolichoderus thoracicus (Smith) (Hymenoptera: Formicidae) menggunakan kaedah akustik di REFERENCES ladang koko. Bachelor of Science (Biology) thesis. University Putra Malaysia,Selangor, Azhar, I. (1985). Natural and cultural control in the Malaysia. Pp 68. management of selected cocoa pests. In Integrated Pest Management in Malaysia Saripah, B. and Azhar, I. (2007). Handling of (Lee, B. S., Loke, W. H., and Heong, K. L., Cocoa Black Ants as a biological control eds). Kuala Lumpur: MAPPS. Pp 255-267. agent against Cocoa Pod Borer in monococoa ecosystem. Proceedings 2007 Conference on Azhar, I. (1989). Towards the development of Plantation Commodities, 3-4 July 2007. integrated pest management of Helopeltis in Kuala Lumpur: Malaysian Cocoa Board. Pp Malaysia. MARDI Research Journal. 17: 55- 48-54. 68. Saripah, B. and Azhar, I. (2003). Penggunaan Azhar, I. (1992). Role of Black Ants in Cocoa Pod Semut Hitam Koko Dolichoderus thoracicus Borer natural control. MAPPS Newletters Smith sebagai agen untuk pengawalan Ulat 16(4): 36-37. Pengorek Buah Koko Conopomorpha cramerella Snellen. Prosiding Bengkel Azhar, I. (1995). An overview on the management Perlindungan Tanaman Koko 2003, 20-21 of key insect pests of cocoa with major October 2003. Tawau: Malaysian Cocoa emphasis on the Cocoa Pod Borer, Board. Pp 17-30.
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ISOLATION OF DNA AND RNA FROM COCOA POD BORER, Conopomorpha cramerella (Snellen) AND CLONING OF ITS GENES
1Goh, L.P.W., 1Chia, Y.C., 2Tan, C.L. 1Fakulti Sains & Sumber Alam, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia. 2Malaysian Cocoa Board, Commercial Zone 1, Norowot Road, 88460 Kota Kinabalu Industrial Park, Sabah, Malaysia.
Malaysian Cocoa J. 8: 31 – 39 (2014) ABSTRACT - Cocoa pod borer (CPB) is a major pest in cocoa cultivation in South-east Asia. It can caused complete crop loss if not control. Despite its importance in cocoa cultivation, it has not been studied extensively, particularly at the molecular and cellular level. This study constitute the first report of isolation of DNA and RNA from cocoa pod borer, Conopomorpha cramerella (Snellen) and cloning of its genes. Two genes essential for growth and morphological development of the insect were cloned, namely aminopeptidase N and chitin synthase. Characterisation of these genes will aid in the development of RNA interference for the control of CPB.
Keywords: Cocoa pod borer, Conopomorpha cramerella, DNA isolation, Aminopeptidase N, Chitin synthase
INTRODUCTION aimed to construct a double-stranded small- interfering RNA (siRNA) of several target genes Total world cocoa production has increased by including acetylcholinesterase, aminopeptidase-N, 13%, from 4.3 million metric tons in 2008 to 4.8 chitin synthase, cytochrome P450, glutathione S- million metric tons in 2012 (WCF, 2014). This transferase and pheromone-binding protein) based represents an average year-over-year production on the cDNA sequences of CPB. The siRNA will increase of 3.1%. This rate of increase may slow be used for RNA interference (RNAi)–mediated in the coming years, as cocoa trees are sensitive to gene knockdown in CPB. The introduced dsRNA changing weather patterns. Periods of drought and can be highly specific to CPB, this approach limits of excessive rain or wind can negatively impact the adverse effects on non-target organisms. yield, and will continue to fluctuate as climate change intensifies. When the gene that encodes the resistance towards certain pesticides or are essential to the Malaysia represents approximately 0.42% growth and development of CPB is knocked down, (20 thousand tons) of cocoa beans production the CPB can be killed easily or suffered from among the world (4.8 million metric tons in 2012). redundant growth. As reported by Bautista et al. This indicates that the production of cocoa beans (2009), when the diamond back moth, Plutella play an important role in the economy of Malaysia. xylostella was fed with synthetic dsRNA against However, there are many problems influencing the CYP6BG1, expression of the P450 gene was production of cocoa beans, including natural suppressed, resulting in reduced larval resistance to disasters, diseases, pests, and many others. An the insecticide, permethrin. RNAi-based gene insect pest, cocoa pod borer (CPB), regulation has been reported in different insect Conopomorpha cramerella is a major problem that orders, including Lepidoptera, Hemiptera, affects the production of cocoa beans in Malaysia. Coleopteran, Diptera, and Hymenoptera (Huvenne The average bean losses in Malaysia due to CPB and Smagghe, 2010). Although RNAi technology were estimated to be at least 15% of annual showed promising results in several studies but potential yields. none have been conducted specifically to target CPB. Thus, this project could serve as a The problem caused by CPB in cocoa preliminary stage in the development of RNAi- field could be solved in many ways. The use of based gene knockdown in CPB. insecticide is the most common approach. Many pesticides used today are broad-spectrum, capable of killing many species including beneficial MATERIAL AND METHOD species. Furthermore, CPB might develop insecticide resistance over time. Therefore, a Materials highly specific targeted approach to overcome this Cocoa pod borer infected pods were collected from problem is extremely needed. Thus, this project is Department of Agriculture, Tuaran, Sabah. The
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pods were wrapped in papers and stored in the of APN1, APN2, APN3 & GST) into the dark. The pods were checked daily for CPB larva pGEM®-T easy vector as shown in Table 3 in that emerged. Larva collected were then immersed a PCR tube then incubated at 4ºC overnight in in RNA Later (Ambion) and stored in -20oC a thermal cycler (BioRad, US). The ligation freezer for later use. products were then subjected to transformation into competent cells. DNA isolation Transformation of the ligation products into About 100 mg of sample was homogenized in 400 competent cells μl of DIGSOL reagent (20 mmol/L edetic acid There are two methods to transform the [EDTA], 50 mmol/L Tris (pH 7.5), 0.4 mol/L NaCl, ligation products into the competent cells, 0.5% sodium dodecyl sulfate [SDS]). Ten including heat-shock method and microliter of proteinase K was added to degrade the electroporation. For heat-shock transformation, proteins in the sample. Subsequently, the sample JM109 or NEB high efficiency competent cells was incubated overnight at 37ºC. The following were used whereas for transformation using day, 100 μl of 5M NaCl was added to the sample electroporation, electrocompetent cells were and incubated for 30 minutes at room temperature. used. Then 500 μl of phenol: chloroform: isopropanol was added into the sample. The sample was then Heat-shock transformation and plating centrifuged at 13,000 rpm for 15 minutes. The supernatant was the transferred into a fresh tube and The JM109 high efficiency competent cells (≥ 450 μl of 100% ethanol was added to precipitate the 1 x 108 cfu/µg) were obtained from -80ºC DNA. The samples were centrifuged again at freezer then put in an ice bath for thawing. 13,000 rpm for 15 minutes. The supernatant was About 2 µl of ligation products was transferred discarded and the DNA pellet was washed with 500 into a microcentrifuge tube containing 50 µl of μl of 70% ethanol and centrifuged for 5 minutes at JM109 high efficiency competent cells. The 13,000 rpm. The supernatant was discarded and the tube containing the mixture was incubated on pellet was air dried. Finally the pellet was re- ice for 20 minutes. After that, heat-shock was suspended in 30 μl TE buffer with RNase. The carried out by placing the tube in 42ºC extracted DNAs were then resolved with 1% waterbath for 50 seconds followed by agarose gel electrophoresis. immediately returning the tube onto ice for 2 minutes. Next, 950 µl of LB medium was Amplification and gel extraction added into the tube and incubated at 37ºC with Polymerase chain reaction (PCR) was performed to shaking (≈ 150 rpm) for 1.5 hours. For each amplify the acetylcholinesterase (Ache), LB agar/ampicillin plates, 100 µl of Aminopeptidase-N (APN), chitin synthase (CHS), transformation culture was placed onto the cytochrome P450 (P450), glutathione S-transferase plate by spreading followed by incubating (GST) and pheromone-binding protein (PBP) genes. overnight at 37ºC. The PCR mixture was prepared as shown in Table 1 and the PCR conditions were shown in Table 2 for Colony PCR for ampicillin-resistant colonies all sets of primers. and broth culture Colony PCR was carried out for colonies that After the amplification of these genes, 1% grow on the LB agar/ampicillin plate. The agarose gel electrophoresis was performed to check colony PCR mixture was mixed (as in Table 1) the quality of the PCR products. The presence of with the exception of the template that was band(s) indicated that PCR amplification was replaced by colonies and subjected to similar performed successfully. The PCR products were cycling condition. then extracted from the agarose gel using silica beads DNA extraction kit (Fermentas). After the PCR amplification, 1% agarose gel electrophoresis was performed and Ligation of the vector and insert each colony suspected of carrying the cloned The pGEM®-T Easy Vector (Promega, USA) gene (APN3 and GST) were then cultured in was a convenient systems for TA cloning of about 5 ml of LB/ampicillin broth in Falcon PCR products. A ligation mixture was tube followed by incubation at 37ºC overnight performed to ligase the insert (PCR products with shaking (≈ 150 rpm).
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Table 1. PCR mixtures for the amplification of CPB genes
Reagent/Solution Working Concentration Volume DNA/cDNA Template ≈ 100 ng/µl 1.0 µl Reverse primer 0.5 µM 4.0 µl Forward primer 0.5 µM 4.0 µl dNTPs mixture 0.2 mM 0.4 µl Taq DNA polymerase 1 U/µl 1.0 µl Standard Taq buffer 1X 2.0 µl
sdH2O 6.4 µl TOTAL 20.0 µl
Table 2. PCR conditions for the amplification of Ache, APN, CHS, P450, GST and PBP genes
Steps Temperature Duration Cycle(s) Activation 95ºC 3 minutes 1 Denaturation 95ºC 60 seconds 35 Annealing 56ºC 60 seconds Extension 72ºC 60 seconds Final extension 72ºC 10 minutes 1 Soak 20ºC ∞ -
Table 3 Ligation mixtures for GST and APN 1-3 PCR products
Component Reaction Volume 2X Rapid Ligation Buffer, T4 DNA Ligase 5.0 µl pGEM®-T Easy Vector 1.0 µl PCR product X µl* T4 DNA Ligase 1.0 µl sdH2O Y µl Final volume 10.0 µl
*Molar ratio of PCR product : vector of 3:1 to 1:3.
Table 4. Restriction enzyme digestion mixture for extracted plasmid DNA
Reagent/Solution Working Concentration Volume Extracted plasmid DNA ≈ 35 ng/µl 4.0 µl NEBuffer 3 1X 1.5 µl BSA 100ug/ml 0.15 µl NotI Restriction Enzyme 2 U/µl 2.0 µl sdH2O 7.35 µl TOTAL 15.0 µl
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Plasmid DNA extraction by Alkaline Lysis NotI restriction enzyme (NEB, UK). Table 4 Method shows the restriction enzyme digestion mixture This protocol is modified from Birnboim and for extracted plasmid DNA and Figure 7 Doly (1979) and Ish-Horowicz & Burke shows the cutting site for NotI restriction (1981). In this protocol, all the centrifugation enzyme. was performed at 12, 000 rpm at 4ºC in a microcentrifuge. Firstly, about 1.5 ml of the c. DNA sequencing and BLAST analysis broth culture was pipette into a The cloned DNA was sequenced at First Base Sdn. microcentrifuge tube then centrifuged for 5 Bhd., Sri Kembangan, Selangor. Similarities of minutes followed by decanting the cocoa pod borer genes with other insects were supernatant. This step was repeated twice. The obtained using Basic Local Alignment Search Tool bacterial pellet was re-suspended in 100 µl of (BLAST) (http://www.nbci..nlm.nih.gov/BLAST). ice-cold Solution I by vortex. Then, about 200 µl of freshly prepared Solution II was added into the mixture and the contents were mixed RESULTS AND DISCUSSION by inverting the tube a few times. Approximately 150 µl of ice-cold Solution III Isolation of CPB DNA was added and mixed by vortex followed by DNA isolated was subjected to 1% agarose gel incubation on ice for 3-5 minutes. electrophoresis to observe the quality of the Centrifugation was carried out for 5 minutes isolated DNA. As observed in Figure 1, the bright and the supernatant was transferred into a new bands above 10,000 bp of the 1kb DNA ladder tube. An equal volume of phenol:chloroform indicated the presence of DNA in all lanes with the was added then centrifuged for 2 minutes. The presence of slight amount of RNA. The isolated upper layer of the mixture was transferred into DNA will be used in the subsequent studies. a new tube and 2 volumes of 100% ethanol were added and the tube was incubated at Isolation of CPB RNA room temperature for 2 minutes. After that, the RNA extraction from CPB was performed using tube was centrifuged for 5 minutes and decant. TRIzol® reagent (Life Technologies, Carlifornia) The pellet was resuspended with 1 ml of 70% according to manufacturer instructions. The ethanol then the supernatant was removed after isolated RNA was electrophorized in a 1.2% centrifugation for 5 minutes followed by air- denaturing agarose gel to observe its’ quality drying the pellet for 10 minutes. Lastly, the (Figure 2). Two distinct bands of 28S and 18S pellet was resuspended in 50 µl of TE buffer were observed with minimal degradation of the (pH 8.0) containing DNase-free RNase (20 RNA. Then, reverse transcription was carried out µg/ml) and stored at -20ºC for subsequent with random hexamers and oligo dT primers as analysis. provided in ThermoScientific RevertAid H Minus First Strand cDNA Synthesis Kit (ThermosFisher Determination of extracted plasmid DNA Scientific, USA) to generate cDNA. Three methods were used to determine whether the extracted plasmid DNA contained the cloned gene. These are PCR amplification, RE digestion, and DNA sequencing. a. PCR amplification for extracted plasmid DNA PCR amplification is one of the ways to check the extracted plasmid DNA containing the gene or not. Method as in Table 2 was used for the PCR amplification. However, the extracted plasmid DNA will be used as template rather than the colonies. b. Restriction enzyme digestion for extracted plasmid DNA Another method to determine whether the extracted plasmid DNA contains the cloned gene is by restriction enzyme digestion using
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M 1 2 3
Isolated DNA
28S
18S
Figure 1 A 1% agarose gel electrophoresis of Figure 2 A 1.2% denaturing agarose gel isolated DNA. M: 1kb DNA Ladder (NEB, electrophoresis. Lane 1 & 2 was loaded with UK); Lane 1 - 3: Sample 1 -3. RNA Sample 1 and Sample 2, respectively.
Polymerase Chain Reaction cloning of optimization is required as the primer sets used CPB genes are degenerate primers. The amplified PCR In this project, only GST and APN were amplified products were extracted and labelled accordingly using genomic DNA (gDNA) and analyzed. No as GST 1 (≈ 700bp), APN 1 (≈ 1800bp), APN 2 (≈ amplification was observed for all primer sets 1500bp) and APN 3 (≈ 700bp) (Figure 3 and 4). when using cDNA (data not shown). Further
Figure 3. PCR amplification of APN gene at different Figure 4. PCR amplification of GST gene annealing temperature (51oC – 56oC). Lane M: 1kb annealing temperature (51oC – 56oC). Lane M: DNA ladder (NEB, UK); Lane 1 – 5 : gDNA amplified 1kb DNA ladder (NEB, UK); Lane 1 – 5 : gDNA except lane 2. amplified.
Transformation of CPB genes into cloning vector Colonies were observed in APN3 and GST plates only (Figure 5), and were subjected to colony PCR for confirmation. APN1 and APN2 were unable to yield any transformants. This might be due to the inefficient ligation of the vector and insert, or the transformation process is inadequate (Figure 6).
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Figure 6. A 1% agarose gel electrophoresis of Figure 5. GST transformants plating on LB PCR products. Lane M: 2-Log DNA ladder agar with ampicillin (100ug/ml). (NEB, UK); Lane 1: APN3; Lane 2 & 3: GST (Colony 4 & 5).
Confirmation of cloned CPB genes PCR Amplification for Extracted Plasmid Restriction enzyme digestion for extracted DNA plasmid The digested products were subjected to 1% The PCR amplification revealed a band of agarose gel electrophoresis for analysis. An approximately 700bp in all lanes, indicating approximately 700bp band was observed that the inserts (APN3 and GST) were (Figure 8), indicates the insertion sequence ligated successfully to the vector (Figure 7) is as expected.
2000 bp
1500 bp
1000 bp 700 bp 500 bp
Figure 7. Plasmid map of pGEM®-T Figure 8. A 1% Agarose gel electrophoresis of digested Easy Vector. plasmids. Lane M: 1kb DNA ladder (NEB, UK), Lane 1: APN3, Lane 2 and Lane 3: GST (Colony 4 & 5)
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DNA sequencing and BLAST analysis database on NCBI (BLAST). It was found to have 86% identity with lepidopteran Aminopeptidase-N insects Trichoplusia ni aminopeptidase-N3a, Aminopeptidases-N (APNs), the receptors 84% identity with Mamestra configurata of Bacillus thuringiensis (Bt) toxin in the aminopeptidase-N and 80% identity with lepidopteran midgut, are involved in the Bt Plutella xylostella aminopeptidase-N (Table pathogen infection mechanism (Lin et al. 5). For the translated protein sequence, it 2014). Bacillus thuringiensis (Bt) crystal was found to have 58% identity with proteins (Cry) bind to aminopeptidase-N lepidopteran insects Epiphyas postvittana receptors on insect midgut membrane aminopeptidase-N, 58% identity with leading to pore formation and subsequent Ostrinia nubilalis aminopeptidase-N3c and death (Ningshen et al., 2013). In our study, 60% identity with Manduca sexta cocoa pod borer aminopeptidase-N gene aminopeptidase-N (Table 6). sequence obtained was searched with the
Table 5. BLAST search of CPB aminopeptidase-N gene sequence
Accession Description Identity
AY836581.1 Trichoplusia ni aminopeptidase-N3a 86%
HM357836.1 Mamestra configurata aminopeptidase-N 84%
GU213042.1 Plutella xylostella aminopeptidase-N 80%
Table 6. BLAST search of CPB aminopeptidase-N translated protein sequence
Accession Description Identity AAF99701.1 Epiphyas postvittana aminopeptidase-N 58% AEO12697.1 Ostrinia nubilalis aminopeptidase-N3c 58% AAP33526.1 Manduca sexta aminopeptidase-N 60%
Chitin synthase database on NCBI (BLAST). It was found Chitin synthase is the key regulatory to have 81% identity with lepidopteran enzyme for chitin synthesis and excretion in insects Ostrinia furnacalis chitin synthase, insects, as well as a specific target of 75% identity with Helicoverpa zea chitin insecticides (Zhuo et al., 2014). It is an synthase and 87% identity with Plutella epidermis-specific expressed gene during xylostella chitin synthase (Table 7). For the the molting stage. Knockdown of chitin translated protein sequence, it was found to synthase gene in the larvae increased the have 78% identity with lepidopteran insects number of non-molting and abnormal Helicoverpa zea chitin synthase, 79% molting larvae. It is a potential target for identity with Ostrinia furnacalis chitin eco-friendly insecticides (Chen et al., 2013). synthase and 77% identity with Helicoverpa Cocoa pod borer chitin synthase gene armigera chitin synthase (Table 8). sequence obtained was searched with the
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Table 7. BLAST search of CPB chitin synthase gene sequence
Accession Description Identity
EU563936 Ostrinia furnacalis chitin synthase 81%
AF229157 Helicoverpa zea chitin synthase 75%
AB271784 Plutella xylostella chitin synthase 87%
Table 8. BLAST search of CPB chitin synthase translated protein sequence
Accession Description Identity AAG09738 Helicoverpa zea chitin synthase 78% Ostrinia furnacalis chitin synthase 79% ACB13821 Helicoverpa armigera chitin synthase 77% AFP50676
CONCLUSION Birnboim, H.C. and Doly, J. (1979). A rapid alkaline extraction procedure for DNA and RNA isolation is the initial step to screening recombinant plasmid DNA. molecular biology study of CPB genes and its Nucleic Acids Res. 24;7(6): 1513-23. functions. The study isolate and characterised two essential growth genes in CPB; aminopeptidase-N and chitin synthase genes. Chen, L., Yang, W.J., Cong, L., Xu, K.K. and This serve as a preliminary step towards using Wang, J.J. (2013). Molecular cloning, RNAi in the control of CPB in cocoa characterization and mRNA cultivation. expression of a chitin synthase 2 gene from the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae). Int. ACKNOWLEDGEMENT J. Mol. Sci. 14(8): 17055-72.
The authors would like to thank the Director- Huvenne, H. and Smagghe, G. (2010). General of the Malaysian Cocoa Board and the Mechanisms of dsRNA uptake in Director of Biotechnology for permission to insects and potential of RNAi for pest publish this paper. control: a review. J. Insect Physiol. 56: 227–235.
REFERENCES Ish-Horowicz, D. and Burke, J.F. (1981). Rapid and efficient cosmid cloning. Bautista, M.A., Miyata, T., Miura, K. and Nucleic Acids Res. 9(13): 2989–2998. Tanaka, T. (2009). RNA interference- mediated knock down of a Lin, P., Cheng, T., Jin, S., Jiang, L., Wang, cytochrome P450, CYP6BG1, from C. and Xia, Q. (2014). Structural, the diamondback moth, Plutella evolutionary and functional analysis xylostella, reduces larval resistance to of APN genes in the Lepidoptera permethrin. Insect Biochem Mol Biol Bombyx mori. Gene 535(2): 303-11. 39: 38–46.
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Ningshen, T.J., Chaitanya, R.K., Hari, P.P., janata. Comp Biochem Physiol B Vimala Devi, P.S. and Dutta- Biochem Mol Biol. 166: 194-202. Gupta,A.(2013). Characterization and WCF (2014). Cocoa Market Update. World Cocoa regulation of Bacillus Foudation, April 2014, 1-11. thuringiensis Cry toxin binding aminopeptidases N (APNs) from non- Zhuo, W., Fang, Y., Kong, L., Li, X., Sima, Y. and gut visceral tissues, Malpighian Xu, S. (2014). Chitin synthase A: a novel tubule and salivary gland: epidermal development regulation gene in Comparison with midgut- the larvae of Bombyx mori. Mol. Biol Rep. specific APN in the moth Achaea 41(7): 4177-86.
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IDENTIFICATION OF GENETIC MARKERS FOR POD BORER RESISTANCE BY SEQUENCING 14 COCOA GENOMES
Roslina, M.S.1, Rosmin, K.1, Lea, J.1, Navies, M.2, Nor Aisyah3, Sumayyah, A.K.3, Zafirah, Z.3, Fahmie, W.3 and Larry Croft3 1Malaysia Cocoa Board, Commercial Zone 1, Norowot Road, Kota Kinabalu Industrial Park, 88460 Kota Kinabalu, Sabah, Malaysia 2Malaysian Cocoa Board, Centre of Cocoa Research and Development, Mile 10, Apas Road, P.O. Box 60237, 91012, Tawau, Sabah 3Malaysian Genomics Resource Centre Berhad (MGRC), 27-9 Level 9, Signature Office, Bandar Mid Valley, 59200 Kuala Lumpur
Malaysian Cocoa J. 8: 40 – 48 (2014) Abstract - We have sequenced and de-novo assembled 14 cocoa genomes, 7 susceptible to pod borers and 7 resistant varieties. Comparison of the genomes revealed multi megabase regions deleted and inserted between varieties. Six million single nucleotide polymorphisms (SNPs) were identified across the genomes. Statistical analysis of SNP distributions between the resistant and susceptible groups identified 6 SNPs that were enriched in the resistant group compared to the susceptible group. Though the SNPs were independently tested, they are tightly genomically clustered in 2 haplotype blocks of 3 SNPs each. Both blocks are statistically significant and the most significant block contains a gene known to regulate plant structure. SNPs in this region are likely to make excellent breeding markers for pod borer resistance.
Keywords : Theobroma cacao L., Conopomorpha cramerella, Cocoa pod borer
INTRODUCTION initial breeding population (Argout et al., 2011; Feltus et al., 2011). The immediate priority of the Cocoa Genome Program is to assist cocoa breeders in identifying Cocoa pod borers (CPB) are moth larvae seedlings that that are most likely to mature into high (Conopomorpha cramerella) which feed on cocoa yielders, possess large bean sizes, good cocoa pods. In Malaysia, they are the primary limitation on flavour and resistance to common pest and diseases the cocoa industry (and can destroy 50% of a crop) using Single Nucleotide Polymorphism (SNP) (Day, 1985). Given the genetic diversity of cocoa and markers. In the case of Malaysia, cocoa genomics the diversity of resistance to pod borers, a search for research is in the preliminary stage which includes genetic markers of resistance seemed feasible (Utro et screening for desirable traits in the germplasm al., 2012; Motamayor et al. 2013). While pod borer collection and development of molecular markers. resistance is a quantitative trait, with varying amounts Breeding populations are currently being established of borer activity in different varieties, the trait can to select clones for desirable traits using conventional also be classified in a binary fashion as “resistant” breeding with molecular tools (Argout et al., 2008; and “susceptible”. A rigorous search for quantitative Kane et al., 2012). Such advanced breeding trait loci would require genetic information from techniques have been successful in low high value upward of 300 plants. By choosing a binary trait, a crops, and it is clear the economics of this approach small number of plants can be used, though greater for high value crops such as cocoa are exceptional. sequencing depth is necessary to identify enough The availability of sequenced cocoa genomes across markers throughout the genome to be able to identify many breeding varieties will create millions of resistance loci. molecular markers directly useful for future breeding improvements (Motamayor et al., 2013). Although critically important, yield, black pod resistant [BP-R], and vascular streak dieback The challenge is to establish superior resistant [VSD-R] are of secondary importance for planting materials that are resistant to pest and the time being. The objective of this study was to disease, and also exhibit high yields, good cocoa sequence complete genome of seven (7) resistant and flavour and high cocoa butter (LoorSolorzano et al., seven (7) susceptible cocoa clones to CPB and to 2012; Motamayor et al., 2013). The focus of the generate enough SNPs to give a good chance of Malaysian Cocoa Genome Programme is to carry out identifying loci implicated in pod borer resistance. genome wide association studies on sets of genetically diverse clones with contrasting traits in order to identify biomarkers for superior traits. The MATERIALS AND METHODS biomarkers will serve as diagnostic tools for screening seedlings with the desirable traits that are Fourteen cocoa leaf samples were received and more likely to mature into superior trees than the extracted with Genomic DNA Plant Kit (Macherey- Nagel, Germany) and the DNeasy Plant Mini Kit 40
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(QIAGEN, Germany). The extracted DNA was then samples were also run on 1% agarose gel to subjected to quality assessment to check the quality, determine the integrity of genomic DNA. Typically, quantity, and integrity of the DNA. The DNA quality at least 1 μg of high quality DNA (as measured by and quantity were measured using NanoDrop 2000 Qubit) is required for TruSeq library preparation. The (Thermo Fisher Scientific Inc., USA) and Qubit 2.0 final sample QC results of the samples selected for DNA Broad Range Assay (Invitrogen, USA). The sequencing are shown in Table 1 and Figure 1.
Table 1. Quality check results of cocoa leaf genomic DNA.
Nanodrop Qubit DNA BR
Sample Vol Conc Total A260/280 A260/23 Reading (ug/ml) Conc Total Name (ul) (ng/ul) (ug) 0 (ng/ul) (ug) C0923 200 33.3 6.66 1.72 1.05 0.164 32.8 6.56 C0924 200 35.1 7.02 1.86 1.95 0.151 30.2 6.04 C0925 50 71.4 3.57 1.67 1.08 0.098 19.6 1.96 C0926 50 65.5 3.28 1.60 0.86 0.230 46.0 2.30 C0927 200 59.6 11.92 1.91 2.01 0.240 48.0 9.60 C0928 50 140.8 7.04 1.81 1.99 0.485 97.0 4.85 C0929 200 26.0 5.20 1.99 2.00 0.108 21.6 4.32 C0930 20 182.8 3.66 1.81 2.01 0.625 125.0 2.50 C0931 60 44.4 2.66 1.92 2.93 0.130 26.0 1.56 C0932 60 11.2 0.67 2.04 2.76 0.041 8.2 0.49 C0933 60 37.1 2.23 1.84 2.30 0.106 21.2 1.27 C0934 30 146.6 4.40 1.82 1.90 0.450 90.0 2.70 C0935 30 229.8 6.89 1.83 2.14 0.802 160.0 4.80 C0936 50 71.9 3.60 1.85 2.03 0.264 52.8 2.64
Figure 1. Gel electrophoresis image of cocoa samples ran on 1% agarose gel, stained with EtBr, Genomic DNA seems intact and passed QC.
Genomic DNA was fragmented using Chip (Agilent, USA) (Table 2). The resulting library Covaris S220 (Covaris Inc, USA) to a targeted size of was sequenced using an Illumina flow cell, and 202 350 – 500 bp. The fragmented DNA was then end- cycles on the Illumina HiSeq 2000 platform repaired, ligated to Illumina TruSeq adapters, and (Illumina, USA). The sequencing run generated a PCR-enriched using TruSeq DNA Sample total of 2 GB of raw data. Preparation Kit (Illumina, USA) according to manufacturer’s protocol. The final sequencing library SNPs were called across the 14 samples was quantified using KAPA kit (KAPA Biosystem, based upon the public reference genome [7,9]. USA) on Agilent Stratagene Mx-3005p quantitative Fisher’s exact test was used to compare SNP PCR (Agilent, USA) and library size was confirmed occurrence between susceptible and resistant groups. using Agilent Bioanalyzer High Sensitivity DNA Any SNP with enrichment in the resistant or 41
Malaysian Cocoa Journal, 8/2014 susceptible group was selected based upon a Fisher’s Enriched SNPs were located on the cocoa exact test p-value cut-off of 5E-7, which corrected for chromosomes and clustering of enriched SNPs was multiple testing of the 6 million identified SNPs. noted.
Table 2. Summary of final library QC for cocoa leaf libraries.
Sample QC info Final Library Multiplexing
Library Average Size Volume Conc. Total Sample conc. base pair range Adapter Index (μl) (ng/μl) (μg) (nM) (bp) (bp) C0923 0.164 32.8 6.56 8.22 581 479-850 AD012 CCTGTA C0924 0.151 30.2 6.04 97.59 644 494-1000 AD004 TGACCA C0925 0.098 19.6 1.96 93.15 440 330-787 AD005 ACAGTG C0926 0.230 46.0 2.30 41.71 495 367-696 AD004 TGACCA C0927 0.240 48.0 9.60 39.03 539 394-779 AD007 CAGATC C0928 0.485 97.0 4.85 33.40 549 426-745 AD008 ACTTGA C0929 0.108 21.6 4.32 32.98 514 405-675 AD009 GATCAG C0930 0.625 125.0 2.50 31.30 576 467-809 AD010 TAGCTT C0931 0.130 26.0 1.56 7.68 602 500-1000 AD002 CGATGT C0932 0.041 8.2 0.49 8.75 493 399-640 AD005 ACAGTG C0933 0.106 21.2 1.27 21.21 481 363-682 AD007 CAGATC C0934 0.450 90.0 2.70 54.24 625 507-971 AD004 TGACCA C0935 0.802 160.0 4.80 79.05 538 419-670 AD005 ACAGTG C0936 0.264 52.8 2.64 165.90 596 470-1000 AD006 GCCAAT
Bioinformatics with a total size of approximately 218Gbp, were Reads were trimmed according to quality score, considered as high quality preprocessed reads and from the 5'-end to the 3'-end, with a threshold were utilised as input for the assembly and SNPs mean score of 20. As shown by Table 3 below, in calling analysis. average around 99% reads for each cocoa genome,
Table 3. Statistical summary of sequencing reads before and after preprocessing analysis for all 14 samples of cocoa genomes. A total of 99.08% of reads retrieved were of high quality. Depth coverage estimation was based on the cocoa matina genome size (approx: 400Mbp).
Raw Reads Pre-Processed Reads Estimated Estimated % High Cocoa Total Total genome Total genome Quality Sample ID Total Reads Bases Bases coverage Reads coverage Reads (Gbp) (Gbp) (400 Mbp) (400 Mbp) C0923 169,203,118 17.09 0.00 167,308,059 16.57 0.00 98.88% C0924 157,534,798 15.91 0.00 156,519,374 15.31 0.00 99.36% C0925 166,503,216 16.82 0.00 165,226,495 16.42 0.00 99.23% C0926 164,086,100 16.57 0.00 162,100,343 16.02 0.00 98.79% C0927 158,208,394 15.98 0.00 156,652,252 15.52 0.00 99.02% C0928 156,159,858 15.77 0.00 154,597,516 15.31 0.00 99.00% C0929 184,647,174 18.65 0.00 183,146,984 18.19 0.00 99.19% C0930 126,344,074 12.76 0.00 125,367,880 12.41 0.00 99.23% C0931 138,564,020 13.99 0.00 136,840,818 13.53 0.00 98.76% C0932 146,467,532 14.79 0.00 145,296,721 14.42 0.00 99.20% C0933 154,846,532 15.64 0.00 153,567,069 15.22 0.00 99.17% C0934 156,125,956 15.77 0.00 154,180,522 14.90 0.00 98.75% C0935 171,170,734 17.29 0.00 169,467,499 16.53 0.00 99.00% C0936 176,998,266 17.88 0.00 176,104,051 17.33 0.00 99.49%
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RESULTS information from paired-end reads) and gap closing were performed after contigs had been produced from Assembly was performed using the MSR-CA-1.4 the overlapping reads. The output results from the Genome Assembler. Further scaffolding (by using assembly are shown in Table 4.
Table 4. Statistical summary of assembly for cocoa contigs for minimum contig size of 1kbp. N90, N50 and N10 represent the length of the smallest contig in the set that contains contigs with combined lengths representing at least 90%, 50%, and 10% of the assembly respectively.
Maximum Size Total Contigs / Average Size N90 N50 (bp) N10 (bp) %Ns %GC (bp) Scaffolds (bp) (bp) 23,032,738 24,386 16,248 5,140 7,557,474 22,025,072 0.00 34.53
The resulting scaffolds / contigs statistical table, the results of the MCB cocoa final assembly assemblies results were compared against the closely were noticeably more improved compared to the related cocoa species, Theobroma cacao L. Cultivar closely related reference cocoa species (Theobroma Matina 1-6. The reference genome was retrieved from cacao L. Cultivar Matina 1-6). More detail the public database of NCBI, USA (Argout et al., summaries for each sample were shown as in Table 6 2011). Table 5 summarises the final draft assemblies and Table 7. comparison between MCB and NCBI. Based on the
Table 5. Comparison of assembly statistics between MCB cocoa scaffolds and Theobroma cacao L. clone Matina 1- 6 scaffolds (from NCBI public databases).
Metric Cocoa T. Matina (MCB) (NCBI) Maximum Length (bp) 23,032,738 10,380,066 Total Scaffolds 23,852 814 Total Size (bp) 397,795,478 344,963,675 Average Length (bp) 16,677 423,788 N90 (bp) 5,376 1,277,569 N50 (bp) 7,557,474 4,345,749 N10 (bp) 22,025,072 9,113,421 %Ns 0.02 4.10 %GC 34.53 34.01 %Q40 98.12% unknown
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Table 6. Summary of total genome sequencing coverage delivered for each plant.
Estimated genome coverage Plant ID Total Reads Total bases (400Mbp)
C0923 169,203,118 17,089,514,918 42.72 x C0924 157,534,798 15,911,014,598 39.78 x C0925 166,503,216 16,816,824,816 42.04 x C0926 164,086,100 16,572,696,100 41.43 x C0927 158,208,394 15,979,047,794 39.95 x C0928 156,159,858 15,772,145,658 39.43 x C0929 184,647,174 18,649,364,574 46.62 x C0930 126,344,074 12,760,751,474 31.90 x C0931 138,564,020 13,994,966,020 34.99 x C0932 146,467,532 14,793,220,732 36.98 x C0933 154,846,532 15,639,499,732 39.10 x C0934 156,125,956 15,768,721,556 39.42 x C0935 171,170,734 17,288,244,134 43.22 x C0936 176,998,266 17,876,824,866 44.69 x
Table 7. Individual genome assembly statistics for each of the 14 cocoa plants.
Largest Total Average Plant ID Pest Genome size N50 %GC contig contigs contig size C0923 Resistant 182,926 34,914 280,051,721 8,021 17,068 33.67
C0924 Resistant 259,603 36,706 284,833,210 7,759 18,099 33.85
C0925 Resistant 284,693 45,341 279,484,953 6,164 11,965 33.75
C0926 Resistant 247,658 39,040 287,061,289 7,353 16,189 33.79
C0927 Resistant 296,958 36,327 295,322,362 8,129 19,261 33.82
C0928 Resistant 715,833 35,286 292,196,550 8,280 19,379 33.82
C0929 Resistant 362,195 32,899 279,530,598 8,496 20,495 33.84
C0930 Susceptible 254,337 39,038 294,468,453 7,543 15,083 33.94
C0931 Susceptible 142,874 33,681 266,999,224 7,927 16,361 33.6
C0932 Susceptible 253,268 39,367 287,504,381 7,303 14,900 33.76
C0933 Susceptible 171,457 42,587 296,162,020 6,954 14,523 33.89
C0934 Susceptible 373,840 38,037 285,527,161 7,506 16,492 33.85
C0935 Susceptible 477,968 27,995 295,957,185 10,571 26,672 33.91
C0936 Susceptible 310,831 34,278 293,493,103 8,562 21,316 33.87
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SNPs Calling disease resistance which is a natural product of genetic diversity. All 14 Samples were mapped against public reference genome of Theobroma cacao L. Cultivar Matina 1-6. While this project used a small sample of 14 SNPs calling analysis was being performed on the plants, it has shown the ease with which genetic aligned reads against the reference genome. In total markers for beneficial traits can be found. More across all 14 samples, 6,313,926 numbers of SNPs subtle quantitative traits, such as improved flavour, were discovered. Fisher’s exact was applied to can also be bred for with these techniques, though identify the significant of the SNPs be found in the such quantitative traits require much larger numbers susceptible or resistant groups. Fisher's exact test was of samples to locate the many genetic loci which used as it is ideal for small samples, non parametric usually regulate quantitative traits. Further improved and is statistically very strong. Table 8 below shown pod borer resistance will be possible using the best SNPs results. quantitative trait and genomic selection techniques along with the markers identified here. Low coverage P-values smaller than 1E-6 are significant genome sequencing is already cost effective to give when corrected for multiple testing. What is sufficient data for such techniques. It is not an particularly exciting is that there is linkage between overstatement to say this technology will vastly the significant SNPs in 2 blocks. We found that a accelerate the breeding methods which we have used tight block on chromosome 1 in a sugar transporter for the last 10,000 years. gene, and a looser linkage block on chr10. One of the SNPs on chr10 is in an open reading frame with no homology to any other proteins. It may be a cocoa CONCLUSIONS specific gene. The SNP with the best P-value (SNP chr1:6,776,244) is within the intron of the gene The two genomically tight clusters of SNPs which are Thecc1EG001379, a sugar transporter. Some sugar enriched in the resistant genomes are highly likely to transporters have been shown to be involved in plant contain loci involved in pod borer resistance. While structural regulation and infection. one gene has homologs implicated in plant structural parameters, the other locus contains a gene of Six million SNPs were identified across the unknown function. A study testing these SNPs in a 14 samples. Six of these SNPs showed enrichment larger number of samples is needed to demonstrate within the resistant group. Four SNPs were found that the SNPs can be confidently used as high quality solely in all 7 resistant samples and none of the 7 breeding markers for pod borer resistance. A susceptible samples, and 1 SNP was homozygous in quantitative trait study is likely to find more genetic all 7 resistant samples and thus showed statistically markers for borer resistance and further enhance the strong enrichment (p-value ~ 1E-8) in the resistant ability to breed resistant cocoa. An analysis of cocoa group. The 6 enriched SNPs were found in 2 genome structural differences and their correlation to genomically tight clusters, demonstrating clear borer resistance is currently underway. linkage disequilibrium.
DISCUSSIONS
Identification of genetic markers for binary traits is a new development, made possible by cheap whole genome sequencing (Allegre et al., 2012; Sveinsson et. al, 2013). With small sample sizes a degree of luck is needed to identify significant SNPs, but as the number of samples increases, the chances of identifying SNPs correlated to a well chosen trait becomes high. The rapidly decreasing cost of genome sequencing has made it possible to enumerate most of the SNPs in a genome, making ultra high resolution genetic mapping possible. Future plant breeding programs will utilise this technology, coupled with genomic selection methodologies, to breed for rapid trait improvements, while also maintaining, or even improving the genetic diversity of superior varieties (Feltus et al., 2011; LoorSolorzano et al., 2012). Genomic selection, based upon millions of SNPs, and measurement of many traits at once, will also create the ability to breed plants to optimal mixtures of multiple traits at once, while improving the innate 45
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Table 8. A list of High Quality SNPs that occur in Chr 1 and 10 against Theobroma cacao L. Cultivar Matina 1-6 reference genome.
#CH POS REF ALT cases:ho control cc_all S_C S_C S_C S_C S_C S_C S_C T_C T_C T_C T_C T_C T_C T_C RO m:het:cnt :hom:h _fishe 0930 0931 0932 0933 0934 0935 0936 0923 0924 0925 0926 0927 0928 0929 M et:cnt rPval ue 10 4,967,647 G C 7,0,14 0,1,1 3.739 1/1 1/1 1/1 1/1 1/1 1/1 1/1 0/0 0/1 0/0 0/0 0/0 0/0 0/0 e-07
10 5,030,235 T G 7,0,14 0,1,1 3.739 1/1 1/1 1/1 1/1 1/1 1/1 1/1 0/0 0/0 0/0 0/0 0/0 0/1 0/0 e-07
10 5,064,478 G C 6,1,13 0,0,0 3.739 1/1 1/1 1/1 1/1 0/1 1/1 1/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 e-07
1 6,775,527 G A 7,0,14 0,1,1 3.739 1/1 1/1 1/1 1/1 1/1 1/1 1/1 0/0 0/1 0/0 0/0 0/0 0/0 0/0 e-07
1 6,775,960 A T 6,1,13 0,0,0 3.739 1/1 1/1 1/1 1/1 0/1 1/1 1/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 e-07
1 6,776,244 A G 7,0,14 0,0,0 2.493 1/1 1/1 1/1 1/1 1/1 1/1 1/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 e-08
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REFERENCES (2012). Transcriptomics and systems biologanalysis in identification of specific Allegre, M., Argout, X., Boccara, M., Fouet, O., pathways involved in cacao resistance and Roguet, Y., Bérard, A., Thévenin, J.M., susceptibility to witches' broom disease. Mol Chauveau, A., Rivallan, R., Clement, D., Biosyst 8(5): 1507-19 Courtois, B., Gramacho, K., Boland-Augé, A., Tahi, M., Umaharan, P., Brunel, D. and Day R.K. (1985) Control of the cocoa pod borer Lanaud, C. (2012). Discovery and mapping (Conopomorpha cramerella), Ph.D. of a new expressed sequence tag-single dissertation, University of London, London, nucleotide polymorphism and simple England. sequence repeat panel for large-scale genetic studies and breeding of Theobroma cacao L. Feltus, F.A., Saski, C.A., Mockaitis, K., Haiminen, DNA Res. 19(1): 23-35. N., Parida, L., Smith, Z., Ford, J., Staton, M.E., Ficklin, S.P., Blackmon, B.P., Cheng, Argout, X., Fouet, O., Wincker, P., Gramacho, K., C.H., Schnell, R.J., Kuhn, D.N. and Legavre, T., Sabau, X., Risterucci, A.M., Da Motamayor, J.C. (2011). Sequencing of a Silva. C., Cascardo, J., Allegre, M., Kuhn, QTL-rich region of the Theobroma cacao D., Verica, J., Courtois, B., Loor, G., Babin, genome using pooled BACs and the R., Sounigo, O., Ducamp, M., Guiltinan, identification of trait specific candidate M.J., Ruiz, M., Alemanno, L., Machado, R., genes. BMC Genomics. 12: 379. Phillips, W., Schnell, R., Gilmour, M., Rosenquist, E., Butler, D., Maximova, S. Kane, N., Sveinsson, S,, Dempewolf, H,, Yang, and Lanaud, C. (2008). Towards the J.Y.,Zhang, D., Engels, J.M. and Cronk, Q. understanding of the cocoa transcriptome: (2012). Ultra-barcoding in cacao Production and analysis of an exhaustive (Theobroma spp.; Malvaceae) using whole dataset of ESTs of Theobroma cacao L. chloroplast genomes and nuclear ribosomal generated from various tissues and under DNA. Am..J Bot. 99(2): 320-9. various conditions. BMC Genomics. 9: 512. LoorSolorzano, R.G., Fouet, O., Lemainque, A., Argout, X., Salse, J., Aury, J.M., Guiltinan, M.J., Pavek, S., Boccara, M., Argout, X., Amores, Droc, G., Gouzy, J., Allegre, M., Chaparro, F., Courtois, B., Risterucci, A,M, andLanaud C., Legavre, T., Maximova, S.N., Abrouk, C. (2012). Insight into the wild origin, M., Murat, F., Fouet, O., Poulain, J., Ruiz, migration and domestication history of the M., Roguet, Y., Rodier-Goud, M., Barbosa- fine flavor Nacional Theobroma cacao L. Neto, J.F., Sabot, F., Kudrna, D., Ammiraju, variety from Ecuador. PLoS One. 7(11): J.S., Schuster, S.C., Carlson, J.E., Sallet, E., e48438. Schiex, T., Dievart, A., Kramer, M., Gelley, L, Shi, Z., Bérard, A., Viot, C., Boccara, M., Motamayor, J.C., Lachenaud, .P, da Silva, E., Mota, Risterucci, A.M., Guignon, V., Sabau, X., J.W., Loor, R., Kuhn, D.N., Brown, J.S. and Axtell, M.J., Ma, Z., Zhang, Y., Brown, S., Schnell, R.J. (2008). Geographic and genetic Bourge, M., Golser, W., Song, X., Clement, population differentiation of the Amazonian D., Rivallan, R., Tahi, M., Akaza, J.M., chocolate tree (Theobroma cacao L). PLoS Pitollat, B., Gramacho, K., D'Hont, A., One 3(10): e3311. Brunel, D., Infante, D., Kebe, I., Costet, P., Wing, R., McCombie, W.R., Guiderdoni, E., Motamayor, J.C., Mockaitis, K., Schmutz, J., Quetier, F., Panaud, O., Wincker, P., Bocs, Haiminen, N., Livingstone, D. 3rd, Cornejo, S. and Lanaud C. (2011). The genome of O., Findley, S.D., Zheng, P., Utro, F., Theobroma cacao. Nat Genet. 43(2):101-8. Royaert, S., Saski, C., Jenkins, J., Podicheti, R., Zhao, M., Scheffler, B.E., Stack, J.C., daHora Junior, B.T., PoloniJde, F., Lopes, M.A., Feltus, F.A., Mustiga, G.M., Amores, F., Dias, C.V., Gramacho, K.P., Schuster, I., Phillips, W., Marelli, J.P., May, G.D, Sabau, X., Cascardo, J.C., Mauro, S.M., Shapiro, H., Ma, J., Bustamante, C.D., GesteiraAda, S., Bonatto, D. and Micheli, F. Schnell, R.J., Main, D., Gilbert, D., Parida,
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L. and Kuhn, D.N. (2013). The genome coverage whole genome shotgun sequencing sequence of the most widely cultivated in cacao (Theobroma cacao L.) and related cacao type and its use to identify candidate species. BMC Genomics. 14: 502. genes regulating pod color. Genome Biol. 14(6): R53. Utro, F., Cornejo, O.E., Livingstone, D., Motamayor, J.C. and Parida, L. (2012). ARG-based Sveinsson, S., Gill, N., Kane, N.C. and Cronk, Q. genome-wide analysis of cacao cultivars. (2013). Transposon fingerprinting using low BMC Bioinformatics. 13 Suppl 19: S17.
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ANTIOXIDANT PROPERTIES OF COCOA PODS AND SHELLS
Azila Abdul Karim1, Azrina Azlan2,3, Amin Ismail2, Puziah Hashim3 and Nur Azilah Abdullah1 1Cocoa Innovation & Technology Centre, Malaysian Cocoa Board, PT12621, Nilai Industrial Area, 71800 Nilai, Negeri Sembilan Malaysia; e-mail: [email protected] 2Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia; 3Halal Product Development Institute, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia
Malaysian Cocoa J. 8: 49 – 56 (2014) ABSTRACT - This study was conducted to compare antioxidant properties of cocoa pods and shells, in terms of its level and activities. The antioxidant level was determined by total phenolic and total flavonoid contents using spectrophotometric method by Follin-Ciocalteau’s reagent and aluminum chloride solution, respectively. Antioxidant activity of these extracts were measured using DPPH and FRAP assays. Cocoa pods contained promising amount of total phenolic and flavonoid content in comparison to cocoa shells. Antioxidant activity found using DPPH assay showed that cocoa pods exhibited highest activity in comparison with the cocoa shells although lower than gallic acid standard. Significant high antioxidant activity of cocoa pod extract was clearly exhibited using FRAP assay in comparison with cocoa shells. Therefore, cocoa pods are potential waste materials that can be used as raw materials for other beneficial application including cosmeceutical product.
Keywords: Cocoa pod, Cocoa shell, Total phenolic content, Total flavonoid content, Scavenging effect
INTRODUCTION other application, cocoa shells were used in particleboard making as its surface layer (Hii, 2006) Abundant of waste from cocoa areas can be turned and as a source of pectin (Mollea, 2008). Cocoa into value-added end products. It was estimated that shells, also known as cocoa hulls, showed positive only 25% from total weight of fresh cocoa fruits were antioxidant activity when evaluated in vitro (Arlorio, used to produce cocoa beans for cocoa and chocolate 2005). Total phenolic content (TPC) of non-roasted processing. Other parts including the pods and shells cocoa husks (cocoa shells) have been measured by are usually discarded. Despite of being thrown away, Ramos (2008) using catechin as standard, of 70% cocoa pods can also be used as sources of fertilizer or methanol-acidic extract after enzymatic hydrolysis animal feed and activated carbon production (Fisal, treatment to obtain dietary fiber. Strong antioxidant 2005). Other potential use of cocoa pods are in activity was exhibited by cocoa shells (Azizah, producing gum (Samuel, 2006), potash in soap- 1999). Recumberri (2007) also evaluated the total making as well as food colorant (Figueira, 1993). antioxidant of cocoa shells along with the The pods contained 45.6–46.4 mg gallic acid determination of dietary fiber composition in cocoa equivalent of soluble phenolic; 32.3% carbohydrate, shells. Amin (2006) determined that total phenolic 21.44% lignin, 19.2% sugars, 8.6% protein and content of cocoa shells was 112.9 + 0.6 mg GAE/g 27.7% minerals (Vriesmann, 2011). Arsel (1992) extract and scavenging activity at 95.9 + 2.0%, which and Fapohunda (2012) reported that cocoa pods was extracted using 70% ethanol. In contrast, extract contained phenolic acid, including caffeic acid, but of 80% ethanol results in total phenolic content at no specific antioxidant measurement was carried out. 23.36 + 1.59 mg GAE/g extract and 2.23 + 0.90 mg CE/g total flavonoid using catechin as standard (John, Cocoa shells, which is enclosed the cocoa 2012). Cocoa shells and cocoa pods extract also seeds is endocarp (Bewley, 2006) and must be exhibited antimicrobial activities in reduction of removed after roasting process to obtain the cocoa Streptococcus mutans and mouth plaque (Srikanth, nibs. Cocoa shells are a potential source of dietary 2008). fibre (Maria, 1994) and fertilizer (Chung, 2003) due to its mono & oligosaccharides, starch, pentosans & Mechanism of skin wrinkles formation was cellulose content, and removal of heavy metal from extensively reported by Mukherjee (2011) and acidic solution in industrial line (Meunier, 2003). In bioactives from plants that exhibited anti-wrinkles
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effects including catechin, epicatechin and gallic comparison purpose, cocoa pods and shells were acid. These compounds have high antioxidant collected by clones, namely, MCB C1 (C1), MCB C3 activity. Addition of extract from plants can delay (C3), MCB C4 (C4), MCB C5 (C5) and PBC123 the oxidation process by radical oxygen species (PBC). In addition, a collection of cocoa pods were (ROS) which is responsible for the formation of labeled MIX representing cocoa pods which has been wrinkles on the skin. Evans (2010) reported that collected in bulk due to the practices of plantation phenolic compounds such as catechin, epicatechin, where the pods where discarded despite their clones. quercetin and kaempferol with antioxidant activity Pods collected were washed thoroughly with tap reduced oxidative stress including skin wrinkles and water to remove debris and chopped into about 1cm2 photoaging (Jung, 2007). Garbisa (2011) proved that in size and sun-dried. Chopped pieces of pods were quercetin and kaempferol inhibit MMP-1 activity, in- placed on the net and exposed to direct sunlight until vitro, and topically (Casagrande, 2006). MMP-1 is a dried. The dried cocoa pod husks were ground into collagenase enzyme involve in degradation of powder form using warring blender. Ground pod collagen resulting the wrinkle formation (Kim, 2006). were sieved using automatic sieve to mesh size of 1 Report by Aburjai (2003) revealed that catechin and mm. Sample of the shells was collected after roasting epicatechin can treat dry skin. Polyphenolics the cocoa beans of fermented said beans as usually substances delayed skin aging, in-vitro (Rutter, 2003) practices in the grinding industry. In this experiment, and exhibited UV-radiation protection (Nichols, cocoa shells, irrespective the clones, was collected 2010). Antioxidant activity of plant phenolic extract from the industry and labeled MIX. The pod and offered an alternative in producing natural anti- shell was labeled as P for pod and S for shell, wrinkles cream (Baumann, 2007; Masaki, 2010). respectively.
This study was carried out to determine total Extraction phenolic and flavonoid content of cocoa pods in Extraction was carried out using aqueous ethanol comparison with the shells. The antioxidant activity (80% v/v). The method described by Catherine was also measured using DPPH and FRAP assays. (2003) was used for the extraction procedure, where Results of this experimental will be used for the 80% ethanol (Sanbogi, 1998) was selected. One selection of materials for anti-wrinkles cosmetic gram of pod powder was soaked in 20 ml of aqueous formulation. ethanol in 50 ml flask with cap. The flask was placed in water bath shaker for 30 minutes, 35oC at 120 rpm. Decanted portion was then filtered using Whatmann MATERIALS AND METHODS filter No.1 to remove remaining floating materials. Solvent was removed using rotary vacuum evaporator Reagents to dryness. Crude extract was re-dissolved in 1-2ml Denatured ethanol (95%) from Hmbg, anhydrous aqueous ethanol (80% v/v). Extracted portion were sodium carbonate from AR, Gallic acid (3,4,5- again filtered and filtered portion was subjected to the Trihydroxybenzoic acid) from Sigma-Aldrich, Folin- analysis. The extraction was carried out in triplicate. Ciocalteau (FC) reagent from Merck, aluminum chloride (AlCl3.6H2O, MW 241.43 g/mol) from Total Phenolic Content (TPC) Determination FriendemannSchmidt, Rutin (rutinoside: 2-(3,4- Kaur (2002) method was used with several dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H1- modifications to measure the total phenolic content in benzopyran-3-yl rutinoside, C27H30O16) from Powder cocoa pod using Folin-Ciocalteau (FC) reagent. Spectrum and Methanol from Merck were used in Total phenolic content of the samples was obtained this experimentation. In addition, 1,2-diphenyl-2- by the calibration curve of standard gallic acid picrylhydrazyl (DPPH, C18H8N5O6, MW measured. Hundred (100) µl of extracted sample (or 394.23g/mol) was purchased from Aldrich and standard) was pipette into vial. Seven point nine dimethylsulfoxide (DMSO) from Merck for total millimeters (7.9ml) of distilled water was added to antioxidant activities measurement. the sample (or standard). Five hundred (500) µl of FC was added to the diluted sample and mix for 8 Sample collection seconds. Then, 1500 µl of sodium carbonate solution Cocoa pod were collected from Cocoa Research & (20% w/v) was added and left in the dark at 40oC for Development Centre (Malaysian Cocoa Board) in 1 hour. Measurement was carried out with UV- Hilir Perak, Perak and Jengka, Pahang. For Spectrophotometer at 765nm, twice, against gallic
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acid standard. The results obtain were expressed in from Benzie (1996), Szollosi (2002), Katalinic mg Gallic Acid Equivalent (GAE)/g sample. (2004), Arnous (2002), Pulido (2000) and Bub (2000). In brief, ferric chloride solution was made by Total Flavonoid Content (TFC) Determination dissolving 3 mM of Ferric chloride in 5 mM citric Measurement of total flavonoids content was carried acid with distilled water. TPTZ solution was out using microplate reader at wavelength of 405 nm prepared by dissolving 1 mM of TPTZ in 0.05M using aluminium chloride colorimetric assay. hydrochloric acid. Standard calibration curve was Method by Chang (2002) was adapted. Solution obtained by different concentration of FeSO4.7H2O containing 2% (w/v) of aluminium chloride was (1.0 – 0.1 mM Fe(II)). To measure the FRAP value, prepared by dissolving 2g of aluminium chloride with 15μl of sample or standard was added to 270 μl of 100 ml of methanol. Extract of sample 0r standard TPTZ solution and measure at 620nm for initial was pipetted at 100 µl into 96-well microplate and reading using microplate reader. Ferric chloride 100 µl aluminium chloride solution was added. solution at 15μl was added and measurement was Incubate the sample for 10 minutes and measure the carried out at immediate. The sample was then absorbance at 405 nm. Total flavonoids content was incubated for 30 minutes (30-37oC) with interval obtained using calibration curve of rutin standard. measurement for 2-3 minutes. The result was The samples were expressed in mg Rutin Equivalent compared with gallic acid or ascorbic acid. EC1 is per gram sample (mg RE/g). effective concentration of the extract required to react with 1 mol Fe (II)/L. Higher value indicated lower Determination of DPPH Scavenging Effect and antioxidant activity, which means high concentration EC50 of extract was needed to reduce 1 mol of Fe (III) to Antioxidant activity was be carried out using DPPH Fe (II) referring to the mechanism of antioxidant assay as described by Patil (2010), Young (2008), compound to FRAP assay. Mustafa (2006) and Kim (2009) methods with some modifications. In brief, the DPPH stock solution was Statistical analysis prepared by dissolving 0.2M of DPPH in ethanol. Standard calibration curves were accepted when the Solution of DPPH was made by diluting 1.2ml of R-squared of the linear regression was higher than DPPH stock solution with 3ml ethanol and 0.5ml 0.999. One-way ANOVA was used to screen the DMSO. For antioxidant activity, samples were made difference value of overall TPC and TFC content of into serial dilution of 1000-7.81 ppm and measure for cocoa pod and shell extract from clones. Two sample comparison. Then, 270µl of DPPH solution was t-test was used to indicate significant difference added to 30µl sample at different concentration, between experimental data in this study and from shake and left it to react for at least 10 minutes before literature, if any. This test was also carried out to see measuring at absorbance 550nm. Gallic acid the difference on DPPH scavenging effect. Level of standard was used as positive control. Ethanol with significant in this study was at level p<0.05, using the addition of DPPH solution was used as blank Minitab Software version 14. (Lee, 2003). Calculation of EC50 (the concentration needed to reduce DPPH by 50%) was obtained using linear regression (Marxen, 2007) on the graph plotted RESULTS & DISCUSSION of remaining DPPH percentage versus concentration ratio of sample to DPPH. Small value EC50 indicates Total phenolic content low concentration of sample required to exhibit Calibration curve for standard gallic acid at 765nm antioxidant activity by this assay. was plotted and regression analysis was carried out, where the equation (1) was obtained (R2=0.999) and Determination of FRAP and EC1 used to predict the total phenolic content (mg GAE/g) Method of conducting FRAP assay for cocoa pod of the samples with the dilution factor. extract was carried out with modification of method
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A765 = -0.003800 + 0.000817*concentration (1)
Where A765 is absorbance value at wavelength 765nm and concentration is in part per millions (ppm) of total phenolic content.
total phenolic of cocoa shell and pod 60
50 shell pod 40
30
20 mgGAE/gextract
10
0 C1 C3 C4 C5 MIX PBC
Figure 1: Total Phenolic Content
Number of sample taken was repeated both type of samples from C1 and C3 has low TPC several times for different samples to obtain low values, measurement of total flavonoid content was relative standard deviation (RSD < 25%) indicating not being carried out for these samples. the data are representative. It was shown in Figure 1 that pod labeled MIX (49.54 + 3.69 mg GAE/g Total flavonoid content extract, n=6) has the highest total phenolic content Calibration curve for rutin standard with known among the selected clones, equally to C4 (47.57 + concentration measured at 405nm obtained equation 3.23 mg GAE/g extract, n=6). Cocoa pod extract (2) with R2= 0.994 and used to predict the total labeled C5 had 43.96 + 1.79 mg GAE/g extract flavonoids (mg RE/g) of the samples. (n=14) followed by PBC (27.57 + 2.89 mg GAE/g extract, n=16), C3 (27.43 + 4.67 mg GAE/g extract, A405 = 0.0670 + 0.00776 concentration (2) n=5) and C1 (19.59 + 2.69 mg GAE/g extract, n=4). High TPC of cocoa pod MIX was not significantly Where A405 is absorbance of extract at 405 different from value obtained by Vriesmann (2011). wavelength and concentration is in part per million (ppm) of total flavonoids content. Figure 2 For shells, C4 (41.35 + 2.23 mg GAE/g summarized the TFC of cocoa pods and shells. extract, n=6) showed significantly highest total phenolic content within the groups, followed by C5 (37.90 + 2.40 mg GAE/extract, n=12), PBC (35.91 + 1.73 mg GAE/g extract, n=12), MIX (32.59 + 0.28 mg GAE/g extract, n=6), C1 (18.63 + 0.67 mg GAE/g extract, n=12) and C3 (16.30 + 1.04 mg GAE/g extract, n=12). TPC value of cocoa shells was varied depending on the source of collection as reported by Amin (2006) and John (2012). Weather, soils and environmental factors contributed to the chemical and physical content of the plants. It was also shown that between pod and shell, within clones, total phenolic content was significantly higher except for C1, where the TPC value was almost equal. Since
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Total flavonoid content vs clones 35
30 SHELL POD 25
20
15
mgRE/gextract 10
5
0 C4 C5 MIX PBC
Figure 2: Total Flavonoid Content
The results showed that pod flavonoid significantly different from the cocoa pod extract content was significantly lower than in the shell for labeled MIX, which however significant at 1000ppm. C4, C5 and PBC. However, pod labeled MIX (22.42 Cocoa pod extract of C4 has lowest scavenging effect + 0.99, n=6) contained significantly highest amount at these concentrations. of total flavonoids, followed by C4 (14.18 + 2.93, n=8), C5 (4.52 + 0.91, n=9) and PBC (3.96 + 0.84, Effective concentration was determined to n=9). For shells, C4 (27.44 + 2.23, n=5) had the understand the antioxidant activity of the sample highest amount of total flavonoid followed by PBC using DPPH assay. Table 1 shows the summary of (17.47 + 3.28, n=6), MIX (10.54 + 0.92, n=6) and C5 the effective concentration (EC50) of the samples. (7.15 +1.13, n=5). Statistically, between pods and Cocoa pod MIX has the lowest EC50 among the shells, the total flavonoid was different, within samples. Although the EC50 the value was two-folds clones. From this result, it was shown that cocoa from the gallic acid, it exhibited antioxidant activity. pods and shells of C5 and PBC has low value of TFC, High antioxidant activity was exhibited in cocoa pods therefore these samples were not tested for their due coloring pigment that might exist in the extract, antioxidant activity as extract with high phenolic which will be determined later. Coloring pigment in compounds contribute to antioxidant activity (Sies, the fruit pods can be the results of bioactive 2004). compound such as chlorophyll, carotenoids and phenolics (Lancaster, 1997). Lowest EC50 was DPPH scavenging effect & EC50 exhibited by cocoa shells MIX which is almost 20- Determination scavenging effect was carried out to folds of gallic acid. The antioxidant activity was potential extract of cocoa pods of C4 and MIX as reduced due to the roasting and fermentation process well as their shell extract. Scavenging effect of cocoa that might degrade the potentially active compound pods and shells in comparison with gallic acid was that exhibited antioxidant activity using DPPH as an summarized in Figure 3. At low concentration (less assay. When calculating the exact concentration than 125ppm), cocoa pods extract exhibited higher needed to scavenging the DPPH radical species by scavenging effect from shells, especially MIX, the extract, it was indicated that high concentration of although lower than gallic acid, significantly. At cocoa shells MIX was needed compared to cocoa 250ppm and 500ppm, both cocoa shells extract has pods of MIX and C4. higher scavenging effect on DPPH, but were not
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DPPH Scavenging (%) vs Concentration (ppm)
100.0 90.0 80.0 70.0 60.0 50.0 40.0
30.0 DPPH Scavenging Scavenging DPPH % 20.0 10.0 0.0 7.8 15.5 31.1 62.3 125 250 500 1000 ppm P/C4 7.0 18.3 26.7 73.0 58.2 67.3 72.3 77.6 P/MIX 11.4 34.2 76.5 87.1 85.0 86.8 84.5 79.6 S/C4 3.9 6.8 13.3 28.2 40.8 87.1 88.2 88.0 S/MIX 3.9 7.2 11.9 23.7 24.3 88.3 84.3 88.3 GALLIC ACID 46.7 88.8 91.7 93.1 92.9 94.5 95.0 96.1
Figure 3: Scavenging Effect of Cocoa Waste Extract (mean value, n=3)
Table 1: Effective Concentration DPPH FRAP Sample EC50 Concentration (mg/ml) at EC50 EC1(ppm) mol Fe(II)/g extract at EC1 Gallic acid 0.106 8.36 73.19 13.66 Pod/MIX 0.258 20.35 12.67 79.96 Pod/C4 0.574 45.26 n.a* n.a* Shell/C4 1.799 141.87 n.a* n.a* Shell/MIX 1.938 152.83 29.76 33.60 *n.a not available
FRAP value & EC1 order to elaborate more on FRAP value obtained by FRAP value of cocoa pod extract and shells at shells and pods extract, the EC1 was determined different concentration were summarized in Figure 4. (Table 1). Pod extract exhibited lowest EC1 value in At low concentration of samples, high FRAP values comparison with gallic acid and shell extract. It were obtained and comparable with ascorbic and showed that 79.96 mol Fe(II) are being reduced from gallic acid as standards. High value of FRAP was Fe(III) in the presence of one gram of cocoa pod exhibited by the cocoa pods and shells, significantly, extract, while only 33.60 mol of Fe(II) produced with from these standards was probably due to the one gram of cocoa shell extract. These values were synergistically effect of antioxidant component in the even higher in comparison with gallic acid as a extracts, yet to be measured using high performance standard (13.66 mol Fe(II)) as well as ascorbic acid liquid chromatography (HPLC) in the future. In (1.5 mol Fe(II)).
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180
160 FRAP value
140 ascorbic acid 120 gallic acid 100 pod 80 shell
60 mM Fe (II)/gmM Fe extract 40
20
0 ppm 7.86 15.56 31.13 62.5 125 250 500 1000 Figure 4: FRAP Value for Cocoa Pod and Shell Extract
CONCLUSIONS Arsel, O. (1992). Study of the phenolic acids of cocoa stems, fruit stalks and beans. In Study of Cocoa pods and shells exhibited antioxidant activity the phenolic acids of cocoa stems. Record with promising amount of total phenolic and Number: 19936796013. pp.30. flavonoid contents. Cocoa pods have higher Azizah, A.H., et al. (1999). Extraction and antioxidant activity in comparison with shells. The characterization of antioxidant from cocoa by- antioxidant activities of the extracts were contributed products. Food Chem., 64: 199-202. of several bioactive compounds that acts Benzie, I.F.F. and Strain, J.J. (1996). The ferric synergistically which suggested to be confirmed by reducing ability of plasma (FRAP) as a measure High Performance Liquid Chromatography (HPLC). of “antioxidant power”: The FRAP assay. Anal. Biochem., 239: 70-76. Bewley, J.D., et al. (2006). The encyclopedia of ACKNOWLEDGEMENT seeds: science, technology & uses. CAB International. The author would like to thank Director General of Bub, A., et al. (2000). Moderate Intervention with Malaysian Cocoa Board (MCB) for his permission to Carotenoid-Rich Vegetable Products Reduces publish this paper. Our appreciation goes to the Lipid Peroxidation in Men. The Journal of MCB’s staff who had directly or indirectly Nutrition, 2200-2206. contributed in carrying out this experiment. Catherine, A.R. and Lester, P. (2003). Flavonoids in Health and Disease. U.S.A: Marcel Dekker. REFERENCES Chang, C.C., et al. (2002). Estimation of total flavonoid content in propolis by two Amin, I., Chew, L.Y. (2006). Antioxidant effects of complementary colorimetric methods. J. Food extracts of cocoa shell, roselle seeds and a Drug Anal., 10(3): 178-182. combination of both extracts on the susceptibility Chung, B.Y., et al. (2003). Compositional of cooked beef to lipid oxidation. J. Food characterization of cacao (Theobroma cacao L.) Technol., 4(1): 10-15. hull. Agric. Chem. Biotehcnol., 46(1): 12-16. Arlorio, M., et al. (2005). Antioxidant and biological Fapohunda, S. and Afolayan, A. (2012). activity of phenolic pigments from Theobroma Fermentation of cocoa beans and antimicrobial cacao hulls extracted with supercritical CO2. potentials of the pod husk phytochemicals. J Food Res Int., 38(8-9): 1009-1014. Phys Pharm Adv, 2(3): 158-164. Arnous, A., et al. (2002). Correlation of pigment and Fisal, A. et al. (2005). Activated Carbons from flavonoid content with antioxidant properties in Cocoa Pod Husks Under Chemical Activation selected aged regional wines from Greek. J. with H3PO4: Effect of CO2 as Oxidizing Agent. Food Comp. Anal., 15: 655-665. Abstracts Malaysian Cocoa International Cocoa
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Conference 2005, 114. Agric. Food Chem., 54: 1151-1157. Hii, C.L. and Chiow, F.S. (2006). Development of Patil, Y., et al. (2010). Evaluation of In Vitro Particle Board from Cocoa Shells. Malaysian Antioxidant Activity of Herbage of Aromatic Cocoa Journal, 2: 22-27. Plants. J. Cell Tissue Res., 10(1): 2125-2129. John, N.A., et al. (2012). Quantification of total Pulido, R., et al. (2000). Antioxidant activity of polyphenolic content and antimicrobial activity dietary polyphenols as determined by modified of cocoa (Theobroma cacao L.) bean shells. ferric reducing/antioxidant power assay. J. Pakistan Journal of Nutrition, 11(7): 574-579. Agric. Food Chem., 48: 3396-3402. Katalinic, V., et al. (2006). Screening of 70 medicinal Ramos, S., et al. (2008). Hypolipidemic effect in plant extracts for antioxidant capacity and total cholesterol-fed rats of a soluble fiber-rich phenols. Food Chem., 94: 550–557. product obtained from cocoa husks. J. Agric. Kaur, C. and Kapoor, H.C. (2002). Anti-oxidant Food Chem., 56: 6985–6993. activity and total phenolic content of some Asian Recumberri, E., et al. (2007). Dietary fibre vegetables. Int. J. Food Sci. Technol., 37: 153- composition, antioxidant capacity and physic- 161. chemical properties of fibre-rich product from Kim, J.H., et al. (2009). Compounds with elastase cocoa (Theobroma cacao L.). Food Chem., 104: inhibition and free radical scavenging activities 948-954. from Callistemon lanceolatus. J Med Plants Samuel, Y.K.C. (2006). Crude Gum from Cocoa of Res, Vol. 3(11): 914-920. Malaysian Origin: Part 1: Rheological Kim, Y.H., et al. (2008). Anti-wrinkle activity of Properties. Malaysian Cocoa Journal, 2: 28-31. Ziyuglycoside I isolated from a Saonguisorba Sanbogi, C., et al. (1998). Antioxidative polyphenols officinalis root extract and its application as a isolated from Theobroma cacao. J. Agric. Food cosmeceutical ingredient. Biosci. Biotechnol. Chem., 46: 454-457. Biochem., 72(2): 303-311. Sies, H. andStahl, W. (2004). Nutritional protection Lancaster, J.E, et al. (1997). Influence of pigment against skin damage from sunlight. Annu. Rev. composition on skin color in a wide range of Nutr., 24: 173-200. fruit and vegetable. J. Amer. Soc. Hort. Sci., Szollosi, R. and Varga, I.S. (2002). Total antioxidant 122(4): 594-598. power in some species of Labiatae (Adaption of Lee, K. W., et al. (2003). Cocoa has more phenolic FRAP method). Acta Biologica Szegediensis, phytochemicals and a higher antioxidant capacity 46(3-4): 125-127. In Proceedings of the 7th than teas and red wine. J. Agric. Food Chem., Hungarian Congress on Plant Physiology, S2- 51: 7292-7295. P24. Maria, A.M., et al. (1994). Cocoa Hull: A Potential Vriesmann, L.C., et al. (2011). Cacao pod husks Source of Dietary Fibre. J. Sci. Food Agric., 66: (Theobroma cacao L.): composition and hot- 307-311. water-soluble pectins. Ind. Crops & Prod., 34: Marxen, K., et al. (2007). Determination of DPPH 1173-1181. radical Oxidation caused by methanolic extracts of some microalgae species by linear regression analysis of spectrophotometric measurements. Sensors, 7: 2080-2095. Meunier, N., et al. (2003). Cocoa shells for heavy metal removal from acidic solutions. Bioresource Technol., 90: 255-263. Mollea, C., et al. (2008). Extraction and characterization of pectins from cocoa husks: A preliminary study. Food Chem., 107: 1353- 1356. Mustafa, O., et al. (2006). Modified 2,2-Azino-bis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS) method to measure antioxidant capacity of selected small fruits and comparison to ferric reducing antioxidant power (FRAP) and 2,2’- diphenyl-1-picrylhydrazyl (DPPH) method. J.
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Short Communication
PRO AND CONS IN APPLYING CANOPY REPLACEMENT METHOD FOR VASCULAR STREAK DIEBACK INFECTED MATURE COCOA
Rozita, O., Nik Aziz, N.M. and Azhar, I. Cocoa Research and Development Center, Malaysian Cocoa Board, P.O.Box 34, Jalan Jengka 23, 28000 Temerloh, Pahang, Malaysia. Email: [email protected]
Malaysian Cocoa J. 8: 57 - 59 (2014) ABSTRACT – Canopy replacement is one of the promising methods to control vascular streak dieback infection in existing mature cocoa. It has been adopted by the farmers who used to plant VSD susceptible clones. However, there are a few pros and cons in adopting the method of canopy replacement. This paper discussed the pros and cons in details.
Keywords: Vascular streak dieback, Canopy replacement, Pro and cons
Canopy replacement has been used in cocoa two stages of grafting where the first grafting point is plantation as one of the method to control vascular between the rootstock (hybrid) with the clonal streak dieback disease in existing mature cocoa. materials and the second stage is the clonal material However, there are also questions that need to be with the clonal materials. We do not know yet answered when applying this management method. whether this kind of grafting will be successful in the One aspect is the graft compatibility between the future. There is no report on the performance of the stock and scion. There are indications that rootstock- two stages of grafting. scion compatibility (probably genetic) may be a factor of grafting success (Bong et al., 2000). The Point and height of grafting per tree also process of graft union between stock and scion in needs to be looked at. Too many points per tree or grafting involves intimate contact between the cell too high point of grafting might have some walls of the two partners. Initially, the walls that are drawbacks. Too many points per tree may cause brought into contact by implantation of the scion competition of nutrient, light and space among the form an extremely bond. This appears to be achieved grafting points, and definitely it will involved source by deposition of large quantities of pectin by both and sink relationship again. Alvim (1977) stated that partners. This bond can form even in incompatible a high yielding crop should have a fast growing interactions, in which the scion will ultimately be canopy, high net photosynthethic rate and an inherent rejected. In compatible interactions, the two partner ability to channel a relatively high proportion of its produce callus tissue from the outer region of their photosynthathes into fruits. Too many points of stem, near the contact point. The two calli grow grafting per tree will also increase the maintenance of together and as they approach, nodules appear on the cocoa cultural practices such as pruning, the canopy outer surfaces of the walls. Where the calli meet, the become too dense and might not be practical. nodules flatten and coalesce, forming what appears to However, if too little grafting point, the recovery be a pectin-rich middle lamella. The pectin is then process from VSD disease might take longer. The degraded in certain areas of a new wall, which most important trait for high productivity is rapid become relatively thin. Plasmodesmata form in the closure of the canopy at the beginning of the growing thin-walled regions, and symplastic continuity season. Until the canopy closes, not all the available between stock and scion is established. The light can be absorbed. After closure, nearly vertical recognition process which governs whether or not a leaves allow more light to penetrate into the canopy, graft will form between two partners is not and when light rays are more or less parallel to the understood. It has been suggested that lectins may be leaves, irradiance per unit leaf area is not high involved but there is little evidence for this (Brett and enough to saturate photosynthesis (Salisbury and Waldron, 1996). In canopy replacement, there will be Ross, 1992).
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Grafting height has yet to be determined. negative perception is only by setting up a Grafting point that is too high might end up with a demonstration plot in the farmer’s field itself. taller tree and difficult to maintain. Other drawback of the higher grafting point is the tree might crack Lacking of VSD resistant or tolerant clones easily when there is a typhoon. Generally the leaves to recommend to the farmers is also a challenge to near the top of the canopy have higher rates of implement this approach. Only a few clones are photosynthesis and become saturated at higher light observed to be tolerant to VSD. This problem is not intensities than those near the bottom of canopy. only faced in Malaysia, similar problem is also faced Such differences are correlated with progressive by other producing country such as Indonesia and decreases in stomatal conductance and mesophyll Papua New Guinea. A more rational mix for a more photosynthetic capacity from top toward the bottom stable polyclonal planting and to ensure a targeted of canopy. Although CO2 concentration, temperature level of production and quality over the long run and humidity differ with the canopy height, the would be 60% or thereabout of the stands be made of largest differences by far are found in the light one or more clones of proven performance in the intensity regime (Jarvis et al., 1976). While grafting locality, 40% of other lesser known clones which are point that is too low may result in the removal of compatible in all or most aspects (Bong et al., 2000). more pods bearing trunk (since most pods bearing at main branches), thus canopy replacement is not In the preliminary research of canopy efficient. Therefore, the correct height of grafting replacement, I found that successful grafting on the should be determined. mature tree was quite difficult to obtain since the trees were already infected by VSD and unhealthy, Finding the right clones to be grafted on the even though after fertilizer application. After a few existing susceptible clonal materials is another factor times of regrafting (three or more times), then the to think about. Instead of tolerance to VSD, the success rate was increased to 70%. It is a challenge to clones to be grafted must also from a moderate vigor the researchers to find other available method to graft plant. Since the grafting point might be more than 1 VSD infected mature tree in order to get satisfied meter height from the ground, it is important to budding success. It is even more challenging when choose the right clones which is not too vigorous as it this method may want to adopt in the farmer’s field. might be too big on the existing tree. Another reason to choose the right clones because the smaller All in all, the canopy replacement existing tree may not be able to support a larger scion management is still one of the convincing method to which causes imbalance in source and sink. adopt in rehabilitation of VSD infected mature cocoa together with other available methods such as If replanting of tolerant clones is to be pruning and timely fertilizer application. With new implemented, the budwoods required is less, only one canopy of tolerant clones which has no suitable budwood need to be grafted to a single seedling. infection site to Oncobasidium theobromae, the However, if canopy replacement is to be susceptible trees can be rehabilitated. Even though implemented, more budwoods are needed because the tolerant clone is still infected, the disease severity more than two budwoods are used per tree. Since is low and the plant can recover faster. Hopefully, if there are not many choices of VSD tolerant clones, it we replace the tops of the infected plant, the plant can seems to be difficult to find more budwoods of the maintain the physiological process at level necessary tolerant clones to be used in this management to sustain life even when exposed to stress including approach. disease stress.
However, among the pro and cons of applying canopy replacement method in relation to ACKNOWLEDGEMENTS the other available means of management approaches, there is another challenges need to be The author would like to acknowledge the Deputy faced. The biggest challenge is the implementation in Director General of Malaysian Cocoa Board for his cocoa field especially in the smallholders’ farm. To constructive comment on this paper. The project was convince the farmers of the new approach of VSD funded with Sciencefund grant, 05-03-13-SF0044. management method is not easy especially for the experienced farmers. To overcome the farmer’s
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REFERENCES Permanent Working Group for Cocoa Pests and Diseases (INCOPED). pp. 123-142. Alvim, P.T. (1977). Cacao. In: Ecophysiological of tropical crops. Ed. Alvim, P.T. and Kozlowski, Brett, C.T. and Waldron, K.W. (1996). Physiology T.T. Academic Press, New York. Pp.279-313 and biochemistry of plant cell walls. 2nd ed. Chapman and Hall. Pages 255. Bong, C.L., Chong, T. C., Lim, K.L. and Lim, G.T. (2000). Integrated crop management of cocoa Jarvis, P.G., James, G.B. and Landsberg, J.J. (1976). clonal plantings for higher productivity and Coniferous forest. In: Vegetation and the efficiency of crop protection in Sabah, atmosphere. Ed. Monteith, J.L. Vol.2. Academic Malaysia. Proc. INCOPED 3rd International press, London, New York. pp. 171-240. Seminar on Cocoa Pests and Diseases, 16-17 October, 2000. Kota Kinabalu: Malaysian Salisbury, F.B. and Ross, C.W. (1992). Plant Cocoa Board (MCB) and International Physiology, 4th Ed. Wadsworth, Inc.
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Short Communication
REPLACE YOUR TOPS WHEN YOU’RE INFECTED!
Rozita, O., Nik Aziz, N.M. and Azhar, I. Cocoa Research and Development Center, Malaysian Cocoa Board, P.O.Box 34, Jalan Jengka 23, 28000 Temerloh, Pahang, Malaysia. Email: [email protected]
Malaysian Cocoa J. 8: 60 – 63 (2015) ABSTRACT – Many attempts have been made to control vascular streak dieback (VSD) infection on mature cocoa trees. However, the methods used did not last long. The best and economical method to control VSD is by using resistant or tolerance clones, but it is not suitable for existing mature cocoa area. In this study, canopy replacement technique was adopted where branches of existing VSD susceptible mature cocoa tree were grafted with those from tolerant clones.
Keywords: Vascular streak dieback disease, Vascular streak dieback management, Canopy replacement
Vascular streak dieback (VSD) disease in cocoa is from VSD. However, none of these fungicides are caused by Oncobasidium theobromae. It is the major widely used in established cocoa plantations (Tan and disease problem in Malaysia and Southeast Asia Tan, 1988, Holderness, 1990, Varghese et al., 1992, including Indonesia and Papua New Guinea. It is Guest and Keane, 2007) and may no be realistic if windborne, leaf-penetrating and vascular pathogen. used solely (Jayawardena et al., 1984). Infection occurs directly into newly flush, undamaged leaves and the fungus grows entirely in Hyphae may emerge from the infected leaf the xylem vessels resulting in wilting of the shoot. scars and develop into a basidiocarp when favorable Oncobasidium theobromae can penetrate cocoa leaf weather exists. In wet weather, this structure is either through leaf lamina or petiole. According to visible as a white, flat, velvety coating over the leaf Prior (1979), it penetrate through smaller, scar and adjacent bark and after evening rainfall, the unhardened leaves, growing directly to the cuticle basidia develop on the basidiocarp. After midnight above veins. It can cause of seedling death if the until the early morning, formation and forcible hyphae penetrate into main stem. Under severe discharge of basidiospores occurs when the infection, it caused death to cocoa seedlings and basidiocarps are sufficiently moist. The killed the branches in mature tree. basidiospores are available only for a few hours in the morning. For the basidiocarps, it will remain In Papua New Guinea, very susceptible fertile for a week if attached at the branches and only types were killed by the vascular streak dieback for a day or two if the branches detached (Guest and disease (Keane, 1981). There are several Keane, 2007). The infected leaves do not showed any management methods to be considered in controlling symptoms for about 3 to 5 months. This incubation VSD disease. This includes quarantine measures to period explains why first symptoms occurs at the restrict transport of the fungus by human, host second or third flush behind the growing tips and resistance to the disease by using less susceptible appear after seasonal rainfall peaks (Prior, 1979). genotype, cultural practices such as planting disease- Under conducive condition; the spores of the fungus free seedlings in the field, the nursery is situated will penetrate the tree through unhardened leaves. away from the infected mature cocoa, pruning of the The fungus grows in the xylem of the leaf and infected branches, shade and canopy management to eventually enters the stem where it produces brown increase aeration and sunlight, biological control by streaks in the wood. In susceptible clones the fungus using epiphytic microbes to reduce leaf infection and spreads throughout the wood and eventually kills it, endophytic fungi and bacteria to protect against but in resistant ones the infection is often contained vascular colonization and fungicides (Guest and within the initially infected branch and the branch Keane, 2000). Musa and Tey (1984) reported that may recover (Prior, 1979). systemic fungicides were able to protect the seedlings
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Under heavily VSD infected areas, where increased flushing and more susceptible sites for the inoculums pressure is high, the current practice as infection and provided a conducive environment for mentioned earlier (pruning and fertilizer applications) peak sporulation of the fungus, it is advised that could not contain the epidemic. Re-emergence of the eradicative pruning should be most effective if disease usually occurs at 4 – 6 month after pruning. carried out immediately prior to disease favourable This is particularly prominent if the susceptible period. Since the global climate change, weather clonal materials such as PBC 130, PBC 131, PBC condition has become unpredictable, thus make the 159 and others are used. The use of resistant or timing of pruning difficult to ascertain. Furthermore, tolerance clones is the most cost effective method of frequent pruning also make field work more establishing cocoa farms in VSD high infected areas. laborious. From the physiological aspect, frequent Prior (1978) also suggested that the main approach to pruning will only interfere the translocation pathway control the disease involves propagation and planting in the sink source relationship. Photosynthesis is of resistant clones. Resistant types still become influenced by the rate of translocation of infected but infections are fewer and the pathogens photosynthetic products from sources to sinks. There grows more slowly and sporulates rarely (Prior, are many cultural practices that influence 1979). Lamin et al., 1996 had done the research on photosynthesis by affecting the activity of sink. the screening, identification and breeding for the Hence, pruning of infected branches may be expected resistant planting materials. They concluded that the to influence photosynthesis directly or indirectly by use of resistant planting materials is the most affecting some type of sink activity (Kozlowski, effective and economical method of controlling the 1992). disease. In a study conducted by Chong and Sidhu (1991) in a very high VSD pressure environment Cocoa leaves as a source and cocoa pods as showed that among the clones evaluated, PBC 123 a sink. When frequent pruning is applied, the tree was considered highly tolerant to VSD and obtained does not have enough energy to make pods, since higher yield. sources leaves supply sinks which they have direct vascular connection was removed. Sugar stored in However, planting of resistant clones are older leaves (source) may be mobilized to become a only suitable for the newly open area. In existing source of food when the newly shoot (sink) need planted area, sidegrafting of these resistant or energy for growth and development of the tolerance clones on the existing affected mature photosynthetic apparatus instead of making new cocoa were introduced. This method could only be pods. In addition, pruning is only suitable for the successfully implemented if the planting materials mature tree rather than cocoa seedlings. are of mixed hybrid origin (seedlings from the seed garden). However, this approach is less successful for ‘If you are infected replace your tops’ existing mature clonal cocoa planting. Sidegrafting refers to a new approach of controlling VSD in on the existing clonal planting material not only existing cocoa clonal planted area. Sidegrafting with cause high incidence of dieback but also involve tolerance clones was made at the selected branches of complete removal of the branches of the bearing the VSD infected plants (Figure 1). The tips of these plant. Farmers are reluctant to adopt this approach if infected branches finally replaced by tolerance clone. the original high yielding infected clonal trees are This approach of canopy replacement would maintain totally replaced. the main branches of the bearing plant and hence rehabilitated the existing VSD infected plant for Pruning of the infected branches is one of improvement and sustaining the productivity of the the methods in minimizing the VSD severity. area. In Malaysia, many efforts have been done to According to Zainal Abidin et al. (1984), pruning is find the best management to control VSD. The most the most popular and practical control measure popular approach is by pruning of infected branches practiced against VSD infection in field plantings. and followed by fertilizer application on the right However, frequency of pruning will contribute to time. However, managing VSD using this method increase in production of new flushes, providing will only last for a few months. The symptoms will more susceptible sites for infection. Since increase in re-appear if the weather condition is favorable. infection was related to a higher rainfall which
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Figure 1: Examples of grafting distances and grafting points established on mature cocoa for canopy replacement.
Compare to the other method that have been constructive comment on this paper. The project was suggested by previous researchers, canopy funded with Sciencefund grant, 05-03-13-SF0044. replacement with tolerance clones may not only reduced the severity of the VSD disease, but also maintain the main trunk of the existing clonal tree REFERENCES: (Figure 2). PBC 130 for example is known of its bigger bean size and higher yield. However, this Alvim, P.T. (1977). Cacao. In: Ecophysiological of clone was very susceptible to VSD. Most of the tropical crops. Ed. Alvim, P.T. and farmers who planted PBC 130 would like to keep Kozlowski, T.T. Academic Press, New York. their trees even though it is susceptible to VSD. In Pp.279-313 canopy replacement approach, the only part of the Bong, C.L., Chong, T. C., Lim, K.L. and Lim, G.T. tree that needs to replace is the canopy itself. The (2000). Integrated crop management of cocoa main trunk will still be maintain. It is not only clonal plantings for higher productivity and maintaining the existing trunk, but at the same time, efficiency of crop protection in Sabah, the farmers also can harvest both pods from the Malaysia. Proc. INCOPED 3rd International existing tree and the newly grafted canopy. Another Seminar on Cocoa Pests and Diseases, 16-17 benefit of canopy replacement management is the October, 2000. Kota Kinabalu: Malaysian trees will bear fruits earlier than replanting. If Cocoa Board (MCB) and International replanting of tolerant clones is adopted, the tree will Permanent Working Group for Cocoa Pests only start bearing fruits at two and a half year after and Diseases (INCOPED). pp. 123-142. planting, while in this management, the tree usually Brett, C.T. and Waldron, K.W. (1996). Physiology bear fruits only after one and a half year of grafting. and biochemistry of plant cell walls. 2nd ed. Chapman and Hall. Pages 255. Chong, C.F. and Sidhu, M. (1991). Performance of ACKNOWLEDGEMENTS PBC clones in Peninsular Malaysia and Sabah. Proc. Interacting cocoa conference challenges The author would like to acknowledge the Deputy in the 90’s. Kuala Lumpur. Director General of Malaysian Cocoa Board for his
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Figure 2: Pod bearing at both existing clone (PBC130) and newly grafted clone (PBC123) at main branches
Guest, D. and Keane, P.J. (2007). Vascular streak Keane, P.J. and Prior, C. (1992). Biology of vascular dieback: A new encounter disease of cacao in streak dieback of cocoa. In: Cocoa Pest and Papua New Guine and Southeast Asia caused Disease Management in Southeast Asia and by the obligate Basidiomycete Oncobasidium Australasia. FAO Plant Production and theobromae. Phytopathology 97 (12): 1654- Protection Paper 112. Food and Agriculture 1657 Organization of the United Nations. 75-83. Holderness, M. (1990). Control of vascular streak Kozlowski, T.T. and Pallardy, S.G. (1997). dieback of cocoa with triazole fungicides and Physiology of woody plants. 2nd ed. Academic the problem of phytotoxicity. Plant Pathology Press. Pg. 411. 39: 286-293. Lamin, K., Lee, M.T., Bong, C.L. Chong, T.C. and Jayawardena, M.P.G., Mohan, E. and Musa, M.J. Phua, P.K. (1996). Breeding for resistance to (1984). Possible method to minimize losses disease in Malaysia with special reference to due vascular streak dieback. Proc. Int. Conf. vascular streak dieback. Ingenic workshop on Cocoa and Coconuts. Pg. 385-395. contribution of disease resistance to cocoa Jarvis, P.G., James, G.B. and Landsberg, J.J. (1976). variety improvement, 25-26th November, Coniferous forest. In: Vegetation and the 1996, Salvador, Brazil. atmosphere. Ed. Monteith, J.L. Vol.2. Layne, D.R. and Flore, J.A. (1992). Photosynthetic Academic press, London, New York. pp. 171- compensation to partial leaf reduction in sour 240. cherry. J. Am. Soc. Hortic. Sci. 117: 279-286 Keane, P.J. (1981). Epidemiology of vascular streak dieback of cocoa. Ann. Appl. Biol. 98: 227- 241.
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Aims & Scope
Malaysian Cocoa Journal aims To create and maintain an active communication network for the exchange of information among scientists and the cocoa industry To support the research and professional activities of researchers in cocoa through publications To publish original research on cocoa and thereby added to the existing database of scientific knowledge.
The Journal will be published once a year initially, subject to the discretion of the Chief Editor. The journal cover original scientific contributions dealing with cocoa, cocoa products and byproducts as well as articles dealing with cocoa extension, licensing, marketing and other activities relating to the cocoa industry.
Manuscript Preparation
Layout of papers Full papers, figures, and tables must be typed double spacing on A4 paper (21.0 cm x 29.7 cm) with margins of at least 4 cm on the left, 2 cm on the right, 4 cm on the top and 4 cm at the bottom.
Abstract Each full paper (research) must include an informative summary not exceeding 250 words. It should contain all essential information regarding objectives, materials and methods, results and conclusions, but excluding figures, sectional headings, tables and references.
Full paper A paper which reports the results of research should be divided into the following sections: (i) Abstract, (ii) Introduction, (iii) Materials and Methods, (iv) Results, (v) Discussions, (vi) Conclusions, (vii) Acknowledgements and (viii) References. No full paper should exceed 3,000 words (except for review papers), figures and graphs. Each table, figure or graph is considered being equal to the number of words, which could have been typed in the equivalent space. Any contribution in excess of this length may not be published in the Journal. Papers are considered to have been submitted in the final form after they have been fully checked for typographical and other errors. The full paper should be in English. Manuscripts are to be written using Times New Romans, font size 10. A full guideline for manuscript writing is obtainable upon request.
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Send three of copies of the manuscript, disk in a heavy-paper envelope, enclosing the copies and figures in cardboard, if necessary, to prevent the photographs from being bent. Place photographs and transparencies in a separate heavy-paper envelope. Manuscripts must be accompanied by a covering letter to the following address,
The Editor, Malaysian Cocoa Journal, Malaysian Cocoa Board, Locked Bag 211, 88999 Kota Kinabalu, Sabah, Malaysia. (c/o Dr. Rosmin Kasran)
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