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Malaysian Cocoa Journal, 8/2014 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 1 Malaysian Cocoa Journal, 8/2014 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.
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