Proceeding of Postgraduate Seminar 2016

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2 Proceeding of Postgraduate Seminar 2016 Hak Cipta Terpelihara Tidak dibenarkan mengeluar ulang mana-mana bahagian artikel, ilustrasi, dan isi kandungan dalam apa jua bentuk dan dengan apa cara sekalipun sama ada secara elektronik, fotokopi, mekanik, rakaman, atau cara lain sebelum mendapat izin bertulis daripada Pengarah, Penerbit UMT, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu, Malaysia All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic, or mechanical including photocopy, recording or any information storage and retrieval system, without permission in writing from Director, Penerbit UMT, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu, Malaysia. ISBN : e

3 CONTENTS MESSAGE FROM THE DEAN 3 ORGANIZING COMMITTEE 4 LIST OF EXTENDED ABSTRACTS 5 EXTENDED ABSTRACTS 7 2

4 MESSAGE FROM THE DEAN Assalamualaikum Warahmatullahi Wabarakatuh and Salam Sejahtera, Alhamdulillah, this year PPSTM once again has organized Postgraduate Seminar. The aim of this annual seminar is to bring together all our postgraduate students to present the progress in their MSc and PhD studies. It is hoped that this seminar will improve their communication skill as well as their knowledge on research methodology. Besides, we hope the seminar will give new ideas and solutions for postgraduate students to improve their studies. I would like to thank the organizing committee to make this seminar a reality. My gratitude also goes to the lecturers for their endless guidance and support to the postgraduate students to help them to finish on time. Last but not least, thank you to all the postgraduate students for their cooperation in realizing this seminar. ASSOC. PROF. DR. AMIZA MAT AMIN DEAN SCHOOL OF FOOD SCIENCE AND TECHNOLOGY UNIVERSITI MALAYSIA TERENGGANU 3

5 ORGANIZING COMMITTEE POSTGRADUATE SEMINAR 2016 SCHOOL OF FOOD SCIENCE & TECHNOLOGY ADVISOR Assoc. Prof. Dr. Amiza Mat Amin CHAIRMAN Assoc. Prof. Dr. Chuah Tse Seng SECRETARIAT Tuan Mohd Najwa Fuad Tuan Man Farah Idayu Abdul Rahman SCIENTIFIC Dr. Ramisah Mohd Shah Dr. Fauziah Tufail Ahmad Dr.Wan Zawiah Wan Abdullah Dr. Nor Idzwana Mohd Idris Dr. Khadijah Saad Dr. Tengku Rozaina Tengku Mohamad DR. Azizah Mahmood PUBLICITY Dr. Aidilla Mubarak En. Ahmad Azman Abdul Malik PUBLICATION Dr. Iffah Hazirah Mohd Nawi Norhafizah Ghazali Siti Noni TECHNICAL Ramlah Awang Mohd Norazka Abdul Malek Rohana A. Wahab Mohd Fauzi Jusoh Zikrie Radjeni FOOD & BEVERAGE Nor Azura Azmi Faridah Mohd Isa GIFTS & SOUVENIRS Armadiana Arifin Norhafizah Ghazali 4

6 LIST OF EXTENDED ABSTRACTS Abstracts Haslinda Wan Hamat, Mohd Nizam Lani, Yusnita Hamzah 2 and Wan Bayani Wan Omar Page 7 Microbiological quality of keropok lekor from selected food premises in Kuala Terengganu and Marang Nurul Farhanah Mohd Aluwi, Nor Hayati Ibrahim and Yusnita Hamzah 10 Properties and stability of salad dressing - type emulsions containing rose cactus (Pereskia bleo) mucilage, xanthan gum and locust bean gum Normardiana Jaafar, Siti Nur afifah Jaafar and Norainy Mahyudin 13 Pilot testing of food safety knowledge, attitude and perception towards food handlers hygiene practice among night market food consumers Siti Fatimah Zakaria, Mohd Nizam Lani, Chuah Tse Seng, Fisal Ahmad and Khairul Mazmi Ahmad 17 Antifungal activity of lactic acid bacteria against pathogenic Sclerotium rolfsi of Capsicum annum plant Norhayati Binti Abd Hadi, Norizah Mhd. Sarbon, Zamzahaila Mohd Zin and Hayati Mohd Yusof 21 Physico-chemical properties and activities of antioxidants compounds in honey bee honey and stingless bee honey Ain Najwa Khairul Anwar, Rosazlin Abdullah, Zuraida Abdul Rahman, Yusnita Hamzah and Wan Zaliha Wan Sembok 27 Effects of different arbuscular mycorrhiza species and its rates on the growth performance of hempedu bumi (Andrographis paniculata Nees) grown on bris soil Muhammad Shahrul Hafiz Elham, Ng Lee Chuen, Sariam Othman and Mohd Razi Ismail 31 Physical barrier mechanism of si-mediated rice against Pyricularia Oryzae Norkaspi Khasim, Adzemi Mat Arshad and Khalid Haron 36 Integration of bamboo (Gigantochloa albociliata) with oil palm and its effect of nutrient cycling on oil palm yield Nura Adila Zahari, Aziz Ahmad and Ng Lee Chuen 40 In vitro study of flavonoid phytoalexin sakuranetin against Pyricularia oryzae 5

7 Wan Nur Suzani Sazleen Wan Shafiin and Chuah Tse Seng 43 Evaluation of pre and post emergence herbicidal activity of 2, 4-di-tertbutylphenol (2,4-DTBP) Haruna Yahaya Rawayau, Adzemi Mat Arshad and Wan Zaliha Wan Sembok Potential impact of organic fertilization on corn growth and yield Naimah Abdul Halim, Chuah Tse Seng and Shamsul Bahri Abd Razak Effects of 2,4-di-tert-butylphenol and selected synthetic herbicides on electrolyte leakage of weeds Rul Hajar Muda, Ng Lee Chuen and Khairulmazmi Ahmad 54 Biocontrol potential of fluorescent pseudomonads on rhizoctonia root rot disease caused by Rhizoctonia solani on chilli Azmi Ismun, Aidilla Mubarak, Shamsul Bahri Abd Razak and Marinah Mohd Ariffin 58 The determination of polyphenols in latex of Hevea brasiliensis and rubber processing effluent via spectrometry, spectrophotometry and chromatography 6

8 MICROBIOLOGICAL QUALITY OF KEROPOK LEKOR FROM SELECTED FOOD PREMISES IN KUALA TERENGGANU AND MARANG Haslinda Wan Hamat 1,2*, Mohd Nizam Lani 2, Yusnita Hamzah 2 and Rozila Alias 3 1 Food Safety and Quality Laboratory, Terengganu Health State Department, Kg. Bukit Tunggal, Kuala Terengganu, 2 School of Food Science and Technology, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu, 3 Institute of Bio-IT Universiti Selangor, Jalan Zirkon A 7/A, Seksyen 7, Shah Alam, Selangor, Malaysia, *Corresponding author gsk2295@umt.edu.my Abstract Microbiological quality is an evaluation of microbiological profiles of food products and it is used to measure the safety of ready-to-eat products. Since keropok lekor is widely consumed, it is important, to study the microbiological quality of keropok lekor from raw materials until ready-to-eat. A total of 176 samples were taken in eight keropok lekor processing premises in Kuala Terengganu and Marang. Aerobic Plate Count (APC) and Enterobacteriaceae counts ranged between <1 to 6.70 log 10 CFU/g and <1 to 6.00 log 10 CFU/g, respectively. Samples were found to have coliform in the range of <1 to 6.00 log 10 CFU/g. Coagulase positive Staphylococci counted 3.30 and 4.10 log 10 CFU/g were isolated from sago starch and fish flesh, respectively. E. coli was present in 12 samples and ranged from 1.1 to 4.7 log 10 CFU/g. Two strains of Salmonella were isolated form fish flesh and dough samples, while Vibrio parahaemolyticus was detected in one fish flesh sample. None of the samples was found to have Vibrio cholerae and Listeria monocytogenes. Further study will be carried out to determine the multiple drug resistant bacteria isolated from various sources of handling, and preparation of keropok lekor. Introduction Keropok lekor or sausage-like fish product is known as a famous traditional Malaysian cuisine especially in the East Coast, Terengganu. This traditional popular product has gained a nationwide potential markets and widely sold in local market, especially for school canteens, night markets and hawker stall. Indicator microorganisms are used as indicators of hygiene and sanitation on food, equipment and environmental surfaces. The most commonly used indicators in the food industry are aerobic plate count, Enterobacteriaceae and coliforms. Examining food products for indicator organisms can provide simple, reliable, and rapid information about processing failure, post-processing contamination, general level of hygiene and presence or the absence of foodborne pathogen to monitor the chain of food production. Raw fish is the main ingredient in production of keropok lekor, its quality is an important factor which influences the quality of end product. Pathogenic bacteria such as Vibrio cholerae, Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella spp. and Escherichia coli are naturally present on live fish. If the raw material is contaminated, the possibility for pathogens to transfer to food processing environment, utensils and cooked food (cross contamination) is significant. Routine application of good sanitation practices should be capable of eliminating pathogens in food processing plant, but if contamination levels are high or the sanitation procedures are inappropriate, the microorganisms may be able to establish themselves, then multiply and become resident. Insufficient hygiene practices in food processing line may also develop the persistence of foodborne pathogens where the pathogens colonised and survived for prolonged period of time in the food processing 7

9 environment and surface. These conditions may influence the microbiological safety of keropok lekor. The objective of this study was to determine the microbiological profile of raw materials, food contact surface, food handlers hand and ready-to-eat keropok lekor. Materials and Methods Sampling Raw fish flesh, sago starch, ice, dough, ready-to-eat keropok lekor, dipping sauce, chilli paste, swabs (sago starch container, freezer internal surface, mixer bowl, mixer hood, chopping board, plastic packaging, display container and food Handler s Hand) from keropok lekor premises were aseptically sampled as explained in Bacteriological Analytical Manual (2003) and ISO 18593:2004(E). All samples were transported to laboratory by maintaining temperature of 0-4 C and analyzed within 24 hours. Microbiological Analysis All samples were analysed for Aerobic Plate Count (AOAC Official Method ), Enterobacteriaceae (AOAC Official Method ), Coagulase Positive Staphylococci (ISO :1999/Amd.1:2003(E)), Coliforms & E. coli (AOAC Official Method ), Salmonella (ISO 6579:2002 (E)), Vibrio cholerae & Vibrio parahaemolyticus (ISO/TS :2007 (E)) and Listeria monocytogenes (ISO :1996/Amd.1:2004(E)). Results and discussion Microbiological status Aerobic Plate Count (APC) in raw materials (raw fish flesh, sago starch, ice, dough and chilli paste) and swabs ranged from < 1 to 6.70 log 10 CFU/g and < 1 to 6.40 log 10 CFU/g, respectively. While, dipping sauce and keropok lekor had APC range of < 1 to 5.1 log 10 CFU/g and 1.80 to 5.50 log 10 CFU/g, respectively. APC in all keropok lekor samples were at the satisfactory level which were less than 6.00 log 10 CFU/g as referred to Food Act 1983 (Act 281) & Regulations 1. Meanwhile, counts of APC above 5.00 log 10 CFU/g in ready to eat (RTE) food were faced as a potential risk due to the presence of pathogens 2. Only one RTE sample which was keropok lekor had APC of 5.50 log 10 CFU/g. Enterobacteriaceae include some of the important human pathogens including Salmonella, Shigella, Yersinia and pathogenic Escherichia coli. Enterobacteriaceae analyses showed results range of < 1.00 to 6.00 log 10 CFU/g and < 1.00 to 5.90 log 10 CFU/g in raw materials and swab samples, respectively. While Enterobacteriaceae in keropok lekor samples were in the range of < 1.00 to 4.30 log 10 CFU/g and less than 1.00 log 10 CFU/g in all dipping sauce samples. Raw materials and swab samples were found to have coliforms in the range of < 1.00 to 6.00 log 10 CFU/g and < 1.00 to 5.90 log 10 CFU/g, respectively. There are less than 1.00 log 10 CFU/g coliforms in all dipping sauce samples. Coliforms count in keropok lekor ranged between < 1.00 to 3.00 log 10 CFU/g. According to Food Act 1983 (Act 281) & Regulations 1, all ready-to-eat (RTE) fish and fish products should have coliforms less than 1.70 log 10 CFU/g. Coliforms in three keropok lekor samples were at the unsatisfactory level which counted 3.00, 2.10 and 2.10 log 10 CFU/g. E. coli were isolated from 12 samples include fish flesh, dough, keropok lekor, keropok display container swab and food handler s hand swab. The detection of E. 8

10 coli in keropok lekor and display container swab indicated fecal contamination in RTE keropok lekor and the contamination was transferred to display containers. Cross contamination like this is common in many food premises which lack of implementation of food hygiene practices. Hand washing procedure may not adequately performed by food handlers due to the detection of E. coli in their hand swabs even though all swabs were sampled after they washed their hands. E. coli is a common inhabitant of the intestinal tract of humans and animals and can be easily disseminated in different ecosystems through the food chain and water. Coagulase positive Staphylococci were reported to be 3.30 and 4.10 log 10 CFU/g and were detected in fish flesh and sago starch samples, respectively. Salmonella was detected in one fish flesh and dough, while Vibrio parahaemolyticus was detected in one fish flesh sample. Vibrio cholerae and Listeria monocytogenes were not detected in all samples tested. Conclusions This study found that 37.5% keropok lekor samples have coliforms count at the unsatisfactory level which could be concluded that sanitation and hygiene practice were not well implemented in those processing premises. E. coli was the most prevalent bacteria in all premises and it is also detected in ready to eat keropok lekor and its display container swab. References 1. Food Act 1983 (Act 281) & Regulations. (2015). Laws of Malaysia. International Law Book Services. p Balzaretti, C. M. and Marzano, M. A. (2013). Prevention of travel-related foodborne diseases: Microbiological risk assessment of food handlers and ready-to-eat foods in northern Italy airport restaurants. Food Control 29:

11 PROPERTIES AND STABILITY OF SALAD DRESSING-TYPE EMULSIONS CONTAINING ROSE CACTUS (Pereskia bleo) MUCILAGE, XANTHAN GUM AND LOCUST BEAN GUM Nurul Farhanah Mohd Aluwi *, Nor Hayati Ibrahim and Yusnita Hamzah School of Food Science and Technology, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu, Malaysia *Corresponding author Abstract In this study, rose cactus (Perekia bleo) mucilage (RCM), xanthan gum (XG) and locust bean gum (LBG) and their ternary blends were used to prepare salad dressing-type emulsions at 2% (w/w) of total emulsion formulation. The emulsions were examined in terms of ph, droplet size, apparent viscosity and creaming stability. Of all emulsions studied, Emulsion D (100% RCM) exhibited the highest ph value (lowest acidity value) compared to others. Two emulsions, Emulsion C (35% RCM: 35% XG: 30% LBG) and Emulsion E (100%XG) showed the smallest droplet diameter and were significantly different (p<0.05) from other emulsions. In terms of viscosity, Emulsion A (25% RCM, 30% XG and 45% LBG), and B (30% RCM, 30% XG,40% LBG), showed significant (p < 0.05) higher viscosity as compared to Emulsion D which contained pure RCM. It was also found that, application of ternary polysaccharide blends significantly (p<0.05) resulted in higher creaming stability of the respective emulsions as compared to pure RCM and XG. This study demonstrated improvement of viscosity and stability of RCM-containing emulsions by blending with other commonly known polysaccharides (XG and LBG) which attributable to their synergism. Introduction Oil-in-water (O/W) emulsions are widely used in many food industrial processes and form the basis of many foods products. Due to energetically unfavourable between the contacting oil and water molecules, emulsions are thermodynamically unstable systems. Mixture of polysaccharides enhances the stability of the emulsion especially towards creaming by improving the viscosity of the aqueous phase.rose cactus (Perekia bleo) mucilage (RCM)is a complex polysaccharide that can be extracted from the plant leaves. Generally, the mucilage could act as stabilizer in the food emulsion conferring long-term emulsion stability. Its performance could be tremendously improved when blended with other thickening polysaccahrides.in our preliminary study, the ternary blends of RCM, xanthan gum (XG) and locust bean gum (LBG) significantly improved the water holding capacity (WHC) of RCM-XG-LBG which varied from % % (unpublished data).thus, it is expected that overall performance of RCM in emulsion system particularly related to WHC (e.g. creaming stablity) could be improved by this simple blending technique. The objective of this study was to determine the effect of RCM-XG-LBG interactions to the physical properties and stability of salad dressing-type O/W emulsions. Materials and Methods Crude mucilage from rose cactus leaves was extracted by using 0.14 M NaOH solution at 70 C and purified with saturated barium hydroxide, followed by drying in the oven overnight at 40 C.The polysaccharide (RCM, XG and LBG) dispersions (2% w/w) were first individually prepared by dispersing polysaccharide powder in deionized water 80 C. Then, blends of 25% RCM:30% XG:45% LBG, 30% RCM:30% XG:40% LBG and 35% RCM:35% XG:30% LBG were prepared. The emulsions 10

12 (125g) were prepared in a lab-scale proportion using 40% oil, 10% water, 4% vinegar, 6% egg yolk and 40% gum blend. The final gum concentration was 2%and emulsification was undertaken using an ultrasonic homogenizer (Cole Panner, CP 505, USA). Each emulsion was determined for ph by a ph meter. Droplet microstructure and droplet size were obtained by using an Olympus BH-2 microscope (Tokyo, Japan) equipped with a Dino-Eye Microscope Eye-Piece Camera (AM423X). Apparent viscosity was measured from flow curves obtained from a rheometer (DHR Series) measurement. The creaming stability at 0 and 15 day were determined under centrifugation force at 3000 rpm. Significant differences among samples were further tested using a One-way ANOVA with Tukey s Multiple Comparison at significant level of p < 0.05 using a Minitab (14) statistical software package. Results and discussion Based on Table 1, RCM seemed to positively modify the lowering effect on ph might be from XG and LBG.The emulsion of ph values were in the range of for freshly prepared emulsions which will directly promote the stability of the emulsions as stated by the previous study 1 that optimum ph were 2.5 to 4.5. From microstructure observation, small droplets were found to be the most abundant in all emulsions. It might be due to the type of homogenizer used i.e. ultrasonic homogenizer which able to generate a very small droplets. Emulsion C (35% RCM: 35% XG: 30% LBG) and Emulsion E (100%XG) had the smallest droplet diameter and they were significantly different (p<0.05) from other emulsions due to hydrophobicity and substantial surface activity of the polysaccharide 2. In addition, emulsion with polysaccharide blends exhibited higher apparent viscosity compared to pure RCM which might be due to the fact that emulsion with a smaller average droplet diameter will constitute of higher viscosity compared to the emulsion with a larger average droplet diameter. As supported by previous study 3, the increase in droplet size will increase the average distance of separation among the droplets and decrease the number of the droplet per unit volume of the emulsions. Table 1: The ph, droplet size and apparent viscosity of different formulation emulsions Type of emulsions ph Droplet size (µm) Apparent Viscosity (Pa.s) A (25% RCM : 30% XG : 45%LBG) 3.17±0.01 ab ±0.33 b 0.57 ± 0.17 bc B (30%RCM : 30% XG : 40% LBG) 3.14 ±0.01 b 12.25± 0.29 b 0.22 ± 0.04 bc C (35% RCM : 35% XG : 30% LBG) 3.12 ±0.03 b ±0.53 c 0.15 ± 0.03 c D (100% RCM) 3.27 ±0.08 a ± 1 ab 0.09 ± 0.02 c E (100% XG) 3 ±0.03 c 9.99 ±1.26 d 1.67 ± 1.45 b F (100% LBG) 2.93 ±0.07 c ±0.29 a 3.87 ± 0.52 a Means are reported from three independent experiments (n = 3). Means with the same superscript within the same column are not significantly different at α = Application of ternary polysaccharide blends significantly (p<0.05) resulted in higher creaming stability of the respective emulsions compared to pure RCM and XG (Table 2). The stored emulsions were easily separated because of weak forces in the systems facilitate the mobility and degree of oil droplet interaction with each other leading to emulsion instability. The results were similar with the emulsion containing 0.2% L.perfoliatum seed gum which less stable during storage at 4 C and showed visible phase separation 4. This is much due to the presence of droplet flocculation as observed in the respective micrograph (data not shown). Upon storage, flocculated droplets tended to coalesce and thus separated into free oil after centrifugation. The emulsion D (100% RCM) and E (100% XG) showed the highest syneresis due to a 11

13 weak structure with the low viscosity of its continuous phase. This is due to the Van der Waals attraction whereby interaction among droplets were weak causing the droplet flocculation.this ultimately contributed to formation of large droplets (coalesce), causing the emulsions separated into free oil after storage and lead to destabilization of the emulsion system. Table 2: The effects of different emulsion formulation on creaming stability at 0 days and 15 days Type of emulsions Creaming stability at 0 days Creaming stability at 15 days A (25% RCM : 30% XG : 45%LBG) ± 0.01 a ± 0.63 b B (30%RCM : 30% XG : 40% LBG) ± 0.01 a ± 0.54 b C (35% RCM : 35% XG : 30% LBG) ± 0.04 b ± 0.09 b D (100% RCM) ± 0.01 c ± 1.13 c E (100% XG) ± 1.71 d ± 0.01 d F (100% LBG) ± 0.01 a ± 0.01 a Means are reported from three independent experiments (n = 3). Means with the same superscript within the same column are not significantly different at α = 0.05.). Conclusions The findings signify that ternary polysaccharides blends of RCM-XG-LBG exhibited improvements in emulsions by contributing to higher viscosity and better stability towards creaming, compared to pure polysaccharides (except for LBG). Both pure RCM and XG emulsions had the lowest viscosity and creaming stability, respectively due a weak network structure which could be overcome by blending them with LBG in ternary system. References 1. Guzey, D., and McClements, D.J. (2006). Formation, stability and properties of multilayer emulsions for aplication in the food industry. Advances in Colloid and Interface Science 128: Sengkhamparn, N., Verhoef, R., and Schols, H.A. (2009). Characterization of cell wall polysaccharide from okra (Abelmoschus esculentus (L.) Moench). Carbohydrate Research 344: Nor Hayati, I., Che Man, Y.B., Tan, C.P. and Nor Aini, I. (2007). Stability and rheology of concentrated O/W emulsions based on soybean oil/palm kernel olein blends. Food Research International 40: Soleimanpour, M., Koocheki, A. And Kadhodaee, R. (2013). Effect of Lepidium perfoliatum seed gum addition on whey protein concentrate stabilized emulsions stored at cold and ambient temperature. Food Hydrocolloids 30(1):

14 PILOT TESTING OF FOOD SAFETY KNOWLEDGE, ATTITUDE AND PERCEPTION TOWARDS FOOD HANDLERS HYGIENE PRACTICE AMONG NIGHT MARKET FOOD CONSUMERS Normardiana Jaafar 1*, Siti Nur afifah Jaafar 1, Nor Ainy Mahyudin 3 1 School of Food Science and Technology, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu, 2 Faculty of Food Science and Technology, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia Abstract *Corresponding author gsk2297@pps.umt.edu.my Recently, food poisoning cases have been increase which has lead to impacts in term of health and economy in developed countries. Despite the current situation, the night market foods still famous among consumers. Street food vendors in night market has been risen in recent years, however this scenario raised public health challenges. The aim of this study is to asses the level of knowledge, attitude and perception on food handlers practice among consumers. This research is beneficial in understanding the curent situation of food safety knowledge, practice and perception towards food handlers practice among consumers besides could be used to improve food safety at night market. At the early stage, pilot test was conducted to test the reliability and validity of the instrument that will be used in the real study. This stage was important to test whether the articulation of the method(s) and instruments selected for use in a research adequate to meet research objectives. The pilot test was conducted at four states in Malaysia; Perak, Perlis, Selangor, Terengganu. A total of 120 consumers were investigated from April to May By using Cronbach Alpha test, the reliability coefficient test was 0.70 and above. The finalized questionnaire consist of 65 items-knowledge (32), attitude (12), practice (15), familiarity (3), and purchase intention (3). Overall, the articulation of the method and instrument designated was satisfactorily reliable, valid and adequate to be used in a research to assess the level of consumers food safety knowledge, consumers attitude towards food safety and perception on food handlers practice among consumers towards night market foods purchase intention with familiarity with night market foods as moderating factor. Introduction Food is the basic need for human survival. Therefore, the growth in the food sector is more impressive nowadays. However, despite this diversity and improvement in food industry, food safety plays an important role as food can be easily contaminated especially during the time of production and consumption. The most common mistakes in food handling were serving foods that already contaminated, getting foods from risky sources, reheating the leftover foods, insufficient cooking, stored and cooled the foods in a wrong ways besides taking too much time exposed the foods at room temperature 1. Food borne illnesses are increasing in numbers in recent years either in developed or in developing countries. Due to increasing in number of cases, foodborne illnesses have attract public attention. In 2007, World Health Organization revealed that the incidence rate of food poisoning was raise at 52.6 per 100,000 people which is one of the top five common disease in Malaysia. In 2006, the incident rate for food poisoning was per 100,000 people as reported by Ministry of Health. In time with the report by WHO, there was found raise in the number of episodes of foodborne disease recorded by several states in Malaysia 2. Despite current situation, the night market foods still famous among consumers. Hence, this study has been conducted to assess the level of knowledge, attitude and 13

15 perception on food handlers practice among night market foods consumers (KAP). A KAP survey is conducted to examine human behaviour regarding certain issue. KAP survey is used to classify what people know (Knowledge), how they feel (Attitude) and what they do (Practice). Before initiating any evaluation towards night market foods consumers, development of a valid and reliable questionnaire is definitely important to study consumers level of knowledge, attitude and perception on food handlers hygiene practices. Hence, this research was conducted in order to develop and access the validity and reliability of the consumers knowledge on food safety, consumers attitude towards food safety and consumers perception on food handlers hygiene practices as independent variable besides familiarity with night market foods as moderating variable and purchase intention of night market foods as dependent variable. Materials and Methods This KAP food safety study was using closed-ended questionnaire among night markets foods consumers. Development of questionnaire was done by adapting measurement items and scales from previous research Tan et al (2013), Sun et al (2012) and Bruhn and Schutz (1999). Some modification was made to the questionnaire in order to make it applicable to Malaysian culture and understanding. Besides that, some items were added regarding food safety knowledge in order to test respondents in many branches of food safety knowledge. Questionnaire was designed bilingual (English and Malay) to obtain information on demographic of respondents, consumers knowledge on food safety (32 items), consumers attitude towards food safety (13), consumers perception on food handlers hygiene practices (15), familiarity with night market foods (3) and purchase intention of night market foods (3). The knowledge items consist of multiple choice items with three answer options; yes, no and do not know. From 32 statements, there are 23 statements require yes and 9 statements with no answers. 1 point was rewarded for the correct answer while 0 point for wrong or do not know answer. 10-point Likert scale was used to measure questions related to consumers knowledge on food safety, consumers attitude towards food safety, consumers perception on food handlers hygiene practices, familiarity of night market foods and purchase intention of night market foods. At early stage, the objectives of research were determined so that the researcher understands the aim and goal of this research. Based on the objective, the variables to measure were identified. Then, items for each part of variable were generalized. Validation of the content of the questionnaire was made by expert panel followed by face validation by potential respondents before proceed to pilot test. Four states with highest rate of food poisoning cases in Malaysia for the year 2012 were selected as study locations i.e.- Perak, Perlis, Selangor, Terengganu 3. Pilot testing was conducted from April to May 2015 to examine the reliability and validity of questionnaire. The reliability of a scale shows how free it is from random error and Cronbach s alpha is the most commonly used statistic which act as indicator of the average correlation among all the items that make up the scale. Meanwhile, the validity of scale refer to degree to which it measure what it is supposed to measure and it involved the collection of empirical evidence concerning its use 4. Data collection was carried out by visiting housing area which is located within 5km radius from night market. Only selected houses were selected and household who consumed night market foods were approached. The purpose and procedure of study was explained thoroughly before respondents started to answer the questionnaire. Respondents free to ask researcher any doubt regarding the statements in the questionnaire. There was no time allocated for the respondents to 14

16 finish answer the questionnaire and they have to complete answer the questionnaire and returned it back to researcher. Results and discussion Among 120 night market foods consumers involved in this pilot test study, 54.2% were female and majority of them lived in suburb area (50.8%). Majority of consumers aged below than 25 years old (55.8%) and single marital status (61.7%). About 82.5% of respondents have income of below than RM1500. This may become the reason why they are interested buying night market foods due to low price of night market foods. Most of the respondents worked in government sector (25.8%). About 22.5% of respondents had attended food safety education while more than half of them never attended or received education regarding food safety. From the amount of the respondent whom get food safety education, the most common types of food safety education was attending class. The consumers knowledge on food safety items of this questionnaire were developed based on the previous study and also from the literature review. In this study, food safety includes aspects on personal hygiene, hygiene practice, disease carried by foods, microorganism, cross-contamination, temperature control, foodborne illness and potentially hazardous foods (PHF). The overall Cronbach s alpha coefficient for consumers attitude towards food safety and perception on food handlers hygiene practice were and respectively. While for the familiarity with night market foods as moderating factors the Cronbach s alpha was For the dependent variable, the Cronbach s alpha of purchase intention was While different levels of reliability are required, depending on the nature and purpose of the scale, Nunnally and Bernstein 5 recommends a minimum level of 0.7. Briefly, all categories in the questionnaire showed that the scale can be considered reliable with the sample as the values of Cronbach s alpha were within the range of recommended value. For the consumers attitude towards food safety, overall of the relationship between the items in this variable consists of weak, moderate and strong relationship when min r=0.184 and max r= There are significant moderate linear relationship between most of the items in this variable and all items represent the variable are valid. For the consumers perception on food handlers hygiene practices there is a significant low to moderate relationship between all the items represent this variable (r=0.180 to r=0.698). All the items in familiarity with night market foods variable show significant moderate to strong relationship between all the items with min r= and max r= The items which has strong significant relationship (out of 3 items) is I have knowledge about night market foods (r = 0.889, p=0.01). Hence all items represent familiarity of night market foods are valid. For the purchase intention of night market foods there is a significant moderate linear relationship between purchase to save money and definitely try night market foods (r= 0.486, p= 0.05). There is weak linear relationship between item buy to save money with the items will consider to purchase night market foods and definitely try night market foods with r=0.328 and r= respectively (has mostly weak relationship between all the items). Hence, all items representing purchase intention except buy to save money are valid. Based on analysis of reliability and validity test, some of the modifications and improvement were done. All the items in consumers attitude towards food safety were retained except item food handlers in night market should wear mask to reduce the risk of food contamination. Besides that, item I will buy foods from food handlers whom prepared a meal in advance also was removed in the variable consumers 15

17 perception on food handlers hygiene practices while the rest were retained. Items I have experienced having night market foods was modified. Modification in term of reconstruct the sentence to I ever have eaten night market foods was made. The finalized questionnaire consist of 65 items- consumers knowledge on food safety (32), consumers attitude towards food safety (12), consumers perception on food handlers hygiene practices (15), familiarity with night market foods (3) and purchase intention of night market foods (3). The initial of 67-items instrument was reduced to a 65-items instrument. Previous study on knowledge, attitude, and practice questionnaire were compared. Study in reliability and validity study of hand hygiene knowledge, attitude and practice among food handlers demonstrated good internal consistency for attitude and practice domain but moderate internal consistency for knowledge (Cronbach s alpha: K=0.696; A=0.965; P=0.713). Validation study on questionnaire regarding food safety knowledge and practice among working women in Egypt 6 had good reliability (Cronbach s alpha: P=0.798). Generally, the internal consistency value for attitude in this current study shows slightly lower than previous study. Meanwhile, the internal consistency value for the practice was similar when compare with the previous study. Conclusion The KAP questionnaire developed in this study to evaluate the consumers knowledge on food safety, consumers attitude towards food safety, consumers perception on food handlers hygiene practices, familiarity with night market foods and purchase intention of night market foods among night market foods consumers is acceptable in terms of validity and reliability. Based on the results of validity and reliability test, some modification were made to improve the effectiveness of the questionnaire as instrument used to measure the variable of this study. It is vital to ensure that the instrument used was applicable and understanding to the target respondent. The score of the knowledge, attitude and perception on food handlers hygiene can be used in determining the level of knowledge and understanding in term of food safety among night market food consumers in Malaysia. References 1. Badrie, N., Gobin, A., Dookeran, S., and Duncan, R. (2006). Consumer awareness and perception to food safety hazards in Trinidad, West Indies. Food Control 17: Sharif, L. and Al-Malki, T. (2010). Knowledge, attitude and practice of Taif university students on food poisoning. Food Control 21: Ministry of Health. (2012). Health Fact Ministry of Health Malaysia. Retrieved on 2014, Feb 19 from: _by_page.pdf 4. Cooper, D. R., and Schindler, P.S. (2003). Business research methods (8 th edn). Boston: McGraw-Hill 5. Nunnally, J.C. and I.H. Bernstein. (1994). Psychometric Theory. 3 rd Edn., McGraw-Hill, Inc., New York. 6. Mohamed, F.F., Mona, M.E. and Mostafa, I.W. (2014). Food safety knowledge and practices among Saudi women. Food Control 47:

18 ANTIFUNGAL ACTIVITY OF LACTIC ACID BACTERIA AGAINST PATHOGENIC Sclerotium rolfsi OF Capsicum annum PLANT Siti Fatimah Zakaria 1, Mohd Nizam Lani 1, Chuah Tse Seng 1, Fisal Ahmad 1 and Khairul Mazmi Ahmad 2 1 School of Food Science and Technology, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu, 2 Department of Plant Protection, Universiti Putra Malaysia, UPM Serdang, Malaysia *Corresponding author gsk1900@pps.umt.edu.my Abstract Sclerotium rolfsii is one of the most destructive soil-borne pathogen of chili plant (Capsicum annum) causing damping-off seedlings, stem rot, root rot, southern blight, and wilt. Those diseases can reduce quality and quantity of produce resulting in economic loss. Lactic Acid Bacteria (LAB) are known to have antifungal activity were used as bio-control agent against S. rolfsii. The aim of this study was to determine the antifungal activity of LAB that responsible towards inhibiting the S. rolfsii causing root and stem rot of chili plant. Identification of LAB from different fermentation products (corn kernel silage and fermented catfish) were done by phenotypic identification (API 50 CHL) and molecular technique (16S rdna sequencing). Cell free supernatant (CFS) of 23 LAB strains (Lactobacillus plantarum, and Pediococcus pentosaceus) were tested for antifungal activity on Potato Dextrose Agar (PDA). Results showed that 15 of the strains LAB from fermented catfish showed greater inhibitory activity (52.9% to 71.4%) against S. rolfsii after incubation at 28ºC for three days compared with the control treatment (S. rolfsii growth in PDA without CFS of LAB). Further study will be focusing on the role of partially purified bacteriocin of LAB in reducing contamination of this pathogen. Introduction Chili is a rotational crop which is best planted in warm and well-drained conditions. Production of chili is very high in Asian countries due to high consumption either as spice, condiment, culinary, supplement, medicine and vegetables. It is a rich source of nutrients, vitamins and fiber for human 1. India is the largest producer of chili in the world (8.5 lakh tones) 1. Chili plant suffers with many fungal, bacterial and viral diseases. Among the fungal disease, root and stem rot of chili caused by S. rolfsii is of major concern and causing the economic losses in chili 2. Today, fungicides such as hexaconazole, tebuconazole and propiconazol always being used to control most of the diseases caused by fungi in chili plant 1. However, problems regarding efficacy of the fungicides residues are causing concern to consumer. Therefore, this issue needs to be solved because fungicides give effects to human health and the environment. Nowadays, consumers are more concerned on staying healthy and eating correctly. Alternatively, antifungal agents produced by microorganisms may be used as bio-control agent against pathogenic fungi 3. Lactic acid bacteria (LAB) is one of the example that produced different types of bioactive molecules, such as organic acids, fatty acids, hydrogen peroxide and bacteriocins that would be able to act as bio-control agent 4 Materials and methods Isolation and identification of Lactic Acid Bacteria LAB were isolates from corn kernel silage and fermented catfish. Corn kernel silage was sampled from the Green House School of Food Science and Technology, 17

19 Universiti Malaysia Terengganu and fermented catfish was prepared by mixed thoroughly the cleaned catfish with salt, brown sugar, roasted rice and spices before ferment at ambient temperature for 15 days. Isolation was done on deman Rogosa Sharpe (MRS) agar (Merck, Germany) and incubated anaerobically for 48 hours at 30ºC. General LAB identification and characterization was carried out by Gram staining, catalase test and oxidase test. Presumptive LAB isolates were confirmed by API 50CHL and 16 rdna sequencing analysis. Pathogenicity test Pathogenicity test was carried out under greenhouse by inoculating chili plants with S. rolfsii obtained from Department of Plant Protection, Universiti Putra Malaysia. Chili seeds were pre-germinated in sterile peat moss for a week than transferred to plastic tray and grown for a week until plant having two leaves. The plants were later transferred to poly bag containing sterilized soil and grown for 30 days or until having six leaves. Chili plant was artificially inoculated by spreading 20 seeds of sorghumbased inoculums with S. rolfsii around the chili stems. The sorghum-based inoculums without S. rolfsii act as control. The plants were water regularly to avoid of any stress. After 10 days of incubation, the plants showing typical root and stem rot symptoms. Plants were pluck off from the soil, washed thoroughly with distilled water and photos were taken. Re-isolation was made from such affected root or stem and culture so obtained was compared with that of original culture. Identification of this pathogen is known as koch s postulate. Antifungal activity of LAB Overnight culture of LAB in MRS broth was centrifuged at 10,000 rpm for 10 min at 4ºC. The 1ml cell free supernatant (CFS) of LAB strains were added in 10 ml molten potato dextrose agar (PDA) and stirred before pour into Petri dish. The 6 mm diameter mycelial plug of 3 days old culture S. rolfsii was cut and transferred to a PDA that had been inoculated with CFS and LAB. Another S. rolfsii mycelial plug was placed on PDA without inoculated with LAB as the control. The plates were incubated at 28ºC. Radius of fungal growth was measured when the growth of the fungus in control plate was full. Calculations of the percentage of inhibition showing the antifungal activity by LAB were obtained. Results and Discussion Identification of LAB Isolates LAB were gram positive, catalase and oxidase negative. Seven strains were isolated from corn kernel silage and 16 strains from fermented catfish. Only one strain has cocci-shaped which being identified as Pediococcus pentosaceus, while other strains were bacilli-shaped. All isolates were sequenced by 16 rdna sequencing analysis of LAB. Pathogenicity test The Sclerotium rolfsii confirmed as pathogen causing root rot of chilli when the same fungus was re-isolated again from infected plant tissue. Pathogen infected the plant at collar and root region. The leaves showed initially pale green and followed by yellowing leaves. The base of the stem turned brown, sunken and sclerotial bodies were formed on infected part. 18

20 Table 1: Confirmation of 23 Lactic Acid Bacteria (LAB) molecularly identified by 16 rdna sequencing analysis. No. Sample Code 1 SA 2 SB 3 SC 4 SD 5 SE 6 SF 7 SG 8 FCN 9 FCM 10 FCO 11 FCP FCK 12 FCE 13 FCA 14 FCH 15 FCG 16 FCL 17 FCI 18 FCB 19 FCD 20 FCC 21 FCJ 22 FCF 23 Identification of LAB strains Lactobacillus plantarum WCFS1 strain WCFS1 Lactobacillus plantarum strain JCM 1149 Lactobacillus plantarum strain JCM 1149 Lactobacillus plantarum strain JCM 1149 Lactobacillus plantarum strain NRRL B Lactobacillus plantarum WCFS1 strain WCFS1 Lactobacillus plantarum strain NBRC Lactobacillus plantarum WCFS1 strain WCFS1 Pediococcus pentosaceus ATCC strain ATCC Lactobacillus plantarum strain NBRC Lactobacillus plantarum WCFS1 strain WCFS1 Lactobacillus plantarum WCFS1 strain WCFS1 Lactobacillus plantarum strain NRRL B Lactobacillus plantarum strain JCM 1149 Lactobacillus plantarum strain JCM 1149 Lactobacillus plantarum WCFS1 strain WCFS1 Lactobacillus plantarum strain JCM 1149 Lactobacillus plantarum WCFS1 strain WCFS1 Lactobacillus plantarum strain JCM 1149 Lactobacillus plantarum WCFS1 strain WCFS1 Lactobacillus plantarum WCFS1 strain WCFS1 Lactobacillus plantarum strain JCM 1149 Lactobacillus plantarum strain JCM 1149 Source Corn kernel silage Corn kernel silage Corn kernel silage Corn kernel silage Corn kernel silage Corn kernel silage Corn kernel silage Fermented catfish Fermented catfish Fermented catfish Fermented catfish Fermented catfish Fermented catfish Fermented catfish Fermented catfish Fermented catfish Fermented catfish Fermented catfish Fermented catfish Fermented catfish Fermented catfish Fermented catfish Fermented catfish %ID Accession No. 97% NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_ % NR_

21 Antifungal activity of LAB against S. rolfsii Efficacy of antifungal activity was tested among different strains of LAB by following poisoned food technique 1. The results showed that there was a significant difference (P<0.05) in percentage of inhibition of S. rolfsii mycelial growth for all the LAB strains tested (Figure 1). The highest (71.4%) mycelial inhibition of S. rolfsii showed by strains L. plantarum strain JCM 1149 (FCF) and L. plantarum WCFS1 strain WCFS1 (FCN) (Figure 1). Others LAB giving greater inhibition were L. plantarum WCFS1 strain WCFS1 (FCC),L. plantarum WCFS1 strain WCFS1 (FCK), both 64.3% and L. plantarum WCFS1 strain WCFS1 (FCP) with 61.4% of inhibition (Figure 1). Results strongly indicate that LAB strains isolated from fermented catfish were greater inhibitor and showing positive antifungal activity compared to LAB strains isolated from corn kernel silage. Figure 1: The percentage of inhibition of LAB against S. rolfsii showing a positive antifungal activity. Conclusions In the present study regarding the bio-control of chili plant diseases caused by S. rolfsii among different LAB strains, strains from fermented cat fish can be recommended as biocontrol agent against pathogenic fungi. The potential LAB strains that had showed greater inhibitory activity against S. rolfsii will be selected for next partial purification of bacteriocinproducing LAB by ammonium sulphate precipitate method. Further test against selected fungi by using in vitro method. Through this purification, the molecular size of partially purified bacteriocin will be determined using sodium dedocyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. References 1. Madhavi, G. B. and Bhattiprolu, S. L. (2011). Integrated disease management of dry root rot of chilli incited by Sclerotium rolfsii (Sacc.). International Journal of Plant Animal and Environmental Science 2: Kalmesh, M. and Gurjar, R. B. S. 2001; Sclerotium rolfsii A new threat to chilli in Rajasthan. Mycology andplant Pathology 31 (2): Chittara, G. S., Breeuwer, P., Nout, M. J. R., Van Aelst, A. C., Rombouts, F. M. and Abee, J.T. (2003). An antifungal compound produced by Bacillus subtilis YM10-20 inhibits germination of Penicilium roqueforti conidio spores. Journal of Applied Microbiology Gerez, C.L., Torino, M.I., Rollán, G. and Valdez, G, F. (2009). Prevention of bread mould spoilage by using lactic acid bacteria with antifungal properties. Food Control 20:

22 PHYSICO-CHEMICAL PROPERTIES AND ACTIVITIES OF ANTIOXIDANTS COMPOUNDS IN HONEY BEE HONEY AND STINGLESS BEE HONEY *Norhayati Binti Abd Hadi, 1,3, Norizah Mhd. Sarbon 1, Zamzahaila Mohd Zin 1 and Hayati Mohd Yusof 1,2 1 School of Food Science and Technology, 2 Institute of Marine Biotechnology Universiti Malaysia Terengganu, 3 Center of Nutrition and Dietetic, Universiti Sultan Zainal Abidin, Kuala Terengganu, Terengganu, Malaysia *Corresponding author norhayatihadi@unisza.edu.my Abstract In the long human tradition honey has been used not only as a nutrient but also as a medicine. Honey is widely used for nourishment, constituting a nutritious supplement with medicinal properties recognized in all over the world. Honey is rich in phenolic compounds, which act as natural antioxidants and are increasingly popular because of their potential role in improving human health. The present study therefore was designed to analyse some physico-chemical characteristics of two different types of honey; namely, honey bee honey (Acacia) and stingless bee honey (Kelulut). Ash, moisture, acidity, hidroxymethyl furfural (HMF), diastase activity and sugar analyses of the honey samples were assessed for chemical characterization. Total phenolic compound, ferric reducing antioxidant capacity (FRAP) and 2,2-diphenyl-1-picryhydrazyl (DPPH) free radical scavenging activity were measured as antioxidant determinants. Honeybee honey (Acacia) shows the high content of ash, sugar, HMF, DPPH, FRAP and total phenolic compared to stingless bee honey (Kelulut). The results also showed that Acacia had low content of moisture, acidity and diastase activity compared to Kelulut. Introduction Honey is defined as a natural food produced by honey bees from the nectar of plants or from secretions of living parts of plants or excretions of plant sucking insects on the living parts of plants. Honey is a semiliquid which contains a complex mixture of carbohydrates,mainly fructose and glucose; other sugars are present astraces, depending on the floral origin. Moreover, organic acids, lactones, amino acids, minerals, vitamins, enzymes, pollen, wax and pigments are present 1. The content and composition of the different types of honey vary with different floral sources as well as climatic and environmental conditions 2,3,4. Several types of honey are found in Malaysia. These are either directly or indirectly introduced in many different foods in Malaysia and have been used as a traditional medicine for the last few decades. The aim of the present study was to determine the physicochemical properties and antioxidant activities of honey bee honey (Acacia) and stingless bee honey (Kelulut). Material and methods Honey: The Acacia and Kelulut honey used in this study was supplied by Agriculture Department of Johor, Malaysia. Determination of Sugar: Modified method of was used for the determination of the sucrose, fructose and glucose composition of the honeys 5. The solution mentioned above was injected to the HPLC (Agilent 1100, USA). The carbohydrate column (5 m and 4.6 mm 250 mm) was used. HPLC conditions were the following: mobile phase, 80% acetone and 20% water; flow rate, 1.4 ml/min; injection volume, 20 L. Refractive index detector (RID) was used and the column temperature was 25 C. Sugars were identified according to their retention times by comparing with sugar standards. The sugar concentration was calculated by using the calibration curve of the each sugar. 21

23 Determination of ph: Five gram of each honey sample was dilutes with 37.5 ml distilled water. The solution was stirred with a magnetic stirrer. Then the ph was measured with a ph meter (WTW Inolab, GmbH, Germany) that was calibrated using ph 4.0 and 7.0 buffers 4. Determination of Ash: Two grams of the sample was ashed by incineration in a furnace at 600 C to a constant weight. Ash percentage was calculated as: % ash = weight of ash x 100 weight of Sample Determination of Moisture Content: A handheld Refractometer (Atago, Japan) was used to determine the water content in honey based on the refractive index. The readings were corrected for a standard temperature of 20 by adding the correction factor of /. The moisture content was calculated by using Wedmore s table referring to the refractive index. Determination of Acidity: The titratable acidity was determined by weighing 10 g of the sample which was diluted to about 100 ml with water and titrated to phenolphthalein end point with 0.1 N NaOH.The result was calculated as milliequivalents per kilogram. Determination of Diastase Activity: The diastase activity was measured using the Phadebas method for α-amylase. Phadebas is a synthetic reagent which produces a blue color when it is hydrolyzed by the diastase. The absorbance at 620 nm is directly proportional to the diastase activity in the honey sample. The diastase activity is calculated as diastase number (DN). Results are expressed in Gothe units per gram of honey, defined as that amount of the enzyme which will convert 0.01 g of starch into the prescribed end oint in 1 h at 40 C under test conditions. Determination of HMF levels by high-performance liquid chromatography (HPLC) method: HMF concentrations were determined using an HPLC method based on the method published by the International Honey Commission (IHC) 1. Ten gram of honey sample were diluted to 50 ml with distilled water, filtered using a 0.45 μm nylon membrane filter and injected (20 μl) into an HPLC system (Waters 2695, Milford, MA, U.S.A.). The HPLC column used was a Merck Purospher Star RP-18e (125 4 mm, 5 μm) fitted with a guard cartridge packed with similar stationary phase (Merck, Germany). The HPLC method included an isocratic mobile phase of 90% water and 10% methanol with a flow rate of 1.0 ml/min. All solvents used were of HPLC grade. The detection wavelength was nm, with specific monitoring at 285 nm. The HMF concentrations in the samples were calculated by comparing the corresponding peak areas of the sample to the HMF standard solutions after correcting for the dilution of the honey samples. A linear relationship (r2 = ) was determined between the concentration and area of HMF peaks and the results are expressed in mg/kg. Determination of total phenolic: Total polyphenol content was determined using a Folin Ciocalteu assay, which measure the formation of blue-green complexes between phenolic compounds and Folin Ciocalteu reagent. First, 100 ml of appropriately diluted samples were added to 0.75 ml of diluted Folin Ciocalteu reagent (Folin Ciocalteu reagent: distilled water, 1:10). Then, the mixture was kept at room temperature for 5 min. Subsequently, 750 ml of 6% (w/v) Na2CO3 was added to it. The solution was allowed to stand at room temperature for 90 min. Absorbance was read at 725 nm using an ultraviolet (UV)-Vis spectrophotometer (SECOMAM, Anthelie Advanced 5, Domont, France). The measurement was compared to a standard curve of prepared gallic acid solutions and expressed as gallic acid equivalents in milligrams. 22

24 Determination of DPPH assay: The scavenging activity against 1,1-diphenyl-2-picrylhydrazil (DPPH; SIGMA, USA) radical was used in this study ml of the honey solution (0.1g/ml) in warm water was mixed with 1.5ml of 0.09mg/ml DPPH in methanol. The mixture was then incubated at 25ºC in a water bath for 5 mins after which the absorbance was measured at 517nm against a blank sample consisting of honey solution with distilled water. The absorbance of a radical blank was also measured using 0.75ml of distilled water. The radical scavenging activity (RSA) of honey was expressed in terms of percentage inhibition of DPPH radical by honey and was calculated as follows: RSA (DPPH. Inhibition, %) = [(AB- AT)/AB] x 100 Where, AB = Absorbance of radical blank (DPPH. without honey) AT = Absorbance of test sample (DPPH. with honey) Determination of FRAP assay: The reducing ability of honey was determined by FRAP assay with some modifications. Briefly, working FRAP reagent was prepared by mixing 10 volumes of 300 mmol/l acetate buffer, ph 3.6 with 1 volume of 10mmol 2,4,6-tripyridyl-s-triazine (TPTZ; SIGMA, USA) in 40mmol/L hydrochloric acid and with 1 volume of 20mmol/L ferric chloride. Two hundred µl of honey solution (0.1g/ml) was added to a test tube containing 1.5ml of freshly prepared FRAP reagent. The mixture was subsequently incubated at 37ºC for 4 mins after which the absorbance value were measured at 593nm against a reagent blank (200 µl of distilled water). The difference between this absorbance and the sample blank (honey solution with distilled water), was calculated to get the final absorbance. Aqueous solutions of known FeII concentration, in the range of µmol/l (FeSO4.7H2O) were used for calibration. The reducing ability of honey was expressed as µm of FeII equivalent/l. M. Result and Discussion Physicochemical analyses Table 1 summarizes the results obtained (mean and standard deviation, SD) from physicochemical analysis of the Acacia and Kelulut. ph All honey samples analyzed were acidic in nature, with ph values varying from 3.4 (Kelulut) and 4.2 (Acacia) (Table 1).The ph values of honeys were in accordance with AOAC. The ph values of the Malaysian honey samples were similar to those reported for Algerian, Brazilian, Bangladeshi, Indian and Spanish honeys (between ph 3.49 and 4.70). This parameter is of great importance during the extraction and storage of honey as it influences the texture, stability and shelf life of honey. Also, floral and geographic origins can cause great variations in honey ph values, as the nectar ph and soil conditions can greatly influence honey physicochemical characteristics. Ash The mineral content in honey is generally small and depends on nectar composition of predominat plants in their formation. The variability in ash contents has been associated in the qualitative way with different botanical and geographical origins of honey. Ash content of Acacia is 0.2 g/100g and Kelulut is 0.1 g/100g. Moisture content Moisture content is an important parameter of honey quality and defines the amount of water present in honey. The moisture content of Acacia ( g/100g) and Kelulut ( g/100g) as shown in Table 1. Moisture is a physico-chemical parameter that is related to the climatic conditions and degree of maturity of honey. Honey moisture content depends on the environmental conditions and the manipulation from beekeepers at the harvest period, and it can vary from year to year. 23

25 Acidity The free acidity of honey may be explained by taking into account the presence of organic acids, particularly gluconic, pyruvic, malic and citric, in equilibrium with their corresponding lactones, or internal esters, and some inorganic ions, such as phosphate and chloride. The value of acidity was found meq/kg (Acacia) and meq/kg (Kelulut) as shown in Table 1. The free acidity of stingless bee honey is generally higher, what justifies the more acidic flavor of this type of honey. Hidroxymethyl furfural (HMF) HMF is an important indicator for honey purity, as HMF content is widely recognized as a parameter that indicates the freshness of honey. High concentrations of HMF in honey are an indicator of overheating and storage in poor conditions. According to the Codex Alimentarius Commission, the HMF concentration in honey should not exceed 80 mg/kg. The HMF content of the honeys analyzed was mg/kg for Acacia and mg/kg for Kelulut (Table 1). The HMF content is indicative of honey freshness. Several factors influence the levels of HMF, such as temperature and time of heating, storage conditions, ph and floral source, thus it provides an indication of overheating and storage in poor conditions 1. Diastase activity Diastase is a natural enzyme of honey. The diastase activity in honey has been used as a freshness indicator over the years. The legislation has set a minimum level for diastase activity; it should not be less than 8 Diastase Number (DN) units, where 1 DN unit hydrolyses 1ml of 1% starch using 1 g of honey for 1 h at 37 C. The DN value for Acacia was and for Kelulut as shown in Table 1. Sugar analysis Sugar composition has been used to discriminate honey samples by botanical origin or geographical origin. The monosaccharides glucose and fructose are the major constituents of honey. Fructose is always the most important sugar quantitatively followed by glucose. In this study, the glucose and fructose content of Acacia was g/100 g and g/100 g respectively (Table 1). Kelulut show the glucose and fructose contents as g/100 g and g/100 g (Table 1). Sucrose, important sugar from a legislative point of view, had low values suggesting an advanced stage of ripening of the honeys, which would encourage the conversion of sucrose into glucose and fructose 4. Total phenolic Many authors have studied the phenolic contents of honey to determine their beneficial effect in human health and whether a correlation exists with floral origins. The mean total phenolic contents of the Acacia and Kelulut honey samples was mg GAE/100 g and mg GAE/100 g (Table 2). The total phenolic compound is sensitive to phenol and polyphenol entities and other electron-donating antioxidants such as ascorbic acid and vitamin E. Flavonoids are low-molecular-weight phenolic compounds that affect the aroma and antioxidant properties of honey. The total flavonoid and total phenolic contents vary between different honey sample depending on the geographical location of the different floral sources, such as Malaysia, Slovenia and Tunisia 3. The determination of the total phenolic content has also been regarded as a promising method of studying the floral origins of honeys. It has been reported that the botanical and geographical region from which the honey is collected not only affects the phenolic and flavonoid concentrations but also pollen distribution and the eventual antioxidant activities of the honey 6. DPPH The radical scavenging activities of the honey samples were determined by using the DPPH radical scavenging assay. DPPH is a stable nitrogen-based radical that has been extensively used to test the free radical scavenging ability of various substances. In evaluating the 24

26 radical scavenging potential of honey, the DPPH assay is frequently used. Usually, a high DPPH scavenging activity confers the high levels of antioxidant activity of the sample.the DPPH radical scavenging activities of all of the honey samples were measured at the following concentrations: 10, 20, 40 and 60 mg/ml. The highest percentage of inhibition was observed at 60 mg/ml for all of the honey samples. The DPPH assay was 49.80% (Acacia) and 48.44% (Table 2). Acacia exhibited the highest percentage inhibition (49.80 %), indicating that it has the highest antioxidant potential. The DPPH radical is one of the few stable organic nitrogen free radicals; it has been widely used to determine the free radical scavenging ability of the various samples 3. FRAP The FRAP assay gives a direct estimation of the antioxidants or reductants present in a sample based on its ability to reduce the Fe3+/Fe2+ couple. The FRAP value of Acacia honey samples was μm Fe [II]/100 g and Kelulut honey was μm Fe [II]/100 g (Table 2). The Acacia exhibited the higher FRAP values confirming its high antioxidant properties. High FRAP values indicate a greater reduction of ferric ions to ferrous ions. Conclusion In conclusion, this is the first study report on the physicochemical and antioxidant activity of Acacia and Kelulut honey. Our results indicate that the antioxidant activity of Acacia honey was higher than Kelulut honey. Due to the nature of antioxidant compounds, honey has an important role in maintaining the health of human body and should be more widely consumed. References 1. Fallico, B., Zappala, M., Arena, E. and Verzera, A. (2004). Effects of conditioning on HMF content in unifloral honeys. Food Chem 85(2): Gheldof, N., Xiao-Hong, W. and Engeseth, N. (2002). Identification and quantification of antioxidant components of honeys from various floral sources. J Agric Food Chem, 50: Aljadi, A. M. and Kamaruddin, M. Y. (2004). Evaluation of the phenolic contents and antioxidant capacities of two Malaysian floral honeys. Food Chem 85(4): Kucuk, M., Kolayli, S., Karaoglu, S., Ulusoy, E., Baltaci, C. and Candan, F. (2007). Biological activities and chemical composition of three honeys of different types from Anatolia. Food Chem 100, Jahanbin, K., Moini, S., Gohari, A. R., Emam-Djomeh, Z. and Masi, P. (2012). Isolation, prufication and characterization of a new gum from Acanthophyllum bracteatum roots. Food Hydrocol 27: Khalil, M. I., Sulaiman, S.A., Alam, N., Moniruzzaman, M. and Bai e, S. (2010). Gamma irradiation increases the antioxidant properties of Tualang honey stored under different conditions. Molecules 17:

27 Table 1 Physicochemical analysis (ph, ash, moisture, acidity, HMF, diastase activity and sugar analysis) of the Acacia and Kelulut honey. Sample ph Ash (g/100g) Moisture (g/100g) Acidity (meq/kg) HMF mg/kg) Diastase Activity (DN) Sugar Analysis Fructose (g/100g) Glucose (g/100g) Sucrose (g/100g) Acacia a a a a a 0 Kelulut b b b b b Means are compared by using Paired Sampels T Test. In each column, values with different letters (superscripts a-b ) indicate significant differences (p<0.05) Table 2 Activities of antioxidants compounds (total phenolic, FRAP and DPPH) in the Acacia and Kelulut honey. Sample Total phenolic (mg GAE/100g) FRAP (%) DPPH (μm Fe [II]/100 g) Acacia a a Kelulut b b Means are compared by using Paired Sampels T Test. In each column, values with different letters (superscripts a-b ) indicate significant differences (p<

28 EFFECTS OF DIFFERENT ARBUSCULAR MYCORRHIZA SPECIES AND ITS RATES ON THE GROWTH PERFORMANCE OF HEMPEDU BUMI (Andrographis paniculata Nees) GROWN ON BRIS SOIL Ain Najwa Khairul Anwar 1 *, Rosazlin Abdullah 2, Zuraida Abdul Rahman 3, Yusnita Hamzah 1 and Wan Zaliha Wan Sembok 1 1 School of Food Science and Technology, Universiti Malaysia Terengganu, 2 Institute of Biological Sciences, University of Malaya, 3 Biotechnology Research Centre, Malaysian Agricultural Research and Development Serdang, Persiaran MARDI-UPM, Malaysia. *Corresponding author gsk2134@pps.umt.edu.my Abstract Thirty six of five-node-cutting of A.paniculata were used in the experiment. Three species of AM which were Glomus sp., Scutellospora sp. and Gigaspora sp. and mixed species with two rates of AM viz. 10g and 50g were arranged in a randomized complete block design (RCBD) with three replicates. Both factors, different species and rates of AM resulted in a significant interaction in increasing number of branches and spores, percentage of root infection and cumulative fresh weight of A.paniculata. Irrespective of AM species, a 50g AM had biggest stem diameter compared to other treatments. In addition, A.paniculata treated with 50g of Scutellospora sp recorded the highest cumulative fresh weight as compared to control plant. No significant interaction was observed between two factors on leaf area and total chlorophyll content of A.paniculata leaves. In conclusion, irrespective of AM species, 50g AM was pronounced in enhancing the growth of A.paniculata planted on BRIS soil. However, no specific species can be deduced in improving the development of A.paniculata as all species had similar effect. Introduction In Malaysia, the herbal industry has become a new source of wealth due to the increased demand in herbal supplements, health functional food, herbs-based energy drinks and skin cares. Currently, there are 11 types of herbs has been identified as highly potential to be commercialized which include tongkat ali, kacip fatimah, misai kucing, dukung anak, hempedu bumi, rosel, pegaga, mengkudu, ginger and belalai gajah. Among these top 11 herbs, hempedu bumi (Andrographis paniculata Nees) is on the rise for consumers demand for health care product, botanical drug, herbal remedies, pharmaceuticals, nutraceuticals, food and beverages. In order to maintain a constant supply of A.paniculata to various industries as abovementioned, the growth and postharvest performances of this herb was investigated. In Terengganu, the cultivation of A.paniculata as a commercial crop is not yet developed which might be due to the occurrence of infertile Beach Ridges Interspersed with Swales (BRIS) soil. This soil has more than 98% sand, excessive drainage, high surface soil temperature, low moisture and nutrient content and covers 67, hectares in Terengganu. In addition, sandy area is not suitable for cultivation due to aforementioned problems. Therefore, this study aimed at evaluating the effectiveness of different species and rates of arbuscular mycorrhizal (AM) in improving the growth and quality of A.paniculata. The suitable species specifically for A.paniculata was also investigated. To the best of my knowledge, the information on pre- and postharvest performance of this local herb planted on BRIS soil is scarce. It is well documented that AM plays a key role in enhancing growth performance of various crops. Besides, the application of AM could also contributes to the short planting period, higher yield and quality of this local herb. Materials and Methods Thirty six of A.paniculata plants were grown in this experiment. The experiment was conducted in a greenhouse at the Universiti Malaysia Terengganu with average temperature of C. Five-node stem cuttings were transferred into polybag containing sterilized 27

29 ( C for 8 hours) BRIS soil which was collected from Stesyen Pembangunan Komoditi Rhu Tapai, Terengganu. The experiment was arranged in a randomized complete block design (RCBD) with three replicates. Single plant represented as one experimental Results and Discussion unit. The treatments include Control (0g of Glomus sp), 10g of Glomus sp., 50g of Glomus sp., 0g Scutellospora sp., 0g of Scutellospora sp., 50g of Scutellopora sp., 0g of Gigaspora sp., 10g of Gigaspora sp., 50g of Gigaspora sp., 0g of mixed sp., 10g of mixed sp., and 50g of mixed sp. All plants experienced similar cultural practices such as irrigation, fertilization, weed, pest and disease control. The experimental period was 77 days (September 29, 2015 November 26, 2015). The preharvest parameter such as stem diameter was recorded at weekly basis, 0, 7, 14, 21, 35, 42, 49, 56, 63, 70 and 77 days after transplanting, (DAT) while spore count and root infection were measured at day 14, 42 and 70 DAT. Meanwhile, postharvest parameters assessed at harvest were fresh and dry weight of leaves and stems, leaf area, chlorophyll content and total phenolic content of A.paniculata leaves. The data were subjected to twoway analysis of variance (ANOVA) using GLM (General Linear Models) procedures and further separated by Tukey for minimum significant difference at P In the present study, different species and rates of AM had a significant interaction in increasing the number of branches, spore and percentage of root infection of A.paniculata (Figures 1, 2 and 3). Inoculation of 50g of Gigaspora sp. on A.paniculata showed the highest number of branches on day 28 and 70 (19 and 132 respectively) as compared to noninoculated A.paniculata (16 and 66 respectively) (Figure 1).This is mainly caused by enhanced cytokinin formation of the plant which leads to more cell division 1. Other than that, on day 42, highest spore number were identified in soil with 50g of mixed species followed by 10g of mixed species and 10g of Gigaspora sp. (160, 86 and 82 spores respectively) (Figure 2). Moreover, on day 70, highest spore number was recorded in soil treated with 10g of mixed species followed by 50g of mixed species and 50g Scutellospora sp (281, 264 and 263 spores, respectively). Apart from that, on day 70, 91.6 % of A.paniculata root was infected with 10g of mixed species followed by 50g of mixed species and Scutellospora sp. and Glomus sp. (85 %, 83.3 % and 83.3 % respectively) (Figure 3) Glo 0g Glo 10g Glo 50g Scu 0g Scu 10g Scu 50g Gig 0g Gig 10g Gig 50g Mix 0g Mix 10g Mix 50g Glo 0g Glo 10g Glo 50g Scu 0g Scu 10g Scu 50g Giga 0g Giga 10g Giga 50g Mix 0g Mix 10g Mix 50g Glo 0g Glo 10g Glo 50g Scu 0g Scu 10g Scu 50g Giga 0g Giga 10g Giga 50g Mix 0g Mix 10g Mix 50g Number of Branches Spore Number Root Infection Percentage (%) Days After Transplanting (DAT) Figure 1: Effects of different species and rates of AM on number of A.paniculata branches. Vertical bar represent HSD value at 5% significant level Days After Transplanting (DAT) Figure 2: Effects of different species and rates of AM on spore number of A.paniculata. Vertical bar represent HSD value at 5% significant level Days After Transplanting (DAT) Figure 3: Effects of different species and rates of AM on percentage of root infection A.paniculata. Vertical bar represent HSD value at 5% significant level. Meanwhile, O.sanctum inoculated with combination of G.mossae and G.versiforme G.mossae and G.versiforme had higher root colonization, chlorophyll content and flower stem length than non-inoculated 2. Higher number of active translocation of minerals along the hyphae produce by AM fungi resulted to more effective fungus to exploit a volume of soil thus increase the uptake of essential elements to the plant. Other than that, no significant interaction was observed between two factors for stem diameter, leaves fresh and dry weight, cumulative fresh weight, leaf area, total chlorophylls and total phenolic content (Figures 6, 7 and Table 1). For stem diameter without regard to 28

30 AM species, diameter size of A.paniculata was only significant on day 77 by the inoculation of different rates of AM. A.paniculata inoculated with 50g of AM showed the biggest stem diameter (7.55mm) as compared to 10g of AM inoculated plants (7.00mm). These results however were comparable with mixed sp. (7.42mm), Gigaspora sp. (7.14mm), Glomus sp. (7.12mm) and Scutellospora sp. (6.78mm). Similarly, different AM species did not enhance the stem diameter of ironwood (Libidibia ferrea) 3. Meanwhile, cumulative fresh weight of A.paniculata increased with the increased of AM level (Table 1). A.paniculata treated with AM at 50g had the highest cumulative fresh weight (25.8g) as compared to control plants (5.8g). However, different species of AM were not noticeable in increasing the A.paniculata cumulative fresh weight. Apart from that, irrespective of AM species, A.paniculata inoculated with 10g AM resulted in significantly higher amount of total phenolic content than control plants (Table 1). The increase in phenolic content is due to AM fungi induce changes in phytohormone level in the host plants 4. Meanwhile, different species of AM paticularly Claroideoglomus etunicatum, Claroideoglomus claroideum and Rhizophagus intraradices were not uniformly affect the major polyphenol content of marigold except for a minor component which was isoharmnetin-malonyl-glucoside 5. In addittion, the inoculation of G.mossae was significantly decrerase the total phenolic content and no significant affect on the total flavonoid content of marjoram 6. Stem Diameter (mm) g AM 10g AM 50g AM Days After Transplanting (DAT) Figure 6: Effects of different rates of AM on stem diameter of A.paniculata. Vertical bar represent HSD value at 5% significant level. Stem Diameter (mm) Giga sp. Glo sp. Mix sp. Scutello sp Days After Transplanting (DAT) Figure 7: Effects of different species of AM on stem diameter of A.paniculata. Vertical bar represent HSD value at 5% significant level. Conclusions In a nutshell, mixed species was pronounced in enhancing the number of spore and root colonization percentage of A.paniculata. However, irrespective of AM species, 50g of AM was pronounced in enhancing the growth and postharvest performance including stem diameter and cumulative fresh weight of A.paniculata. 29

31 Table 1. Effects of three rates and four species of arbuscular mychorriza on fresh and dry weight, cumulative fresh weight, leaf area, total chlorophyll and total phenolic content of A.paniculata grown on BRIS soil Factor Leaf fresh weight (g) Leaf dry weight (g) Cumulative leaves fresh weight (g) Leaf area (cm 2 ) Total chlorophylls Total phenolic AM rates (R) a 1.56 a b a 0.95 a 0.05 b a 2.22 a a a 0.88 a 0.16 a a 2.05 a a a 1.02 a 0.06ab F-test NS NS * NS NS * significance AM species (S) Gigaspora 6.12 a 1.96 a a a 0.87 a 0.03 a sp. Scutellopora 6.02 a 2.08 a a a 0.81 a 0.10 a sp. Glomus sp a 2.22 a a a 1.14 a 0.07 a Mixed sp a 1.52 a a a 0.97 a 0.02 a F-test NS NS NS NS NS NS significance Interaction, RxS NS NS NS NS NS NS NS, *: non-significant or significant at P <0.05. Mean separation within columns and factors followed by the same letter are significantly different by HSD at P References 1. Ramakrishnan, K. and Selvakumar, G. (2012). Influence of AM fungi on plant growth and nutrient content of tomato (Lycopersicum esculentum Mill). International Journal of Research in Botany. 2(4): Mohammad Saharkhiz, J., Mohammad, M., Mehdi, Z. and Jaime A. Teixiera, D.S. (2011). Responses of Ocimum sanctum to inoculation with arbuscular mycorrhizal fungi and fertilization with different phosphate sources. Medicinal and Aromatic Plant Science and Biotechnology. 5(2): Silva, F.A., Ferreira, M.R.A., Soares, L.A.L., Sampaio, E.V.S.B. Silva, F.S.B. and Maia, L.C. (2016). Arbuscular mycorrhizal fungi increase gallic acid production in leaves of field grown Libidibia ferrea. Journal of Medicinal Plant Research. 8(36): Toussaint, J.P., Smith, F.A. and Smith, S.E. (2007). Arbuscular mycorrhizal fungi can induce the production of phytochemicals in sweet basil irrespective of phosphorus nutrition. Mycorrhiza, 17: Engel, R., Szabo, K., Abranko, L.. Rendes. K., Fu zy, A. and Takacs, T. (2016). Effect of arbuscular mycorrhizal fungi on the growth and polyphenol profile of marjoram, lemon balm, and marigold. Journal of Agriculture and Food Chemistry. 64: Hristozkova, M., Geneva, M., Stancheva, I., Boychinova, M. and Djonova, E. (2015). Aspects of mycorrhizal colonization in adaptation of sweet marjoram (Origanum majorana L.) grown on industrially polluted soil. Turkey Journal Biology. 39:

32 Physical Barrier Mechanism of Si-mediated Rice Against Pyricularia oryzae Muhammad Shahrul Hafiz Elham 1*, Ng Lee Chuen 1, Sariam Othman 2 and Mohd Razi Ismail 2 1 School of Food Science and Technology, 2 Institute of Oceanography and Environment, Universiti Malaysia Terengganu, 21030, Kuala Terengganu, Terengganu *Corresponding author erulelham@yahoo.com Abstract Rice blast caused by fungus Pyricularia oryzae is most devastating and prevalent diseased in rice which can caused yield lost up to 70%. The severity of several plant caused by pathogenic fungi are dramatically decreased in agronomic and horticultural crop. The application of silicon have improved the resistance of plant host against pathogenic fungus. In spite of recent advance linking silicon to host resistance via genomic and proteomic, the exact mechanism of silicon increasing the resistance of plant required further investigation. This study aimed to investigate the best silicon sources which reduce the disease severity assessment (DSA) of blast disease. We also provide the cytological evidence on the accumulation and formation of silicon for both rice variety MR219-4 (partial resistance to P. oryzae) and MARDI Aerob1 (resistance to P. oryzae). Rice varieties MARDI Aerob 1 and MR219-4 were cultivated in the greenhouse to determine the effectiveness of Si through evaluation of rice blast disease development. Rice seedlings (14 days after sowing) from both varieties were foliar sprayed with Silicon Dioxide (SiO), Potassium Silicate (KSiO 4) and Sodium Silicate (NaSiO 4) at various concentrations: 3, 6, 9 mg L -1.The treatments were arranged in randomized completely block design (RCBC). The formation of the Si cuticle layer and the deposition of Si in the rice leaf will be determined using Scanning Electron Microscope with Energy Dispersive X-ray (EDX). From this study, Calcium Silicate at 9mg L -1 shows the lowest value of DSA for MR 219-4, 1.87% and MARDI Aerob 1, 0.04% compared to control 22.58% and 2.395% respectively. Through EDX, MARDI Aerob 1 shows the highest silicon content (5763 count/s) compared to MR219-4 (5218 count per second) for control. This result of this study strongly suggests the formation of Silicon play an active role in the resistance of rice to blast disease. Introduction Rice blast diseased is caused by fungus Pyricularia oryzae. This disease is one of the most significant disease which causes serious yield lost. According to Hajano et al. (2011), blast disease have reported over 85 countries and can be very disastrous under favourable condition 2. The major strategy nowadays is by using a fungicide and producing resistance variety. However, fungicide in an environmental friendly and can effecting the cultivation system. Besides, producing a resistance cultivar is just a short term of remedy as blast diseased spread very fast Another approach gaining interest in preventing this diseased is by using the application of silicon. Rice is known as a silicon accumulator which take up silicon up to 10% of dry basis in the form on monosilicic acid 4. Many studies have been conduct to elucidate how this nonessential element providing a resistance against P. oryzae. To date, 2 hypotheses have been proposed which is biochemical interaction and physical barrier mechanism produces by the application of silicon. Despite the abundance of studies of physical barrier mechanism in preventing the penetration of P. oryzae in plant, the mechanisms have not been well establish and inconclusive. There is still no direct evidence on how the application of this element enhance the resistance of rice against P. oryzae The objective of this study is to determine the best silicon sourced reducing the blast diseased. We also want to elucidate the effect of silicon in providing physical barrier mechanism for rice variety MARDI Aerob 1 (resistance variety) and MR219 line 4 mutant (susceptible variety) 31

33 Methodology Pathogenicity testing The P. oryzae isolate was obtained from Division of Agrotechnology and Bioscience, Malaysian Nuclear agency (Nuclear Malaysia). The isolate were first cultured in 100ml of Potato Dextrose Broth (PDB) with marble. The marble were used to break the mycelia. A fully growth mycelia were mixed with PDA at ratio 8:2 and kept it growth in petri dish. After the whole surface fully with mycelia, the mycelia mats were gently scrap with spatula and were incubate under wet cheese cloth for 2 days with continuous light to induce the sporulation. The spore with concentration of 1x10 6 (15ml/pot) were used as sourced of inoculation. Rice plant 14 days after sowing (DAS) were inoculated during evening and kept in dark with % humidity for 24 hours. After 7 days, the plant was observes for any symptom growth. Silicon Treatment Silicon oxide, Potassium silicate and Sodium silicate were used as a silicon sources in this study. The silicon sources were first diluted with distilled water to form stock solution 100mg/L. the silicon was applied as foliar spray at rate 3,6 and 9 mg/l with tween 20 (0.05%, v/v) as a surfactant 14 days after sowing Diseased Severity Assessment and Area under Disease Progressive Curve (AUDPC) Blast severity was evaluated 6 days after inoculation based on visual assessment caused by P. oryzae into scale provided by IRRI standard 3. The diseased severity was calculated based on the equation below Disease severity (%) = [Σ(r nr)/(9 Nr)] 100 r indicates rating value (0 9) nr indicates the number of infected leaves with a rating of r Nr indicates the total number of leaves tested for each replication. Data from blast severity was used to calculate the area under blast progress curve (AUBPC) for each leaf of each plant per replication. The Formation and Localization of Silicon in Rice The localization of silicon deposition in leaves was determined using SEM with EDX. For this experiment, plant were treated with 9 ml/l of Calcium silicate The mid-portion of sample were taken in 7 th day after Si treatment, The samples were cut in squares from a mid-portion of the youngest fully extended leaves at the 4- leaf growth stage. The specimen were fixed postfixes and dehydrated as described for SEM. Result and Discussion Diseased Severity Assessment. All type of Si treatment shows a significant reduction (α: 0.05) in disease severity compared to non-treated plant for both variety, MR219-4 and MARDI Aerob 1 (figure 4.4a 4.4b). Sodium silicate for rice variety MR219-4 at 9mg/l significantly reduced the blast severity compared to others silicon sources (1.87%). However there is no significant different between 6mg/L and 9mg/L of calcium silicate. Then, the trend is followed by Silicon oxide at 9mg/L (7.9987%) and 6mg/L. In this study, Sodium silicate shows the lowest in reducing blast disease even in high concentration ( %). Similar pattern also can be observed for variety Aerob 1 with sodium Silicate shows the lowest disease severity by 0.04% compared to the control 2.397% at 15days. The trend then followed by Silicon dioxide and Sodium Silicate. Plant treated with Calcium silicate at 9 ml/l shows lowest AUDPC for both variety, MR219-4 (88.00%) and MARDI Aerob 1 (98.25%). 32

34 Figure 1: Disease severity assessment for rice variety MARDI Aerob 1 Figure 2: Disease severity assessment for rice variety MR219-4 Silicon deposition and localization The corresponding EDX spectre compared with SEM images shows a significant different of silicon content between treated and untreated plant for both variety. Silicon content untreated MR219-4 (6080 count/s) is low compared to untreated Aerob 1 (7610 count/s). The silicon content in increasing by 7451 count/s (Aerob 1) and 5250 count/s (MR219-4) after treated with Calcium silicate at 9 mg/l. The deposited silicon is most likely to become prevalent in the apoplast of rice plant when the plant become matured 4. Another study showed, Si application and P. oryzae inoculation result in higher silicon deposit in rice leave and more silicon papilla can be found in a guard cell of stoma from the area fungus grew in both susceptible and resistance cultivar 1. 33

35 In this study, a Calcium silicate shows the best silicon sources in reducing blast disease and the blast disease assessment kept decreasing when increasing the silicon concentration. Through microscopic and EDX, MARDI Aerob 1 control shows the highest silicon deposition Conclusion compared to MR This result shows that silicon enhance the resistance by providing physical barrier to prevent appresorial penetration. Further study need to be determined on the interaction of this element with Pyricularia oryzae in term of plant biochemical reaction and physical barrier mechanism especially for different susceptibility against blast disease. References 1. Cai, K., Gao, D., Luo, S., Zeng, R., Yang, J., and Zhu, X. (2008). Physiological and cytological mechanisms of silicon-induced resistance in rice against blast disease. Physiol Plant 134(2): doi: /j x 2. Hajano, J. U., Pathan, M. A., Rajput, Q. A., and Lodhi, M. A. (2011). Rice Blast mycoflora, symtomatology and pathogenicity. International journal for agro veterinary and medical sciences 5: IRRI. (2008). World Rice Statistic. from 4. Mitani, N., Ma, J. F., and Iwashita, T. (2005). Identification of the silicon form in xylem sap of rice (Oryza sativa L.). Plant Cell Physiol 46(2): doi: /pcp/pci Sang, G. K., Ki, W. K., Eun, W. P., and Choi, D. (2002). Silicon-induced cell wall fortification of rice leaves: a possible cellular mechanism of enhanced host resistance to blast. Phytopathology 92(10):

36 Rice variety MR219-4 EDX Image EDX graft Control Silica bodies 5218 count/s Silica cell MR count/s Treated MARDI Aerob 1 Control 5763 count/s MARDI Aerob count/s Treated Figure 3 The microimage of electron dispersive x-ray (EDX) with the silicon distribution on the adaxial part of leave. The highest distribution can be observed in MR219-4 treated with silicon (11680 count/s) followed by MARDI Aerob 1 treated (12945 count/s 35

37 INTEGRATION OF BAMBOO (Gigantochloa albociliata) WITH OIL PALM AND ITS EFFECT OF NUTRIENT CYCLING ON OIL PALM YIELD Norkaspi Khasim 1*, Adzemi Mat Arshad 2, Khalid Haron 1 1 Crop and Livestock Integration Unit, Malaysia Palm Oil Board, Kajang, Selangor, 2 Food Crop Science Unit, School of Food Science and Technology, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu. Corresponding author kaspi@mpob.gov.my Abstract Bamboos (Gigantochloa albociliata), also known as woody bamboos, belong to family Graminae. They are divided into two types, i.e. monopodial and sympodial. They occur naturally in the tropical, subtropical and temperate region of all continents. In Malaysia, bamboo is found in abundance and widely scattered in about 5% of the total forest reserve area. It multiples of economic uses such as the shoot for food and the culms for building peasant houses and handicraft. Besides higher biomass, bamboo has other advantages over wood as carbon stock. Bamboo generates 30% more oxygen than trees. It helps reduce carbon dioxide gases blamed for global warming. Preliminary observation for 2 year in MPOB Research Station indicated that bamboo shoot thrived well in the oil palm plantation area. It survival rate was higher at about 90% and has a greater potential to be integrated with oil palm. Crops integration in the oil palm area is in-line with the government s policy to increase land productivity, food production and to increase the income of oil palm growers. For this reason, there is a need to conduct a thorough study on integration of bamboo shoot with oil palm. The study is being conducted at MPOB Research Station in Keratong, Pahang. The objective of the study are to measure the growth performance of bamboo planted with oil palm, to evaluate the soil chemical and physical properties in bamboo and soil (N, P, K, Ca, Mg, Si and total carbon), to assess the impact of bamboo on oil palm yield and to measure growth performance of oil palm in integrated plot. Preliminary result indicated that bamboo shoot integration in double avenue oil palm planting system with 136 palms/ha survives well and no mortality rate was recorded. The vegetative growth of bamboo shoot at 1 st year after planting in double avenue oil palm was satisfactory good compared to normal oil palm planting. Introduction Bamboos (Gigantochloa albociliata), commonly known as woody bamboos, belong to family Graminae. They are divided into two types, i.e. monopodial and sympodial. They occur naturally in the tropical, subtropical and temperate region of all continents. It was introduced in Europe from China and Japan during the 19 th century and the first bamboo gardens were established and became known to the public 2. In Malaysia, bamboo is found in abundance and widely scattered in about 5% of the total forest reserve area 1. It has, however, received comparatively little research attention. Most bamboos species found in the local forests are Gigantochloa sp., Schizostachyum zollingerii and Dendrocalamus pendulus 3. Bamboo is a natural product from the forest in Malaysia that has been used traditionally to construct various types of living facilities including various agricultural and kitchen tools, shelter, fence, raft, bridge, animal traps, weapon, etc. Meanwhile, its shoot is used for food which presently have been developing as one of important industry in agricultural sector in Malaysia. Due to it being strong, tough and cheap of wood, bamboo is also used as structural material at construction sites in China, India, Malaysia and other countries 5. Commercially, bamboo can be converted into charcoal and activated carbon via carbonisation followed by activation 4,5,6. An observation was made on the plot of the integration of bamboo and oil palm that had been established in the MPOB Research Station 36

38 in Kluang, Johor and MPOB Research Station in Hulu Paka, Terengganu. Preliminary observation on the two years old bamboo indicated that it thrived well under the oil palm population in the both areas. The growth performance of the integrated bamboo was comparable to the conventionally cultivated bamboo without oil palm. Its survival rate was higher at about 90% and has a greater potential to be integrated with oil palm. For this reason, there is a need to conduct a thorough study on the integrated cultivation of bamboo with oil palm. Objectives of this study are to determine the growth and yield of Buluh Madu/Buluh Aura (Gigantochloa albociliata) grown in combining with oil palm for production of bamboo shoots and to determine the growth and yield effects of bamboo integration on the oil palm yield. Results and Discussion About 300 seedlings at the age of 6 months were gathered from Supplier in Jitra, Kedah. After selection, about 242 seedlings were planted to the field on November to December 2014 with the planting distance of 2m, 4m and 6m in 1 row planting system. The growth of bamboo in 6 months after planting was satisfactory good. No mortality was recorded in 6 months after planting (MAP). The vegetative growth of bamboo shoot at the age of 6 MAP and 1 YAP was recorded and shown in Table 1. Table 1 showed the data of vegetative growth of honey bamboo at 1 year after planting (YAP) for the treatment of 2m,4m and 6m. For plant height, treatment 4m showed the highest average with the value of m compared to other treatments. For culm circumference at ground surface and breast height (1.3 m) treatment 4m showed the highest average of 9.83 cm and 6.55 cm respectively. For number of node, treatment 4m showed the highest average with the figure of 22.8 and for number of branches, treatment 4m was also showed the highest average with the figure of All data showed not significantly difference at 1 year after planting (1 YAP). Shoot Production. About 242 plants have been planted in double-avenue oil palm planting system started in November until December Every clump starts producing new shoot at 2-3 months after field planting. However, at the early stage, the new shoots produced will not be harvested because it must be retained as single culm until 8-10 culms for one clump for the purpose of new shoot (Rebung) production. Harvesting programme for Rebung was started in January 2016 that was 1 year after field planting. Several bamboo clumps started producing the Rebung at the average of 1-2 shoots per clump. It was observed that Rebung will be progressively and consistently produced in moderate raining season especially in the 1st quarter (Jan-April) and 3rd quarter (Sept- Dec) in every year. Due to long drying period in the year 2016 started from January until April 2016 has affected Rebung production in research plots. It was observed that bamboo clumps progressively started to produced Rebung in Mei Until Jun 2016, 15 Rebung was harvested with the total weight of 5.88 kg (fresh) and 4.99 kg (after processed). The average weight of Rebung after processed was 332 g/rebung. Oil Palm Yield For oil palm, yield recording was started in March 2015 (8 years after field planting). One year harvest will be completed in February 2016 and proceeding for second year harvest in The FFB yields were not much different between the bamboo-oil palm integrated plots and control plot until June 2016 (Table 2). 37

39 TABLE 1: VEGITATIVE GROWTH OF BAMBOO AT 1 YEAR AFTER PLANTING INTEGRATED IN DOUBLE AVENUE OIL PALM PLANTING SYSTEM AT MPOB STATION IN KERATONG, PAHANG. Treatment 2 m 4 m 6 m Parameter Replicate month 6 month 6 month month month month Height (cm) Average Culm circumference (5 cm from ground surface) Culm circumference (1.3 m from ground surface) No. of nodes No. of branches Average Average Average Average TABLE 2 : FFB YIELD IN BAMBOO PLOT INTEGRATED WITH PALM AT MPOB STATION, KERATONG, PAHANG. Tonne/Ha/Yr Plot 2015/2016 (Mac 15 Feb 16) *2016/2017 (Mac 16 Feb 17) Bamboo integrated (D. Avenue,136 palms/ha) Control (Normal, 136 palms/ha) Control (D.Avenue, 136 palms/ha) Notes : * FFB yield until June

40 Conclusion Based on the data recorded, it s indicated that bamboo shoots integration in double avenue oil palm planting system survives well and no mortality was recorded. The vegetative growth of bamboo shoots at 1 year after planting in research plot at MPOB Station in Keratong, Pahang was satisfactory good comparable to commercial planting. References 1. Abd. Latif Mohmod. (1987). Guideline on the manufacturing of blinds and satay sticks. Forest Research Institute Malaysia Technical Information 2. 8 pp 2. Azmy, H. M. (2004). Landscaping with bamboo. Perpustakaan Negara Malaysia Cataloguing-in-Publication Data. ISBN Azmy, H. M. and Abd. Razak, O. (1991). Field Identification of Twelve Commercial Malaysian Bamboos. FRIM Technical Information No.25. Forest Research Institute of Malaysia, Kepong, Malaysia. 12 pp. 4. Chan K, Jae-Wook, L, Jong-Hyu, K. and Kap-Seung, Y. (2006). Feasibility of bamboo-based activated carbons for an electrochemical supercapacitor electrode. Korean Journal of Chemical Engineering 23: Hameed, B. H., Din, A. T. M. and Ahmad, A. L. (2007). Adsorption of methylene blue onto bamboo-based activated carbon; kinetics and equilibrium studies. Journal of Hazardous Materials 141: Young, C. B., Kwang, J. C. and Joo, H. C. (2005). Production and CO 2 adsorption characteristics of activated carbon from bamboo by CO 2 activation method. Korean Engineering Research 43:

41 IN VITRO STUDY OF FLAVONOID PHYTOALEXIN SAKURANETIN AGAINST Pyricularia oryzae Nura Adila Zahari 1*, Aziz Ahmad 2 and Ng Lee Chuen 1 1 School of Food Science and Technology, 2 School of Fundamental Science, University Malaysia Terengganu, Kuala Terengganu, Terengganu, Malaysia. *Corresponding author gsk2303@pps.umt.edu.my Abstract The importance of phytoalexin for disease resistance has been demonstrated in a study which showed that after inoculation with P. oryzae, phytoalexins accumulated more quickly and to a higher extent in resistant rice than in susceptible rice, inducing severe restriction of fungal growth. Only one phenolic phytoalexin has been found in rice so far, the flavanone sakuranetin, which is formed in rice in response to UV irradiation or blast infection. The objective of the study is to determine in vitro testing of phytoalexin against P. oryzae. The inhibitory activity of sakuranetin to mycelium growth of P. oryzae was determined using a potato dextrose agar (PDA). The mycelium growth of P. oryzae was inhibited by sakuranetin with the concentration of 0.1 mm, 0.2 mm and 0.3 mm in the PDA medium. A time-course observation indicates that the growth of P. oryzae (in diameter) was significantly inhibited in PDA containing sakuranetin at 0.3 mm observed at 2 to 5 day post. The inhibition rate of P. oryzae rise with the increase of sakuranetin concentration in PDA. Introduction Phytoalexins have been demonstrated to accumulate rapidly at the site of attempted infection in sufficient quantities to inhibit the in vitro growth of fungi and bacteria. Sakuranetin was first identified from the cortex of the bark of a cherry tree (Prunus spp.) as an aglycone of sakuranin and also found in rice and several other plant species. Sakuranetin cannot be found in healthy rice leaves and its biosynthesis is rapidly induced by both biotic and abiotic stresses, such as infection with phytopathogens such as M. oryzae, Xanthomonas oryzae, and Ditylenchus angustus 2. Materials and Methods Chemical Commercial standard of sakuranetin for grade of high performance liquid chromatography (HPLC) was purchase from Sigma Aldrich. Determination of Antifungal Activity of Sakuranetin In Vitro Testing Four blast mycelium plugs (0.5cm each) were placed on each potato dextrose agar (PDA) containing sakuranetin at various concentrations: 0.1, 0.2, and 0.3 mm while PDA without sakuranetin (0 mm) serve as a control. There are five replications for each concentration. The diameter of mycelium colony will be measured after 0, 1, 2, 3, 4 and 5 days of incubation at 25 C in the dark 3. Results and Discussion Determination of Antifungal Activity of Sakuranetin In Vitro Testing The inhibitory activity of sakuranetin to mycelium growth of Pyricularia oryzae was devaluated in vitro using a potato dextrose agar (PDA). The diameter of four blast mycelium plugs of P. oryzae for each PDA plate was measured for 5 day post incubation (dpi) (Figure 1 and Figure 3). The mycelium growth of P. oryzae was inhibited by sakuranetin with the concentration of 0.1 mm until 0.3 mm in the PDA medium compared to the control without sakuranetin. A time-course observation indicates that growth of mycelium colony (in 40

42 diameter) of P. oryzae with sakuranetin at 0.3 mm was significantly inhibited from the observation at 2 to 5 dai which is 0.1 to 1.0 cm compare to 0.2, 0.1 mm concentrations and control which are 0.5 to 1.5 cm, 0.9 to 1.8 cm and 1.3 to 2.8 cm respectively (Figure 2). The inhibition rate of P. oryzae rise with the increase of sakuranetin concentration in PDA. Hasegawa et al. 3 also reported the inhibition rate by 0.3 mm sakuranetin became slightly higher than by 0.1 mm thereafter, and 51 and 36% of the growth was inhibited by 0.3 and 0.1 mm at 5 dpi, respectively. The phytoalexins have been shown to be effective in inhibiting in vitro growth by the blast fungus M. grisea and they accumulate more rapidly and to larger quantities in the incompatible interaction with the pathogen than in the favourable interaction 1. Figure 1: Diameter of mycelium colony of Pyricularia oryzae on PDA containing sakuranetin at 5 dpi Figure 2: Time course analysis on the spread mycelium colony of Pyricularia oryzae on PDA containing sakuranetin at Mm. 41

43 0.0 mm 0.1 mm 0.2 mm 0.3mM Figure 3: Inhibitory effect of sakuranetin against Pyricularia oryzae on PDA containing sakuranetin at mm. Conclusion In vitro study have shown that the flavonoid phytoalexin, sakuranetin significantly inhibit the growth mycelium of P. oryzae for five days of incubations. Reference 1. Dillon, V. M., Overton, L., Grayer, R. J., and Harborne, J. E Differences in phytoalexin response among rice cultivars of different resistance to blast Phytochemistry 44: Shimizu, T., Lin, F., Hasegawa, M., Okada, K., Nojiri, H., & Yamane, H Purification and identification of naringenin 7-O-methyltransferase, a key enzyme in biosynthesis of flavonoid phytoalexin sakuranetin in rice. Journal of Biological Chemistry, 287(23), Hasegawa, M., Mitsuhara, I., Seo, S., Okada, K., Yamane, H., Iwai, T., & Ohashi, Y Analysis on blast fungus-responsive characters of a flavonoid phytoalexin sakuranetin; Accumulation in infected rice leaves, antifungal activity and detoxification by fungus. Molecules, 19(8),

44 EVALUATION OF PRE AND POST EMERGENCE HERBICIDAL ACTIVITY OF 2, 4-DI-TERT-BUTYLPHENOL (2,4-DTBP) Wan Nur Suzani Sazleen Wan Shafiin* and Chuah Tse Seng School of Food Science and Technology, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu, Malaysia *Corresponding author Abstract Development of natural herbicide has become current interest among researchers nowadays due to increased numbers of herbicide-resistant weeds tremendously. Natural compound of 2,4-Di-tert-butylphenol (2,4-DTBP) was previously proven to have herbicidal property but its potential to be developed as herbicide remain unknown. This study aimed to characterize herbicidal activity of 2,4-DTBP on four bioassay species of weeds, Eleusine indica, Leptochloa chinensis, Hedyotis verticillata and Asystasia gangetica under glasshouse conditions. The results showed that 2,4-DTBP had poor post emergence herbicidal activity with only 15 to 65% suppression even at an application rate of as high as 7 kg ai/ha on seedlings of E. indica, L. chinensis, H. verticillata and A. gangetica at 3-to 4 leaf stage. For pre emergence herbicidal test, L. chinensis was the most sensitive towards 2,4-DTBP treatment, with 53% reduction of emergence at 2.5 kg ai/ha. Shoot growth reduction of H. verticillata, L. chinensis and E. indica were ranged from 40 to 60% at the similar application rate while the root growths of these bioassay species were inhibited by 50 to 80%. On contrary, A. gangetica was tolerant to 2,4-DTBP even the application rate of 2,4-DTBP was increased to 5 kg ai/ha. This finding has revealed the potential application of 2,4-DTBP as pre emergence natural herbicide for weed control. Introduction Natural herbicide derived has become a great interest in present day to overcome development of herbicide-resistant weeds. 2, 4-Di-tert-butylphenol is one of allelochemicals present in plants such as Pereskia bleo Kunth (6). Previously, 2,4-DTBP has been proven to have herbicidal property but its potential to be developed as herbicide remain unknown. Thus, 2,4-Di-tert-butylphenol (2,4-DTBP) has been used as pre and post application herbicide tested on several selected weeds species such as Eleusine indica, Leptochloa chinensis, Hedyotis verticillata and Asystasia gangetica as its phytotoxicity against some weedy plant has been proven previously. Nevertheless, pre and post application of 2,4- DTBP as soil applied herbicide were not yet discovered. Materials and Methods Materials Seeds of Eleusine indica were collected from roadside of Gong Badak, Terengganu, Leptochloa chinensis were collected on rice fields of Pengkalan Maras, Terengganu while Hedyotis verticillata and Asystasia gangetica seeds were sampled from an oil palm plantation of Kuala erang, Terengganu. Renggam soil series was collected from Maras, Terengganu. 2,4-Di-tert-butylphenol (99% purity) was purchased from Sigma Chem. Co. Post Emergence Herbicidal Test Seeds of each bioassay species were sown in paper cups (4.5 diameters by 7 cm height) with 5 holes at the bottom of each cup. The cups were then placed in a tray (15x10x8 cm) containing water at 1 cm deep to provide irrigation from the bottom. The cups filled with 50 g Renggam soil series and placed in a glasshouse and maintained at a relative humidity of 78-80% and a temperature of C with 12h photoperiod. The 2, 4-DTBP treatments at the rates of 0, 7, and 56 kg ai/ha were prepared by dissolving the compound in 0.3% dimethylnsulfoxide (DMSO) and 0.03% saponin. Homogeneous seedlings of each bioassay 43

45 species at 2-leaf stage were sprayed with a hand sprayer to provide 800 μl of liquid per cup. After 21 days of treatments, the above ground parts of the plant tissues were harvested. Shoot fresh weight and root length of the seedlings were determined and data were expressed as percentage of control. Pre emergence herbicidal test A total of 25 seeds of each L. chinensis, E. indica, H. verticillata as well as 5 seeds of A. gangetica were sown separately in a paper cup of 4.5 diameters by 7 cm height filled with 50 g Renggam soil series and placed in a glasshouse as described above. The compound of 2,4-DTBP was dissolved in 0.24% DMSO and % saponin in 4 rates; 0.625, 1.25, 2.5 and 5 kg ai/ha. 2,4-DTBP at each application rate was then applied onto soil surface with a micropipette to provide 800 μl of liquid per cup one days after sowing the seeds. The cups were then placed in the tray to provide irrigation as described above. Non-treated weed seeds was used as control with 0.24% DMSO and % saponin. Seedling were considered emerged when attained a plumule length of 2 mm. After 21 days of treatments, the emerged seedlings were recorded as a percentage of total number of viable seeds used in each replication. The root length and shoot fresh weight of emerged seedlings were measured and recorded. The data were expressed as percentage of control. Statistical analysis For post emergence herbicidal test, the experiments were arranged in completely randomized factorial design with five replications where two factors namely 2, 4-DTBP application rate and weed species were involved. The percentage data were checked for homogeneity of variance and normality before being subjected to two-way ANOVA. Mean treatments were separated using the Tukey test at 5% of significant level by excluding percentage data of control. For pre emergence herbicidal test, the experiments were arranged in completely randomized factorial design with five replications where two factors namely concentration of 2, 4-DTBP and weed species were involved. Log10 transformation and square root (x + 0.5) transformation were performed on percentage data of shoot fresh weight and root length, respectively, before being subjected to two way ANOVA. Tukey test was used to compare the mean among the treatments at 5% of significant level by excluding percentage data of control. Results and Discussions The inhibitory effect of 2,4-DTBP on shoot fresh weight of all tested bioassay varied with application rates indicating that 2, 4-DTBP significantly reduced the shoot fresh weight (Figure 1). At the lowest application rate, 7 kg ai/ha (equivalent to 70 µm), E. indica was most susceptible recording 39% suppression of the shoot growth as compared to L. chinensis and H. verticillata with 22 and 15 % suppression, respectively 4. Dry weight of Trifolium alexandrinum L. was reduced by 37 % and Physalis ixocarpa Brot. by 22% when treated with at 60 µm of Tricolorin A, a natural compound derived from Ipomoea tricolor Cav. Shoot fresh weight of both L. chinensis and E. indica were inhibited by 80% at the highest application rate of 56 kg ai/ha. At the same application rate, 60 and 45% reduction of shoot fresh weight were recorded for H. verticilata and A. gangetica, respectively. On the other hand, glyphosate and paraquat provided complete control of H. verticillata seedlings at 0.80 kg a.i/ha 1. These previous studies clearly shows that the effective rate of synthetic herbicide for controlling tested bioassay, is less than 1 kg ai/ha regardless of any active ingredients. 44

46 Shoot fresh weight (% of control) Application rate of 2,4-DTBP (kg ai/ha) Figure 1: Post emergence application of 2, 4-Di-tert-butylphenol (2,4-DTBP) on shoot fresh weight of Leptochloa chinensis ( ), Eleusine indica ( ), Hedyotis verticillata ( ) and Asystasia gangetica ( ) 21 days after treatment. Vertical bar represent standard deviation (SD) of the mean. Average across 2,4-DTBP rate, root length of L. chinensis was the most sensitive to 2,4- DTBP treatment, with 38% of nontreated control, followed by E. indica, H. verticillata and A. gangetica, being 75 % of nontreated control, suggesting that root length of E. indica, H. verticillata and A. gangetica seedlings were less affected by the 2, 4-DTBP. Therefore, 2,4- DTBP gave a strong reduction of bioassay species root length (63-57 % of nontreated control) at application rates ranging from kg ai/ha when average across weed species. Data analysis showed an interaction between pre emergence application rate of 2, 4-DTBP and weed species for emergence, shoot fresh weight and root length. The emergence of L. chinensis, E. indica and H. verticillata declined progressively with the increase rate of 2, 4 DTBP. Leptochloa chinensis was the most sensitive towards 2, 4 DTBP treatment, with 53% reduction at 2.5 kg ai/ha to 67% reduction at the highest application rate of 5 kg ai/ha. Meanwhile, both E. indica and H. verticillata were similarly inhibited by approximately 12% at 2.5 kg ai/ha 2, 4-DTBP. Likewise, the emergence of L. chinensis was recorded to be approximately 50 % when applied dibutyl phthalate as pre emergence at 2.4 kg a.i./ha, where dibutyl phthalate is a natural compound extracted from Chrysopogon serrulatus Trin. 2. This result suggests that 2,4-DTBP exhibited similar promising pre emergence herbicidal activity as dibutyl phthalate on inhibition of L. chinensis emergence. On the other hand, A. gangetica was the most tolerant species because the emergence remained 100% even at the highest application rate. The 2, 4-DTBP inhibited shoot fresh weight of tested weeds in a dose dependent manner with the phytotoxic activity being significantly more apparent on H. verticillata L. chinensis, E. indica than A. gangetica. At the lowest application rate of kg ai/ha, the shoot growth of H. verticillata, L. chinensis and E. indica were only reduced by 6-14%. Interestingly, the reduction of shoot growth was evident and increased to 40-58% when the application rate was increased to 2.5 kg ai/ha. A higher rate of coumarin at 3.8 kg ai/ha was needed to inhibit shoot growth of grassy weed, Sorghum halepense L. Pers. by 50%. Increased application rate to 5 kg ai/ha resulted in further reduction of shoot growth by 48 73% 5. However, shoot fresh weight A. gangetica seedlings were inhibited by 10% only although 2,4-DTBP was applied at the highest rate of 5 kg ai/ha. Root length of L. chinensis, E. indica and H. verticillata were % of nontreated control at kg ai/ha and decreased progressively with increased rate of application. It is noted that 22, 44 and 53% of nontreated control were recorded for the root lengths of L. chinensis, H. verticillata and E. indica seedlings, respectively, when being subjected to 2.5 kg ai/ha of 2,4-DTBP treatment. These results implies that L. chinensis was the most susceptible towards 2, 4 DTBP treatment. On the other hand, root length of A. gangetica seedling was less affected and the effects were negligible across the application rate. The respond of A. gangetica to 2,4-DTBP differs from other three bioassay 45

47 species might be due to evolutionary differences in tolerance to allelopathic compounds among the target species 3. Figure 4.3: Pre emergence application of 2,4-Di-tert-butylphenol (2,4-DTBP) on emergence(a), shoot fresh weight (B) and root length (C) of Leptochloa chinensis ( ), Eleusine indica ( ), Hedyotis verticillata ( ) and Asystasia gangetica ( ) 21 days after treatment. Vertical bar represent standard deviation (SD) of the mean. Conclusion In general, 2, 4-DTBP was able to suppress emergence, shoot and root growth of L. chinensis, E. indica and H. verticillata effectively at 2.5 kg ai/ha by %. However, 2,4- DTBP has poor post emergence herbicidal activity because an application rate of as high as 7 kg ai/ha was needed to inhibit the shoot growths of L. chinensis, E. indica, H. verticillata and A. gangetica seedlings by % only. Therefore, it is suggested that 2,4-DTBP has potential to be developed as novel pre emergence natural herbicide. References 1. Chuah, T. S., Noor-Zalila, M. R., Cha, T. S. and Ismail, B. S. (2005). Paraquat and glyphosate resistance in woody borreria (Hedyotis verticillata) growing at oil palm plantations in Terengganu, Malaysia. Malaysian Applied Biology 34(2): Chuah, T. S., Oh, H. Y., Habsah, M., Norhafizah, M. Z. and Ismail, B. S. (2014). Potential of crude extract and isolated compounds from golden beard grass (Chrysopogon serrulatus) for control of sprangletop (Leptochloa chinensis) in aerobic rice systems. Crop and Pasture Science. 65: Hunter M. E. and Menges, E. S. (2002). Allelopathic effects and root distribution of Ceratiola ericoides (Empetraceae) on seven rosemary scrub species. American Journal of Botany, 89(7): Lotina-Hennsen, B., King-Díaz, B., Pereda-Miranda, R. (2013). Tricolorin A as a natural herbicide. Molecules 18: Nazemi, A. H., Asadi, G. A. and Ghorbani, R. (2015). Herbicidal activity of coumarin when applied as a pre-plant incorporated into soil. Notulae Scientia Biologicae 7(2): Sri Nurestri, A. M., Sim, K. S., Norhanom, A. W. and Hashim, Y. (2009). Cytotoxic components of Pereskia bleo (Kunth) DC. (Cactaceae) leaves. Molecules 14:

48 POTENTIAL IMPACT OF ORGANIC FERTILIZATION ON CORN GROWTH AND YIELD Haruna Yahaya Rawayau*, Adzemi Mat Arshad, and Wan Zaliha Wan Sembok School of Food Science and Technology, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu, Malaysia *Corresponding author Abstract A greenhouse experiment was conducted at the School of Food Science and Technology, Universiti Malaysia Terengganu (UMT) to determine the potential impact of organic fertilization on corn grown on Rhu Tapai and Rasau soil series. The treatments included were different types of organic manure (cattle, horse and chicken manure) either combined with arbuscular mycorrhizal (AM) or without AM. A total of 80 corn plants were cultivated two types of soils as aforementioned. The application of the chicken manure and AM significantly affected the corn crop planted on Rasau soil series. The better growth performances of Rasau soil series grown corn crop can be seen clearly than Rhu Tapai soil series grown corn crop. Thus, the combination of chicken manure and AM will be used for the next experiment at Bukit Kor, UMT. The result of this study will help local farmers to explore the market of organic corn production, which many nations especially USA, India in countries in South East Asia are benefitting due to its nutritional and medicinal value. Introduction In the developing world, rapid population growth is bringing more competition for agricultural land of which the consequence is declining fertility of the soil, the food security of the affected nations can only be assured by taking measures of replenishing the soil with its lost nutrients with continuous reduction in yield 1. The third worlds better means of achieving soil fertility improvement is organic fertilizer being it affordable and readily available among the dwellers, of which animal droppings is the more available. The use of organic manure has helped farmers from total reliance on synthetic materials in soil fertility improvement 2. In trying to make the world green and free of in-organic sources of plant nutrients, the available nutrients of organic source has to be renewed for proper soil fertility management 3. Many research have been carried with promising outcomes in inoculating the soil with arbuscular mycorrhizal (AM) fungi that improves the performance of plant 4. AM is known to improve phosphorus nutrition by accessing large surface area of soil, improvement of soil acidity and making phosphate available to plants. The weakness of AM fungi is its nonsignificant role in N nutrition which has to do with mobility nature of nitrate 5. Soil amendment with organic fertilizer and bio-fertilizer forms part of new soil fertility management. Integration of biochar with AM fungi has shown some positive result as the biochar serves as medium for AM fungi hyphae and also prevent any attack from soil microorganisms that may be harmful to their colony as such aid the symbiosis between plant host and the fungus, it is also affirm that biochar amendment positively affects plant growth and yield as it is a source of nutrient which it is capable of releasing, and aid in holding the soil nutrients together 6. The research is aimed towards finding the combined effects of AM fungi and different sources of organic manure (Biochar inclusive) on the growth performance of corn plant, as a way of exploring organic farming that is gaining popularity due to its significance in soil fertility management and safe consumption of produces. Material and Methods The experiment was arranged as according to the Randomized Complete Block Design (RCBD) with four replications. The factors were; Organic Manure [OM1=Control (no OM), 47

49 OM2=Chicken Manure, OM3=Cattle Manure, OM4=Horse Manure and OM5=Biochar and AM Fungi (M1= with and M2= without). All these treatments were applied on corn planted on two different soil series namely S1:Rasau soil series and S2: BRIS soil series). Sweet corn (Zea mays saccharata L.), Var F1 HY Thai Super Sweet corn was obtained from the seeds collection of the School of Food Science and Technology. At the beginning, 3 seeds were sown in 30kg pot and later thinned to one. Both soil series were then amended with organic manure as according to the treatments two (2) weeks prior to planting. Meanwhile, the amount of AM fungi was 10g/pot (with spores/g). All the growth parameters viz plant height, stem girth, leaf length, leaf width, leaf area and leaf number were measured at 2, 4, 6, 8 and 10 Weeks After Planting (WAP). The data collected were statistically analysed using Two-Way Analysis of Variance (ANOVA) and the treatment means were further separated using Duncan Multiple Range Test (DRMT) at p 0.05 (SAS software 9.4). Results and Discussions There was significant difference (p=<0.05) in almost all the growth parameters as affected by organic manure amendment, Arbuscular Mycorrhizal inoculation and soil type. Table 1 shows the Analysis of Variance (ANOVA) which indicates a significant difference (P<0.001) in all the parameters for main effects of OM, AMF and SOI, the means were separated and presented in Table 2. Fig 1: AMF colonisation on inoculated corn root Chicken manure (OM2) with highest (61.71) means value for plant height was significantly (P<0.001) different statistically from other organic manure sources, and that shows it helps in improving the growth of corn plant which also agree with Odedina et al 1 and as well for leaf width (4.249), leaf length (42.63), stem girth (6.1), leaf area (137.76) but was not significantly different (P<0.05) to OM5 (biochar) with mean for leaf number. Apart from leaf number, AM fungi inoculation enhances the growth performance of corn plant significantly in all other parameters with higher mean value compared to without AM inoculation (Table 2). As expected, AM fungi are known to improve plant nutrition from its symbiotic relationship with its host 4. Rasau soil as well significantly improves the growth performance of corn plant in all the growth parameters at 4 weeks after planting except leaf number where it didn t differ statistically with BRIS soil as presented in table 2, it has a mean value of for plant height while BRIS has 45.70, leaf width 3.60, leaf length 37.71, stem girth 5.28, leaf area were all significantly different from the mean values obtained in BRIS soil, the reason might be because of the nutrient content from the two different soil types (mineral and BRIS) as reported by Senjobi et al 2. 48