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Page 1: Proceeding of Postgraduate Seminar 2016 - PPSTMppstm.umt.edu.my/wp.../04/PROCEEDING-OF-POST-GRADUATE-SEMI… · 4 ORGANIZING COMMITTEE POSTGRADUATE SEMINAR 2016 SCHOOL OF FOOD SCIENCE
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Proceeding of Postgraduate Seminar 2016

Hak Cipta Terpelihara © 2016. 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, 21030 Kuala Terengganu, Terengganu,

Malaysia.

© 2016 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, 21030 Kuala Terengganu, Terengganu, Malaysia.

ISBN : e 978-967-0962-78-8

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CONTENTS

MESSAGE FROM THE DEAN 3

ORGANIZING COMMITTEE 4

LIST OF EXTENDED ABSTRACTS 5

EXTENDED ABSTRACTS 7

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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

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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 Fatimah @ 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

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LIST OF EXTENDED ABSTRACTS

Abstracts Page

Haslinda Wan Hamat, Mohd Nizam Lani, Yusnita Hamzah2 and Wan Bayani Wan Omar

Microbiological quality of keropok lekor from selected food premises in Kuala Terengganu and Marang

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Nurul Farhanah Mohd Aluwi, Nor Hayati Ibrahim and Yusnita Hamzah Properties and stability of salad dressing - type emulsions containing rose cactus (Pereskia bleo) mucilage, xanthan gum and locust bean gum

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Normardiana Jaafar, Siti Nur’afifah Jaafar and Norainy Mahyudin Pilot testing of food safety knowledge, attitude and perception towards food handlers’ hygiene practice among night market food consumers

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Siti Fatimah Zakaria, Mohd Nizam Lani, Chuah Tse Seng, Fisal Ahmad and Khairul Mazmi Ahmad Antifungal activity of lactic acid bacteria against pathogenic Sclerotium rolfsi of Capsicum annum plant

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Norhayati Binti Abd Hadi, Norizah Mhd. Sarbon, Zamzahaila Mohd Zin and Hayati Mohd Yusof Physico-chemical properties and activities of antioxidants compounds in honey bee honey and stingless bee honey

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Ain Najwa Khairul Anwar, Rosazlin Abdullah, Zuraida Abdul Rahman,

Yusnita Hamzah and Wan Zaliha Wan Sembok Effects of different arbuscular mycorrhiza species and its rates on the growth performance of hempedu bumi (Andrographis paniculata Nees) grown on bris soil

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Muhammad Shahrul Hafiz Elham, Ng Lee Chuen, Sariam Othman and Mohd Razi Ismail

Physical barrier mechanism of si-mediated rice against Pyricularia Oryzae

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Norkaspi Khasim, Adzemi Mat Arshad and Khalid Haron

Integration of bamboo (Gigantochloa albociliata) with oil palm and its effect of nutrient cycling on oil palm yield

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Nura Adila Zahari, Aziz Ahmad and Ng Lee Chuen In vitro study of flavonoid phytoalexin sakuranetin against Pyricularia oryzae

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Wan Nur Suzani Sazleen Wan Shafiin and Chuah Tse Seng

Evaluation of pre and post emergence herbicidal activity of 2, 4-di-tert-butylphenol (2,4-DTBP)

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Haruna Yahaya Rawayau, Adzemi Mat Arshad and Wan Zaliha Wan Sembok Potential impact of organic fertilization on corn growth and yield

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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

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Rul Hajar Muda, Ng Lee Chuen and Khairulmazmi Ahmad

Biocontrol potential of fluorescent pseudomonads on rhizoctonia root rot disease caused by Rhizoctonia solani on chilli

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Azmi Ismun, Aidilla Mubarak, Shamsul Bahri Abd Razak and Marinah Mohd Ariffin

The determination of polyphenols in latex of Hevea brasiliensis and rubber processing effluent via spectrometry, spectrophotometry and chromatography

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MICROBIOLOGICAL QUALITY OF KEROPOK LEKOR FROM SELECTED FOOD PREMISES IN KUALA TERENGGANU AND MARANG

Haslinda Wan Hamat1,2*, Mohd Nizam Lani2, Yusnita Hamzah2 and Rozila Alias3

1 Food Safety and Quality Laboratory, Terengganu Health State Department, Kg. Bukit Tunggal, 21200 Kuala Terengganu, 2School of Food Science and Technology, Universiti

Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, 3Institute of Bio-IT Universiti Selangor, Jalan Zirkon A 7/A, Seksyen 7, 40000 Shah Alam, Selangor, Malaysia,

*Corresponding author email: [email protected]

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 log10 CFU/g

and <1 to 6.00 log10 CFU/g, respectively. Samples were found to have coliform in the

range of <1 to 6.00 log10 CFU/g. Coagulase positive Staphylococci counted 3.30 and

4.10 log10 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 log10 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

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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 990. 12), Enterobacteriaceae (AOAC Official Method 2003.01), Coagulase Positive Staphylococci (ISO 6888-1:1999/Amd.1:2003(E)), Coliforms & E. coli (AOAC Official Method 991.14), Salmonella (ISO 6579:2002 (E)), Vibrio cholerae & Vibrio parahaemolyticus (ISO/TS 21872-1:2007 (E)) and Listeria monocytogenes (ISO 11290-1:1996/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 log10 CFU/g and < 1 to 6.40 log10 CFU/g, respectively. While, dipping sauce and keropok lekor had APC range of < 1 to 5.1 log10 CFU/g and 1.80 to 5.50 log10 CFU/g, respectively. APC in all keropok lekor samples were at the satisfactory level which were less than 6.00 log10 CFU/g as referred to Food Act 1983 (Act 281) & Regulations1. Meanwhile, counts of APC above 5.00 log10 CFU/g in ready to eat (RTE) food were faced as a potential risk due to the presence of pathogens2. Only one RTE sample which was keropok lekor had APC of 5.50 log10 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 log10 CFU/g and < 1.00 to 5.90 log10 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 log10 CFU/g and less than 1.00 log10 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 log10 CFU/g and < 1.00 to 5.90 log10 CFU/g, respectively. There are less than 1.00 log10 CFU/g coliforms in all dipping sauce samples. Coliforms count in keropok lekor ranged between < 1.00 to 3.00 log10 CFU/g. According to Food Act 1983 (Act 281) & Regulations1, all ready-to-eat (RTE) fish and fish products should have coliforms less than 1.70 log10 CFU/g. Coliforms in three keropok lekor samples were at the unsatisfactory level which counted 3.00, 2.10 and 2.10 log10 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.

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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 log10 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 305.

2. 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: 202-207.

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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, 21030 Kuala Terengganu, Terengganu, Malaysia

*Corresponding author email:[email protected]

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 3871.7% - 3977.87% (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

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(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 2.93 – 3.27 for freshly prepared emulsions which will directly promote the stability of the emulsions as stated by the previous study1 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 polysaccharide2. 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 study3, 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±0.01ab

10.36 ±0.33b 0.57 ± 0.17bc

B (30%RCM : 30% XG : 40% LBG) 3.14 ±0.01b 12.25± 0.29b 0.22 ± 0.04bc C (35% RCM : 35% XG : 30% LBG) 3.12 ±0.03b

11.25 ±0.53c 0.15 ± 0.03c

D (100% RCM) 3.27 ±0.08a 10.34 ± 1ab 0.09 ± 0.02c E (100% XG) 3 ±0.03c 9.99 ±1.26d 1.67 ± 1.45b F (100% LBG) 2.93 ±0.07c 11.03 ±0.29a 3.87 ± 0.52a

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.

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 separation4. 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

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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) 100.00 ± 0.01a 91.98 ± 0.63b B (30%RCM : 30% XG : 40% LBG) 100.00 ± 0.01a 91.88 ± 0.54b C (35% RCM : 35% XG : 30% LBG) 93.16 ± 0.04b 92.18 ± 0.09b D (100% RCM) 82.35 ± 0.01c 79.09 ± 1.13c E (100% XG) 62.29 ± 1.71d 42.73 ± 0.01d F (100% LBG) 100.00 ± 0.01a 100.00 ± 0.01a

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: 227-248.

2. Sengkhamparn, N., Verhoef, R., and Schols, H.A. (2009). Characterization of cell wall polysaccharide from okra (Abelmoschus esculentus (L.) Moench). Carbohydrate Research 344: 1824-1832.

3. 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: 1051-1061.

4. 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): 292-301

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Normardiana Jaafar1*, Siti Nur’afifah Jaafar1, Nor Ainy Mahyudin3

1School of Food Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala

Terengganu, Terengganu, 2Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

*Corresponding author email: [email protected]

Abstract

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 2015. 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 temperature1. 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 26.04 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 Malaysia2. 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

PILOT TESTING OF FOOD SAFETY KNOWLEDGE, ATTITUDE AND PERCEPTION TOWARDS FOOD HANDLERS’ HYGIENE PRACTICE AMONG

NIGHT MARKET FOOD CONSUMERS

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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, Terengganu3. 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 use4. 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

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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 0.889 and 0.704 respectively. While for the familiarity with night market foods as moderating factors the Cronbach’s alpha was 0.817. For the dependent variable, the Cronbach’s alpha of purchase intention was 0.691. While different levels of reliability are required, depending on the nature and purpose of the scale, Nunnally and Bernstein5 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=0.808. 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= 0.511 and max r= 0.701. 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= 0.119 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’

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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 Egypt6 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 17:370-377.

2. Sharif, L. and Al-Malki, T. (2010). Knowledge, attitude and practice of Taif university students on food poisoning. Food Control 21: 55-60.

3. Ministry of Health. (2012). Health Fact 2012. Ministry of Health Malaysia.

Retrieved on 2014, Feb 19 from:

http://www2.moh.gov.my/images/gallery/stats/heal_fact/health_fact_2012_page

_by_page.pdf

4. Cooper, D. R., and Schindler, P.S. (2003). Business research methods (8th edn). Boston: McGraw-Hill

5. Nunnally, J.C. and I.H. Bernstein. (1994). Psychometric Theory. 3rd 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: 427-435.

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ANTIFUNGAL ACTIVITY OF LACTIC ACID BACTERIA AGAINST PATHOGENIC Sclerotium rolfsi OF Capsicum annum PLANT

Siti Fatimah Zakaria1, Mohd Nizam Lani1, Chuah Tse Seng1, Fisal Ahmad1 and Khairul Mazmi Ahmad2

1School of Food Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala

Terengganu, Terengganu,2Department of Plant Protection, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia

*Corresponding author email: [email protected]

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 human1. 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 chili2. Today, fungicides such as hexaconazole, tebuconazole and propiconazol always being used to control most of the diseases caused by fungi in chili plant1. 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 fungi3. 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 agent4 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,

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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 sorghum-based 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.

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Table 1: Confirmation of 23 Lactic Acid Bacteria (LAB) molecularly identified by 16 rDNA sequencing analysis.

No. Sample Code Identification of LAB strains

Source %ID Accession No.

1 SA Lactobacillus plantarum WCFS1 strain WCFS1

Corn kernel silage 97% NR_075041.1

2 SB Lactobacillus plantarum strain JCM 1149

Corn kernel silage 96% NR_117813.1

3 SC Lactobacillus plantarum strain JCM 1149

Corn kernel silage 96% NR_117813.1

4 SD Lactobacillus plantarum strain JCM 1149

Corn kernel silage 97% NR_115605.1

5 SE Lactobacillus plantarum strain NRRL B-14768

Corn kernel silage 97% NR_042394.1

6 SF Lactobacillus plantarum WCFS1 strain WCFS1

Corn kernel silage 97% NR_075041.1

7 SG Lactobacillus plantarum strain NBRC 15891

Corn kernel silage 95% NR_113338.1

8 FCN Lactobacillus plantarum WCFS1 strain WCFS1

Fermented catfish 97% NR_075041.1

9 FCM Pediococcus pentosaceus ATCC 25745 strain ATCC 25745

Fermented catfish 97% NR_075052.1

10 FCO Lactobacillus plantarum strain NBRC 15891

Fermented catfish 97% NR_113338.1

11 FCP Lactobacillus plantarum WCFS1 strain WCFS1

Fermented catfish 96% NR_075041.1

12 FCK Lactobacillus plantarum WCFS1

strain WCFS1 Fermented catfish

96% NR_075041.1

13 FCE Lactobacillus plantarum strain

NRRL B-14678 Fermented catfish

98% NR_042394.1

14 FCA Lactobacillus plantarum strain

JCM 1149 Fermented catfish

97% NR_117813.1

15 FCH Lactobacillus plantarum strain

JCM 1149 Fermented catfish

98% NR_117813.1

16 FCG Lactobacillus plantarum WCFS1

strain WCFS1 Fermented catfish

98% NR_075041.1

17 FCL Lactobacillus plantarum strain

JCM 1149 Fermented catfish

98% NR_117813.1

18 FCI Lactobacillus plantarum WCFS1

strain WCFS1 Fermented catfish

95% NR_075041.1

19 FCB Lactobacillus plantarum strain

JCM 1149 Fermented catfish

97% NR_117813.1

20 FCD Lactobacillus plantarum WCFS1

strain WCFS1 Fermented catfish

98% NR_075041.1

21 FCC Lactobacillus plantarum WCFS1

strain WCFS1 Fermented catfish

95% NR_075041.1

22 FCJ Lactobacillus plantarum strain

JCM 1149 Fermented catfish

96% NR_117813.1

23 FCF Lactobacillus plantarum strain

JCM 1149 Fermented catfish

98% NR_117813.1

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Antifungal activity of LAB against S. rolfsii

Efficacy of antifungal activity was tested among different strains of LAB by following poisoned food technique1. 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 bio-control 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 bacteriocin- producing 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: 31-37.

2. Kalmesh, M. and Gurjar, R. B. S. 2001; Sclerotium rolfsii – A new threat to chilli in Rajasthan. Mycology andPlant Pathology 31 (2): 261

3. 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 94-159

4. 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 20:144-148.

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PHYSICO-CHEMICAL PROPERTIES AND ACTIVITIES OF ANTIOXIDANTS

COMPOUNDS IN HONEY BEE HONEY AND STINGLESS BEE HONEY

*Norhayati Binti Abd Hadi,1,3, Norizah Mhd. Sarbon1, Zamzahaila Mohd Zin1 and Hayati Mohd Yusof 1,2

1School of Food Science and Technology, 2Institute of Marine Biotechnology

Universiti Malaysia Terengganu, 3Center of Nutrition and Dietetic, Universiti Sultan Zainal Abidin, 21300 Kuala Terengganu, Terengganu, Malaysia

*Corresponding author email: [email protected]

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 honeys5. 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.

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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 buffers4. 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 0.00023/℃. 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 200–450 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 = 0.9997) 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.

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Determination of DPPH assay: The scavenging activity against 1,1-diphenyl-2-picrylhydrazil (DPPH; SIGMA, USA) radical was used in this study 3. 0.75ml 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 100-1000 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 (19.2 + 0.8 g/100g) and Kelulut (29.5 + 1.5 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.

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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 68.9 + 0.5 meq/kg (Acacia) and 164.5+ 3.5 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 54 + 4 mg/kg for Acacia and 76 + 4 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 conditions1. 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 6.7 +0.2 and 9.2 +0.2 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 35.7 + 0.4 g/100 g and 40.53 + 1.5 g/100 g respectively (Table 1). Kelulut show the glucose and fructose contents as 25 + 0.7 g/100 g and 21.4 + 1.4 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 fructose4. 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 52.15 mg GAE/100 g and 32.78 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 Tunisia3. 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 honey6. 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

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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 samples3. 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 659.80 μM Fe [II]/100 g and Kelulut honey was 606.18 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): 305–313.

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: 5870–5877.

3. 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): 513–518.

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, 526–534.

5. 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: 14–21.

6. 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: 674-687.

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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 4.2 + 0.2a 0.2 + 0.0 19.2 + 0.8 a 68.9 + 0.5 a 54 + 4 6.7 + 0.2 40.53 + 1.5a 35.7 + 0.4 a 0

Kelulut 3.4 + 0.2b 0.1 + 0.0 29.5 + 1.5 b 164.5 + 3.5 b 76 + 4 9.2 + 0.2 21.4 + 1.4 b 25 + 0.7 b 2 + 0.5

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 52.15 + 1.34 a 659.79 + 1.78 a 49.8 + 1.48

Kelulut 32.78 + 0.97 b 606.18 + 6.88 b 48.4 + 0.45

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

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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 Abdullah2, Zuraida Abdul Rahman3, Yusnita Hamzah1 and

Wan Zaliha Wan Sembok1

1School of Food Science and Technology, Universiti Malaysia Terengganu, 2Institute of Biological Sciences, University of Malaya, 3Biotechnology Research Centre, Malaysian Agricultural Research

and Development Serdang, Persiaran MARDI-UPM, Malaysia.

*Corresponding author email: [email protected] 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 67,582.61 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 35 – 45 °C. Five-node stem cuttings were transferred into polybag containing sterilized

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(90 - 110 °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

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 two-way analysis of variance (ANOVA) using GLM (General Linear Models) procedures and further separated by Tukey for minimum significant difference at P ≤ 0.05. 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 non-inoculated A.paniculata (16 and 66 respectively) (Figure 1).This is mainly caused by enhanced cytokinin formation of the plant which leads to more cell division1. 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). 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-inoculated2. 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

Results and Discussion

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.

0

50

100

150

200

250

300

350

14 42 70

Sp

ore

Nu

mb

er

Days After Transplanting (DAT)

Glo 0g Glo 10g Glo 50g

Scu 0g Scu 10g Scu 50g

Giga 0g Giga 10g Giga 50g

Mix 0g Mix 10g Mix 50g

0

20

40

60

80

100

120

14 42 70

Ro

ot

Infe

ctio

n P

erce

nta

ge

(%)

Days After Transplanting (DAT)

Glo 0g Glo 10g Glo 50gScu 0g Scu 10g Scu 50gGiga 0g Giga 10g Giga 50gMix 0g Mix 10g Mix 50g

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.

0

20

40

60

80

100

120

140

160

180

200

0 7 14 21 28 35 42 49 56 63 70 77

Nu

mb

er o

f B

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ches

Days After Transplanting (DAT)

Glo 0g Glo 10g Glo 50g Scu 0g

Scu 10g Scu 50g Gig 0g Gig 10g

Gig 50g Mix 0g Mix 10g Mix 50g

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.

Figure 4: Effects of different species and

rates of AM on spore number of

A.paniculata. Vertical bar represent HSD

value at 5% significant level.

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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 plants4. 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-glucoside5. In addittion, the inoculation of G.mossae was significantly decrerase the total phenolic content and no significant affect on the total flavonoid content of marjoram6. 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.

0

1

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4

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9

10

0 7 14 21 28 35 42 49 56 63 70 77

Ste

m D

iam

eter

(m

m)

Days After Transplanting (DAT)

Giga sp. Glo sp. Mix sp. Scutello sp.

Figure 7: Effects of different species of AM on stem diameter of A.paniculata. Vertical bar represent HSD value at 5% significant level.

0

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6

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0 7 14 21 28 35 42 49 56 63 70 77

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m)

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0g AM 10g AM 50g AM

Figure 6: Effects of different rates of AM on stem diameter of A.paniculata. Vertical bar represent HSD value at 5% significant level.

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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 (cm2)

Total chlorophylls

Total phenolic

AM rates (R) 0 4.77 a 1.56 a 14.85 b 30.32 a 0.95 a 0.05 b 10 6.28 a 2.22 a 22.70 a 46.74 a 0.88 a 0.16 a 50 6.37 a 2.05 a 22.79 a 47.08 a 1.02 a 0.06ab F-test significance

NS NS * NS NS *

AM species (S)

Gigaspora sp.

6.12 a 1.96 a 20.12 a 46.47 a 0.87 a 0.03 a

Scutellopora sp.

6.02 a 2.08 a 20.41 a 45.38 a 0.81 a 0.10 a

Glomus sp. 6.81 a 2.22 a 23.38 a 47.18 a 1.14 a 0.07 a Mixed sp. 4.28 a 1.52 a 16.54 a 26.51 a 0.97 a 0.02 a F-test significance

NS NS NS NS NS NS

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≤0.05.

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): 24-26.

2. 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): 114-118.

3. 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):1110-1115.

4. 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: 291−297.

5. Engel, R., Szabo, K., Abranko, L.. Rendes. K., Fuzy, 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: 3733−3742.

6. 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: 461-468.

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Physical Barrier Mechanism of Si-mediated Rice Against Pyricularia oryzae

Muhammad Shahrul Hafiz Elham1*, Ng Lee Chuen1, Sariam Othman2 and Mohd Razi Ismail2

1School of Food Science and Technology, 2Institute of Oceanography and Environment, Universiti Malaysia Terengganu, 21030, Kuala Terengganu, Terengganu

*Corresponding author email: [email protected]

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 (KSiO4) and Sodium Silicate (NaSiO4) 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 condition2. 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 acid4. Many studies have been conduct to elucidate how this non-essential 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)

Abstract

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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 1x106 (15ml/pot) were used as sourced of inoculation. Rice plant 14 days after sowing (DAS) were inoculated during evening and kept in dark with 98-100% 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 standard3. 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 7th 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 (8.223839%). 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%).

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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 matured4. 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 cultivar1.

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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

compared to MR219-4. 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): 324-333. doi: 10.1111/j.1399-3054.2008.01140.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: 53-63.

3. IRRI. (2008). World Rice Statistic. from http://www.irri.org/science/ricestat 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): 279-283. doi: 10.1093/pcp/pci018

5. 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): 1095-1103.

Conclusion

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Rice variety

EDX Image EDX graft

MR219-4 Control

MR219-4 Treated

MARDI Aerob 1 Control

MARDI Aerob 1 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

5218 count/s

12945 count/s

5763 count/s

11680 count/s

Silica bodies

Silica cell

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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 Haron1

1 Crop and Livestock Integration Unit, Malaysia Palm Oil Board, 43000 Kajang, Selangor, 2 Food Crop Science Unit, School of Food Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala

Terengganu, Terengganu.

Corresponding author e-mail: [email protected]

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 19th century and the first bamboo gardens were established and became known to the public2. In Malaysia, bamboo is found in abundance and widely scattered in about 5% of the total forest reserve area1. It has, however, received comparatively little research attention. Most bamboos species found in the local forests are Gigantochloa sp., Schizostachyum zollingerii and Dendrocalamus pendulus3.

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 countries5. Commercially, bamboo can be converted into charcoal and activated carbon via carbonisation followed by activation4,5,6. An observation was made on the plot of the integration of bamboo and oil palm that had been established in the MPOB Research Station

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 1st year after planting in double avenue oil palm was satisfactory good compared to normal oil palm planting.

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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 359.2 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 18.1. 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 2014. 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 2016. 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 2017. The FFB yields were not much different between the bamboo-oil palm integrated plots and control plot until June 2016 (Table 2).

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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.

Parameter Replicate

Treatment

2 m 4 m 6 m

6 month 12

month 6 month

12 month

6 month 12

month

Height (cm)

1 371.8 323.0 394.8 381.6 393.7 360.8

2 363.8 368.0 358.9 338.3 342.8 294.3

3 290.2 306.9 343.2 357.8 273.8 262.7

Average 341.9 332.6 365.6 359.2 336.8 305.9

Culm circumference

(5 cm from ground surface)

1 8.74 8.35 9.48 9.60 8.82 9.00

2 8.82 9.00 10.23 9.80 9.78 9.40

3 7.10 8.30 7.42 10.10 8.46 7.80

Average 8.22 8.55 9.04 9.83 9.02 8.73

Culm circumference

(1.3 m from ground surface)

1 5.40 5.70 5.76 6.52 5.50 6.45

2 5.48 5.80 5.92 6.35 6.08 6.70

3 3.62 5.25 3.52 6.80 4.80 4.50

Average 4.83 5.58 5.07 6.55 5.46 5.88

No. of nodes

1 24.6 22.0 25.6 23.7 24.9 22.1

2 24.6 23.0 23.4 22.1 22.4 22.3

3 22.3 18.5 23.0 22.6 22.6 15.0

Average 23.8 21.2 24.0 22.8 23.3 19.8

No. of branches

1 15.9 17.3 18.9 18.3 20.3 18.7

2 13.0 15.9 17.5 17.9 15.0 15.0

3 14.3 17.1 9.5 18.0 12.4 15.1

Average 14.4 16.8 15.3 18.1 15.9 16.3

TABLE 2 : FFB YIELD IN BAMBOO PLOT INTEGRATED WITH PALM AT MPOB

STATION, KERATONG, PAHANG.

Plot Tonne/Ha/Yr

2015/2016 (Mac’15 – Feb’16)

*2016/2017 (Mac’16 – Feb’17)

Bamboo integrated (D. Avenue,136 palms/Ha)

20.48 2.39

Control (Normal, 136 palms/Ha)

7.54 0.13

Control (D.Avenue, 136 palms/Ha)

13.74 1.48

Notes : * FFB yield until June 2016.

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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 983-41679-0-3. 3. 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: 592-594.

5. 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 141:819-825.

6. Young, C. B., Kwang, J. C. and Joo, H. C. (2005). Production and CO2 adsorption characteristics of activated carbon from bamboo by CO2 activation method. Korean Engineering Research 43: 146-152.

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IN VITRO STUDY OF FLAVONOID PHYTOALEXIN SAKURANETIN AGAINST Pyricularia oryzae

Nura Adila Zahari 1*, Aziz Ahmad 2 and Ng Lee Chuen 1

1School of Food Science and Technology, 2School of Fundamental Science, University Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia.

*Corresponding author email: [email protected]

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 angustus2. 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 dark3. 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

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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 interaction1.

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 0.0- 0.3 Mm.

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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 0.0-0.3 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. 1997. Differences in

phytoalexin response among rice cultivars of different resistance to blast Phytochemistry 44:599-603.

2. Shimizu, T., Lin, F., Hasegawa, M., Okada, K., Nojiri, H., & Yamane, H. 2012. 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), 19315-19325.

3. Hasegawa, M., Mitsuhara, I., Seo, S., Okada, K., Yamane, H., Iwai, T., & Ohashi, Y. 2014. 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), 11404-11418.

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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, 21030 Kuala Terengganu,

Terengganu, Malaysia

*Corresponding author email: [email protected]

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 28-30 ˚C with 12h photoperiod. The 2, 4-DTBP treatments at the rates of 0, 7, 14. 28 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

Abstract

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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 0.024 % 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 0.024 % 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, respectively4. 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/ha1. 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.

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Figure 1: Post emergence application of 2, 4-Di-tert-butylphenol (2,4-DTBP) on shoot fresh weight of ), Hedyotis verticillata ( ) and Asystasia Leptochloa chinensis ( ), Eleusine indica (

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 14 - 56 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 0.625 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 83 - 88 % of nontreated control at 0.625 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

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species might be due to evolutionary differences in tolerance to allelopathic compounds among the target species3.

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 50 – 80 %. 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 15-40 % 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):43–49.

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. 65:461–469

3. 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):1113–1118.

4. Lotina-Hennsen, B., King-Díaz, B., Pereda-Miranda, R. (2013). Tricolorin A as a natural herbicide. Molecules 18:778–788.

5. 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): 239–243.

6. Sri Nurestri, A. M., Sim, K. S., Norhanom, A. W. and Hashim, Y. (2009). Cytotoxic components of Pereskia bleo (Kunth) DC. (Cactaceae) leaves. Molecules 14:1713

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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, 21030 Kuala Terengganu, Terengganu, Malaysia

*Corresponding author email: [email protected]

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 yield1. 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 improvement2. 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 management3. Many research have been carried with promising outcomes in inoculating the soil with arbuscular mycorrhizal (AM) fungi that improves the performance of plant4. 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 non-significant role in N nutrition which has to do with mobility nature of nitrate5. 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 micro-organisms 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 together6. 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),

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.

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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 20-25 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 al1 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 host4. 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 52.93 for plant height while BRIS has 45.70, leaf width 3.60, leaf length 37.71, stem girth 5.28, leaf area 108.31 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 al2.

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Table 1: A two way ANOVA table showing mean values of different growth parameters at 4 Week After Planting (WAP) Treatments Plant height Leaf Width Leaf length Stem Girth (mm ) Leaf Area (cm2) Leaf Number (cm) OM ** ** ** ** ** ** AMF ** ** ** ** ** ns SOI ** ** ** ** ** ** Interactions OM*AMF ns ns ns ns ns ns OM*SOI ns ns ns ns ns ns AMF*SOI ns ns ns ns ns ns OM*AMF*SOI ns ns ns ns ns ns CV 6.41 12.52 11.38 7.63 24.65 11.96 NB: OM= Organic Manure, AMF= Arbuscular Mycorrhizal Fungi, SOI= Soil, CV= Coefficient of Variation, ns= not significant, *=significant at 0.05 level, **= significant at 0.01 level Table 2: Effects of Arbuscular Mycorrhizal inoculation, soil type and different sources of organic manure on growth of corn at 4 weeks after planting Treatments Plant height Leaf Width Leaf length Stem Girth (mm ) Leaf Area (cm2) Leaf Number (cm) Organic Manure (OM) OM 1 (control 28.99e 1.90c 20.14d 2.84d 29.78c 5.56c OM 2 (Chicken Manure) 61.71a 4.24a 42.63a 6.10a 137.76a 7.44a OM 3 (Cattle Manure) 52.28c 3.57b 37.70bc 5.25b 102.77b 6.31bc OM 4 (Horse Manure) 47.31d 3.40b 35.93c 4.76c 96.41b 6.89ab OM 5 (Biochar) 55.86b 3.78b 40.15a 5.60b 115.64b 7.31a Arbuscular Mycorrhizal Fungi (AMF) AMF 1 (with) 53.15a 3.72a 38.80a 5.30a 114.68a 6.75a AMF 2 (without) 45.48b 3.03b 31.82b 4.52b 78.27b 6.65a Soil type (SOI) SOI 1 (Rasau) 52.93a 3.60a 37.71a 5.28a 108.31a 7.12a SOI 2 (BRIS) 45.70b 3.15b 32.91b 4.54b 84.63b 6.27b NB: Means with the same letter within same column of either of the treatments are not significantly

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Conclusion The outcome of this research indicates that Rasau soil series amended with chicken manure and Arbuscular mycorrhizal inoculation improves the growth performance of corn plant. References

1. Odedina, J. W., Odedina S. A., & Ojeniyi S. O. (2011). Effect of types of manure on growth and yield of cassava (Manihot esculenta Crantz). Researcher, 3 (5): 1-8.

2. Senjobi, B. A., Peluola C. O., Senjobi C. T., Lawal I. O., Ande O. T. and Salami B. T. (2010). Performance of Cochorus olitorius as influenced by soil type and organic manure amendments in Yewa North Local Government Area, Ogun State. African Journal of Biotechnology, 9(33): 5309-5312.

3. King, L. D. (1990). Soil nutrient management in the United States. In sustainable Agricultural system, Ed. C.A. Edwards et al., St. Lucie Press. FL., Pp: 89-106.

4. Jeffries P. (1987). Use of mycorrhizae in agriculture. Crit. Rev. Biotech. 1987; 5: 319-348.

5. Tarafdar, J. C.; Marschner, H. (1994). Phosphatase Activity in the Rhizosphere and Hyphosphere of VA Mycorrhizal Wheat Supplied with Inorganic and Organic P. Soil Biology Biochemistry 26: 387–395.

6. Lehmann, J., Gaunt, J. & Rondon, M. (2006). Bio-char sequestration in terrestrial ecosystems – A review. Mitigation & Adaptation Strategies for Global Change, 11, 395–419.

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EFFECTS OF 2,4-DI-TERT-BUTYLPHENOL AND SELECTED SYNTHETIC HERBICIDES ON ELECTROLYTE LEAKAGE OF WEEDS

Naimah Abdul Halim*, Chuah Tse Seng, Shamsul Bahri Abd. Razak

School of Food Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu,

Terengganu, Malaysia

*Corresponding author email: [email protected]

Abstract There is an increasing evidence that allelochemical, 2,4-di-tert-butylphenol (2,4-DTBP) can be an ideal novel herbicide candidate. 2,4-DTBP could result in electrolyte leakage by inducing oxidative stress through the generation of reactive oxygen species, which leads to lipid peroxidation and membrane damage in root tissues and chloroplast in leaf tissues. However, the dependence of 2,4-DTBP on light for its phytotoxicity remains unknown. Thus, this study aimed to examine phytotoxic effect of 2,4-DTBP in comparison with selected synthetic lipid peroxidation herbicides on electrolyte leakage of weedy plants, Hedyotis verticillata and Leptochloa chinensis under dark and light conditions within 24 h of incubation period. Laboratory assay revealed that leaf discs of H. verticillata and L. chinensis treated with 2,4-DTBP, paraquat (photosystem I inhibitor), and dinoterb (uncouplers) showed an increasing of electrolyte leakage after 3 h of dark incubation period and the effect was further enhanced during 6 h of light exposure. On the other hand, glufosinate (glutamine synthetase inhibitor) caused a marked amount of electrolyte leakage for both bioassay species after 3 h of dark conditions and sustained for the following incubation period. In contrast, herbicide which inhibits photosystem II (diuron), carotenoid biosynthesis (e.g., fluridon, isoxaflutole, and clomazone) and protoporphyrinogen oxidase (oxyfluorfen) gave minor or negligible effect on electrolyte leakage within 24 h of incubation period. The results of this study suggest that 2,4-DTBP causes ion leakage irrespectively of light or dark conditions and its bioactivity is comparable to paraquat and dinoterb. Introduction Recently, there is increasing evidence that allelochemicals, natural plant products derived from higher plants, may be ideal herbicide candidates. For example, 2,4-di-tert-butylphenol (2,4-DTBP), a phenolic compound isolated from plant tissues, has been demonstrated to have herbicidal potential2. 2,4-DTBP have been identified in the culm plus leaf extracts of Napier grass (Pennisetum purpureum)2. The study revealed that 2,4-DTBP induced oxidative stress by generating reactive oxygen species (ROS), thus leading to lipid peroxidation and membrane damage in root tissues and in chloroplast leaf tissues in weedy plants. However, its phytotoxic activity was not compared with other herbicides known to cause lipid peroxidation due to ROS such as diuron, paraquat, fluridone, isoxaflutole, clomazone, oxyfluorfen, glufosinate, and dinoterb. Notable electrolyte leakages have been detected in plants treated with lipid peroxidation herbicides3. Electrolyte leakage is one of the stress responses due to loss of membrane integrity, particularly with regards to lipids and may indirectly cause tissue damage3. Any process that causes destruction of cell membranes can occur with or without rupturing the membrane. To date, the exact mechanism of action of 2,4-DTBP remains unclear. Thus, the objective of this study was to examine the phytotoxic effects of 2,4-DTBP on membrane integrity of the weedy plants, H. verticillata and L. chinensis, compared to selected lipid peroxidation herbicides under light and dark conditions. Materials and Methods Experiments were performed according to the previous study with some modifications3. Briefly, the lamina of the second fully expanded leaf of H. verticillata and L. chinensis were, respectively, punched out with a cork borer in order to obtain a disc with a 5 mm diameter.

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Three discs of H. verticillata and L. chinensis were each placed in 5 mL of test compound at a concentration of 75 mg/L in 50 x 10 mm on Petri plates. Distilled water was used as a treatment control and control tissues were exposed to the same amount of acetone as treated tissues, but without the test compounds. The final concentration of acetone in the dishes was 1% (v/v). Plates were incubated in darkness for 18 h prior to exposure to light (100 µmol m−2 s−1) as photosynthetically active radiation (PAR) in a growth chamber at 25°C. Electrolyte leakage was measured using an electrical conductivity meter and measured at predetermined time intervals (up to 24 h). Beginning 3 h after the dark incubation period, a second measurement was made after 18 h (overnight), at which time the samples were placed in the light and a final measurement was made 6 h after light exposure. Each experiment was repeated twice and arranged in a Complete Randomized Design (CRD) with three replicates. To evaluate the effects of compounds on membrane integrity, the maximum conductivity was measured by boiling three leaf discs from each treatment for 20 min at 95°C. Electrolyte leakage values were expressed as a percent of the electrolyte leakage observed in control treatments. Results and Discussions Herbicides that inhibit PS II interrupt the electron flow by competing with the binding of plastoquinone at its Qb binding site called D1 protein on PS II. Then, this competition blocks the electron flow through PSII because of displacement of Qb and effectively generates ROS. Interestingly, diuron did not induce any electrolyte leakage for both H. verticillata and L. chinensis during 24 h of incubation period. Similarly, previous study demonstrated that no notable ion leakage on cotyledon discs of cucumbers occurred when treated with PSII inhibitors such as atrazine, diuron, and bentazon3. Under normal circumstances, plastocyanin transfers its electron through a series of steps to ferrodoxin, and finally on to NADP in PS I. PS I inhibitor, generates highly reactive free radicals after accepting an electron from PS II during the electron flow of photosynthesis. In this study, paraquat induced notable ion leakage during the dark period of incubation. This ion leakage was further enhanced when leaf discs were exposed to 6 h of the light period, with H. verticillata being more sensitive than L. chinensis. Similar findings have been documented by previous study where maize green tissues treated with paraquat at different concentrations (1, 10, and 100 µM) showed relatively low levels of electrolyte leakage within 12 h of dark period incubation, but a dramatic loss of membrane integrity during the following light incubation period5. Carotenoids play important roles in protecting against the destruction of chlorophyll by light (photooxidation). In the plants, some of the synthesized chlorophylls are transformed from the short-lived singlet form to the longer-lived triplet form after the chlorophylls have been electronically excited by absorbing light photons. Carotenoids are able to reduce the energy of triplet chlorophyll, including singlet oxygen produced from triplet chlorophyll, when generated under high light intensity. Singlet oxygen, and possibly triplet chlorophyll, initiate detrimental responses leading to membrane damage4. Surprisingly, the results of present study showed that carotenoid inhibitors such as fluridone, isoxaflutole, and clomazone did not exert any effects on electrolyte conductivity within the 24 h incubation period. These results are in agreement with previous findings using cotyledon cucumber discs as the bioassay species3.

Enzyme protoporphyrinogen oxidase (PROTOX) is the primary herbicide target in chlorophyll biosynthesis and is responsible for converting protoporphyrinogen IX to protoporphyrin IX. Accumulation of protoporphyrinogen IX occurs when protoporphyrin IX cannot be formed because PROTOX is inhibited. Once protoporphyrinogen IX leaks out of the chloroplast into the cytoplasm, it is oxidized to protoporphyrin IX and then reacts with oxygen and light to form singlet oxygen in the presence of light. However, oxyfluorfen had little and negligible

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effect on ion leakage during 24 h of incubation period for both bioassay species. On the contrary, PROTOX inhibitors such as acifluorfen and sulfentrazone caused rapid electrolyte leakage in cucumbers in the presence of light at 1000 µmol m−2 s1 3.

Glufosinate is the only commercial inhibitor of glutamine synthetase in the nitrogen assimilation pathway of plants, a key enzyme in glutamine biosynthesis. Formation of glutamine and glutamate is inhibited by glufosinate, which disrupts many important nitrogen metabolism and synthetic reactions in plants. In the present study, glufosinate elicited dramatic electrolyte leakage within the first 3 h of the dark incubation period. This effect was sustained during the subsequent dark and light periods. However, previous study reported that glufosinate caused only a small amount of ion leakage on cucumber leaf discs during the 24 h incubation period3.

Dinoterb, a phenol herbicide, is a weak acid known to induce the uncoupling of photooxidative phosphorylation and oxidative phosphorylation6. These uncoupling reactions can occur in both mitochondria and chloroplasts1. Dinoterb has a multifaceted mechanism of action involving the inhibition of photosynthesis and ATP synthesis. In this study, dinoterb induced notable ion leakage during the dark incubation period. The effects were further enhanced during the subsequent light period. Similarly, previous study reported that dinoterb caused electrolyte leakage on cotyledon discs of cucumbers during the dark incubation period and this effect was most pronounced after light exposure at 1000 µmol m−2 s1 3.

It has been proven that 2,4-DTBP induces oxidative stress through the generation of ROS in weedy plants2. In this study, 2,4-DTBP caused ion leakage during a 24-h incubation period. Similarly, paraquat, dinoterb and 2,4-DTBP induced ion leakage after incubating the leaf discs for 3 h in the dark. These effects were further enhanced over the following 15 h dark period and 6 h light period. H. verticillata was considerably more sensitive than L. chinensis to dinoterb and 2,4-DTBP treatment. Conclusions It can be concluded that the 2,4-DTBP, paraquat, and dinoterb caused dramatic ion leakage during the 24 h incubation period. The results of this study suggest that 2,4-DTBP has potent light-independent herbicidal activity comparable to that of dinoterb, causing the loss of membrane integrity. The results of this study suggest that 2,4-DTBP causes ion leakage irrespectively of light or dark conditions and its bioactivity is comparable to paraquat and dinoterb. References

1. Belbachir, O., Matringe, M., Chevallier, D. and Tisut, M. (1980) Physiological actions of dinoterb, a phenol derivative: 2. Effects on isolated plant mitochondria and chloroplasts. Pesticide Biochemistry and Physiology, 14, 309-313.

2. Chuah, T.S., Norhafizah, M.Z. and Ismail, B.S. (2015) Evaluation of the biochemical and physiological activity of the natural compound, 2,4-ditert-butylphenol on weeds. Crop and Pasture Science, 66, 214.

3. Dayan, F.E. and Watson, S. B. (2011) Plant cell membrane as a marker for light-dependent and light independent herbicide mechanisms of action. Pesticide Biochemistry and Physiology, 101, 182-190.

4. Hess, F.D. (2000) Light-dependent herbicides; an overview. Weed Science, 48, 160-170.

5. Kim, J. S., Choi J. S, Kim T. J, Hur, Y. and Cho, K. Y. (2001) Differential effects of herbicidal compounds on cytoplasmic leakages of green - and white - maize leaf segments. Journal of Photoscience 8, 61-66.

6. Terada, H. (1990) Uncouplers of oxidative phosphorylation. Environmental Health Perspectives, 87, 213-218.

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BIOCONTROL POTENTIAL OF FLUORESCENT PSEUDOMONADS ON RHIZOCTONIA ROOT ROT DISEASE CAUSED BY RHIZOCTONIA SOLANI ON CHILLI

Rul Hajar Muda1*, Khairulmazmi Ahmad2 and Ng Lee Chuen1

1School of Food Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu,

Terengganu, 2Department of Plant Protection, 2 Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Corresponding author email: [email protected]

Abstract The biocontrol potential of fluorescent pseudomonads isolated from soil rhizosphere of chilli was evaluated against Rhizoctonia solani in causing root rot disease on chilli. A total of 50 isolates out of 115 potential fluorescent pseudomonads were selected based on its percentage inhibition radial growth (PIRG) for more than 65% from dual culture assay. All 50 isolates were further screened for production of volatile organic compound, hydrogen cyanide, phosphate solubilization and indole acetic acid. Forty isolates were selected based on their in vitro screenings results using hierarchical cluster analysis in IBM SPSS Statistics 20. Only 34 isolates were identified as Pseudomonas spp. using 16s rRNA sequencing. The identified species are Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas balearica, Pseudomonas monteilli, Pseudomonas mosselii, Pseudomonas putida and Pseudomonas stutzeri. A total of 13 most potential Pseudomonas spp. were further tested for seedlings vigor index. Pseudomonas putida strain SKPf10 (isolate B5C1), Pseudomonas aeruginosa strain IBBPo16 (isolate B3C56) and Pseudomonas putida strain KB9 (isolate B5C7) were selected with the most prominent in promoting plumule and radicle growth and significantly higher than the control at p < 0.05. The bio-efficacy of these fluorescent Pseudomonas spp. will be evaluated in glasshouse conditions in suppression of R. solani and enhancing plant growth of chilli seedlings. Introduction In chilli, Rhizoctonia solani is a major soil-borne pathogen causing root/stem rot and damping-off of seedlings in young transplants1. Chemical control is not advisable to control the disease on vegetable crops due to the environmental and health hazards. Therefore, the biological control is gaining attention. Fluorescent pseudomonads are well known to suppress fungal root diseases of agronomic crops, such as diseases caused by R. solani by producing various antifungal metabolites. This study aimed to isolate fluorescent Pseudomonas spp. from rhizosphere of healthy chilli and to screen the most potential isolate through in vitro screenings as a biocontrol agent against Rhizoctonia root rot disease caused by R. solani and also in promoting the early growth of chilli. Materials and Methods

Isolation of fluorescent pseudomonads The soil samples collected from healthy chilli rhizosphere zone were isolated for fluorescent pseudomonads. Representative colonies were selected, purified and maintained. In vitro screening test – antagonistic activity Dual culture assay was conducted as the first screening to antagonistic activity. The effect of volatile compound produced by fluorescent bacteria towards the growth of R. solani was studied according to paired petri dish technique. For hydrogen cyanide (HCN) production assay, filter paper disc soaked in picric acid solution (0.5% picric acid and 2% Na2Co3 in 100 ml of distilled water) was placed in the lid of the plate containing the Tryptic Soy agar (TSA) medium supplemented with 4.4 g/L glycin fluorescent bacteria. After 3 days of incubation,

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the colour changes of filter paper disc was observed and read at 625 nm wavelength using a UV Spectrophotometer. In vitro screenings - Plant growth promoting performance To determine the phosphate solubilizing ability, single colony of fluorescent bacterial culture was spot-inoculated on Pikovskaya’s agar (PVK) medium and incubated at 28 ± 2°C for 3 days. The phosphate solubilizing ability was determined by measuring the clear zone formed around the bacterial colony. For the formation of indole acetic acid (IAA), fluorescent bacteria were grown in nutrient broth (NB) containing 0.2% L-tryphtophan and incubated at 28 ± 2°C for 48 h. Two ml of the supernatant were mixed with 80 µl of orthophosphoric acid and 4 ml of Salkowski’s reagent (50 ml of 35 % perchloric acid + 1 ml of 0.5 M FeCI3) and kept in the dark at room temperature for 20 min. A colorimetric technique was performed to determine the quantitatively IAA production. The colour density was read at 530 nm using a UV Spectrophotometer. Seed germination Chili seeds from Kulai variety were soaked in 1 % sodium hypochlorite (NaCIO3) for 1 min and rinsed thoroughly in sterilized distilled water. The seeds were then soaked in bacterial suspension (109 cfu/ml) at room temperature for 24 h. After 24 h, treated seeds were placed in petri dish containing wet filter papers. The plates were incubated in growth chamber at 32ºC in light for 12 h and 24ºC in dark for 12 h. At 10 days of incubation, the Vigour Index-I2 was calculated.

Identification of fluorescent Pseudomonads using 16s ribosomal RNA sequencing The DNA was extracted using Wizard® Genomic DNA Purification Kit, PROMEGA by following the manufacturer’s protocol. Bacterial 16S rRNA gene sequences were amplified by PCR technique using the universal primer pair 27F_ (5’-AGA GTT TGA TCC TGG CTC AG-3’)3 and 1492R (5’-TAC GGY TAC CTT GTT ACG ACT T-3’)3. PCR reaction mixtures were run using a thermal cycler by following cycling conditions: One cycle of initial denaturation at 95°C for 2 min, followed by 33 cycles of denaturation at 95°C for 30 sec, annealing at 52.6°C for 50 sec and extension at 72°C for 1 min and the final extension at 72°C for 5 min. DNA of the amplified PCR products were separated by gel electrophoresis on a 2 % agarose gel. The genomic DNA was sent to a sequencing service provider to get the sequences. The sequences were blasted in NCBI (National Center for Biotechnology Information) database website using the nucleotide basic local alignment for identification. Analysing the data All the data obtained from in vitro screenings and seed germination results were analysed using IBM SPSS Statistic 20. Results Isolation of fluorescent pseudomonads From the seven sampling sites, a total of 115 fluorescent colonies were successfully isolated, purified and maintained on new King’s B (KB) agar. In vitro antagonistic screening of fluorescent pseudomonads against Rhizoctonia solani Fifty out of 115 isolates (43.5%) of fluorescent bacteria successfully inhibited R. solani with PIRG values more than 65% in dual culture assay (DC). In volatile compound production (VOC) assay, 19 out of these 50 isolates were found to inhibit the growth of R. solani. Meanwhile, in HCN assay, there were 10 isolates detected to produce hydrogen cyanide through the colour changes of filter paper which was moisten previously with picric acid solution.

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In vitro promoting performances of fluorescent pseudomonads Only 5 isolates were able to solubilize phosphate which indicated by the formation of clear zone around the bacterial colonies on PVK medium. Seventeen isolates successfully produce indole acetic acid (IAA) which indicated by the appearance of red colour in bacterial culture after being treated with FeCl3-HClO4 reagent. Identification of the fluorescent pseudomonads using 16s ribosomal RNA sequencing All the results of 50 isolates were analyzed by hierarchical cluster analysis in IBM SPSS Statistic 20 where isolates having the same characteristics were grouped together. There were 40 isolates were selected to be identified in 16s rRNA sequencing. Single fragment of 1,500 bp PCR product were successfully visualized under UV translluminator for all the 40 fluorescent bacterial isolates. The nucleotide sequences were identified as Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas balearica, Pseudomonas monteilii, Pseudomonas mosselii, Pseudomonas putida, Pseudomonas stutzeri, Burkholderia sp., Burkholderia ambifaria, Burkholderia anthina, Burkholderia cepacia, and Burkholderia metallica with similarity between 84% - 100% to the sequences in the GeneBank. There were 34 isolates identified as Pseudomonas spp. and 6 isolates were Burkholderia spp. Burkholderia spp. were eliminated for the subsequent study. Seed germination Only 13 isolates of fluorescent Pseudomonas spp. were tested for seed germination after being ranked in a scoring index. The most potential of fluorescent Pseudomonas strains were selected based on the mean comparison of early growth performance of chilli seeds (Table 1). The top three bacterial isolates (Pseudomonas putida strain SKPf10, Pseudomonas aeruginosa strain IBBPo16 and Pseudomonas putida strain ACO-140) were significantly different from the control in plumule length, radicle length and vigor index. These isolates met the requirement for high antagonistic activity in suppressing R. solani which was more than 80% of PIRG value derived from dual culture assay result. However, there was no significant difference in germination rate. Discussions The current study somewhat similar to a previous study by Ahmadzadeh & Tehrani4 where, a total of fluorescent pseudomonads derived from soil rhizosphere of wheat and common bean has successfully inhibited the growth of R. solani in in vitro with high level of antagonistic activity and produced traits involved in growth promoting. The effectiveness of fluorescent pseudomonads in inhibiting the growth of R. solani may associated to the production of antifungal metabolites such as siderophore, protease, hydrogen cyanide and diacetylphloroglucinol antibiotics4. The present study revealed the potential of fluorescent pseudomonads in growth promoting to wider type of crop. Previous study by Ameer-Basha et al.2 found that seed inoculation with fluorescent Pseudomonas spp. significantly enhanced seed

germination and seedling vigor of sorghum seed over the non-treated control. Thus, it is agreed that bacterial inoculants of fluorescent pseudomonads help to increase plant growth, germination rate, improve seedling emergence and protect plants from disease5,6.

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Table 1: The early growth performance of chilli inoculated with the selected fluorescent Pseudomonas spp. isolates.

Bacterial isolates

Name Plumule

(mm) Radicle (mm)

Germination rate

(%) Vigour index

Control 10.8 cd 16.0 bcd 87.1 ab 2328.7 bcd

B5C1 Pseudomonas putida strain SKPf10 20.3 a 30.5 a 92.7 ab 4684.9 a

B3C56 Pseudomonas aeruginosa strain IBBPo16 18.1 ab 32.7 a 91.7 ab 4657.3 a

B5C7 Pseudomonas putida strain KB9 18.3 ab 30.7 a 90.0 ab 4401.0 a

B4C5 Pseudomonas putida strain ACO-140 13.2 bc 20.1 b 98.3 a 3292.5 ab

B3C19 Pseudomonas mosselii strain PHS21 13.2 bc 17.4 bc 86.7 ab 2726.2 bc

B4C1 Pseudomonas monteilii strain DD029 12.5 c 17.7 bc 85.0 ab 2587.5 bc

B1C3 Pseudomonas mosselii strain DASE-WM2 10.3 cd 15.6 bcd 93.3 ab 2464.5 bcd

B4C3 Pseudomonas putida strain ACO-140 8.7 cde 13.7 bcde 76.7 ab 1843.0 bcde

B3C61 Pseudomonas aeruginosa strain ZH1 6.5 def 12.1 bcde 88.3 ab 1630.0 bcde

B3C4 Pseudomonas stutzeri strain 40/D/Mac2 6.6 def 9.4 cde 76.7 ab 1389.2 cde

B3C37 Pseudomonas mosselii strain PHS21 3.8 ef 6.7 de 66.7 b 839.5 de

B3C48 Pseudomonas aeruginosa strain ZH1 1.5 f 4.4 e 81.7 ab 507.7 e

B3C43 Pseudomonas aeruginosa strain C2 1.6 f 4.2 e 76.7 ab 460.5 e

Means followed by the same letters within column are not significantly different from each other by Duncan test at P ≤ 0.05

Conclusion Isolate of Pseudomonas putida strain SKPf10, Pseudomonas aeruginosa strain IBBPo16 and Pseudomonas putida strain ACO-140 were selected as the most potential fluorescent pseudomonads in controlling root rot disease caused by R. solani and also promote plant growth of chilli. References

1. Rini, C.R. and K.K. Sulochana. 2006. Management of seedling rot of chilli (Capsicum annuum L.) using Trichoderma spp. and fluorescent pseudomonads (Pseudomonas fluorescens). Tropical Agriculture. 44(1&2): 79-82.

2. Ameer-Basha, S., G. Raghavendra, M.V. Nagesh-Kumar, K. Dharma Reddy and R. Sudhakar. 2013. Performance of native fluorescent Pseudomonas on in vitro seed germination and seedling vigour of Sorghum bicolor (L.) Moench. International Journal of Bio-resource and Stress Management. 4(4): 487-491.

3. Lane, D.J. 1991. 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics. 115–175. Edited by E. Stackebrandt & M. Goodfellow. New York: Wiley.

4. Ahmadzadeh, M., and A.S. Tehrani. 2009. Evaluation of fluorescent pseudomonads for plant growth promotion, antifungal activity against Rhizoctonia solani on common bean, and biocontrol potential. Biological Control. 48(2): 101-107.

5. Lugtenberg, B., T. Chin-A-Woeng and G. Bloemberg. 2002. Microbe-plant interactions principles and mechanisms. Antonie van leeuweenhoek. 81: 373-383.

6. Ashrafuzzaman, M., F. Akhtar-Hossen, M. Razi-Ismail, Md. Anamul-Hoque, M. Zahurul-Islam, S.M. Shahidullah and S. Meon. 2009. Efficiency of plant growth-promoting rhizobacteria (PGPR) for the enhancement of rice growth. African Journal of Biotechnology. 8(7): 1247-1252.

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The Determination of Polyphenols in Latex of Hevea brasiliensis and Rubber Processing Effluent via Spectrometry, Spectrophotometry and Chromatography

Azmi Ismun1*, Shamsul Bahri Abd Razak1, Marinah Mohd Ariffin2 and Aidilla Mubarak1

1School of Food Science and Technology, 2School of Marine and Environmental Science, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia.

*Corresponding author email: [email protected]

Abstract Hevea brasiliensis is one of the most important commodity crops in Malaysia. Despite being a main rubber-growing country, the information on polyphenol composition in latex of Hevea brasiliensis is still scarce. Polyphenols possesses diverse roles in plants via plants’ secondary metabolism such as ultraviolet protection and resistance compounds towards fungi infection. To date, there is no report on the content of polyphenols and other related valuable compounds in rubber processing effluents. Knowing the composition of valuable compounds in the waste can further generate profit from the industry. The objective of this study is to determine the presence of polyphenol compounds in C-serum and effluent using Fourier transform infrared spectroscopy (FTIR) analysis. This study also aimed to quantify total phenolics with Folin Ciocalteu’s assay via UV-Vis spectrophotometer, and to profile specific phenolics on High Performance Liquid Chromatography (HPLC). Results from the FTIR analysis show the presence of the polyphenol in both latex and effluent of rubber processing. Total phenolic content was found to be higher in latex than in effluent. An optimal method for determining polyphenol from the selected samples on HPLC has been determined. HPLC analysis showed detection of several polyphenol peaks in both latex and effluent when compared to aunthentic polyphenol standards. The current achievement in this study marks the potential of understanding polyphenol composition in latex of Hevea brasiliensis and effluent from rubber processing which has not been explored before. Introduction Hevea brasiliensis is a tall plant natively from Brazil and produce valuable latex. Rubber is one of world’s industrial commodity and Malaysia is one of the top world’s rubber producers and currently exported to over 190 countries worldwide. This sector contributes RM 14.7 billion to nation’s income in 2013. The valuable latex yielded from rubber tree contains several components including rubber fraction, C-serum, B-serum proteins, sugars, lipids and water.Many studies have explored the pharmacological benefits of polyphenols including studies on polyphenols acting as health-promoting properties1. However, polyphenols are also crucial in regulating plant metabolism and defense, which polyphenol plays a role in defense mechanism on tapping-wound of rubber plant. The effluents potentially increased the biochemical oxygen demand (BOD) index of the water if the waste is released untreated. Higher BOD number determines the consumption of oxygen by microorganisms to oxidize besides its contribution to air pollution via malodour2. Understanding the polyphenol composition in the effluent from the rubber processing is a valuable knowledge that can generate possible profit from otherwise a waste product. This study is an initiative to contribute towards a fundamental understanding on rubber plant secondary metabolites and enrich the information on rubber plant, besides to assess any potential purification of polyphenol, which is important in the aspect of waste management from the processing industry. Materials and Methods Polyphenol extraction of C-Serum latex and rubber processing effluent Latex of H. brasiliensis was collected from rubber estate located at Lingai, Tepuh, Kuala Terengganu and the rubber-processing effluent was obtained from Pond 1, MARDEC at

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Kuala Berang, Terengganu. The collected latex was centrifuged using high speed centrifugation to obtain the desired C-serum protein. The effluent also underwent the same process in effort to obtain its supernatant. Then, the C-serum and effluent were freeze-dried before extracted via Solid-Phase extraction (SPE) using C-18 sorbent cartridge and elute by ethylacetate3. The eluent then concentrated by nitrogen gas before redissolved in 1 mL of HPLC-grade water for chromatography analysis using High Performance Liquid Chromatography (HPLC). Determination and quantification of Polyphenol Content in C-serum and effluent Detection of polyphenol on FTIR was done based on the peak value in the region of infrared spectrum and compared with correlation table5. Total phenolic concentration was measured in C-serum of latex using UV-Vis spectrophotometer. Gallic acid with concentrations between 0 to 100 mg/L was used as standard in this assay. Optimization of High Performance Liquid Chromatography Method for Polyphenol separation HPLC analysis have been done with UV detector at 280 nm. Stationary phase that has been used was C-18 column. Four methods have been tested to determine the best separation of polyphenols standard mixture. Methanol and acetic acid were used as mobile phases for Method A and B while for Method C and D, acetonitrile and phosphoric acid were applied. Different ratios due to gradients according to specific methods. Five standards have been applied including chlorogenic acid (CGA), gallic acid (GA), naphthoic acid (NA), quercetin (QUE) and rutin. The best separation of the standards mixture was applied on C-serum and effluent samples. Results and discussion Determination of polyphenol in latex and effluent from rubber processing via FTIR The FTIR spectrum obtained from the analysis of C-serum shows detection of polyphenols that represent O-H group and supported by the presence of peaks of C-O group as shown in Figure 1 and 2. For the effluent, the FTIR spectrum obtained detected both same peaks which represent O-H phenolic group. The other peaks that were also present from the effluent analysis were of amine and C-X halogen and fluoride group, which represent the smell and odour characteristic of the effluent. Several studies have applied FTIR as a tool to analyze and determine polyphenols in plants and crops4. FTIR also was considered as a new tool to classify cancer cells that treated with polyphenols5.

Fig 2: FTIR spetrum of rubber processing effluent

Fig 1: FTIR spetrum of C-serum of H.brasiliensis

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Determination of total polyphenol content in latex and effluent from rubber processing Total phenolic content was determined via the Folin-Ciocalteu’s assay as display in Figure 3. Total

phenolic concentration was measured in C-serum of latex using UV-Vis spectrophotometer, which the

absorbance values were measured against gallic standard curve. Polyphenol content for C-serum

was found to be higher than in effluent (0.0393 and 0.0099 g galic acid/mL respectively). A lower

value of total phenolic content in the effluent is expected as a result from various processing steps of

the rubber before having the effluent release to the collection pond. However, the total phenolic

content in the effluent was considered to be valuable since it is a source of waste from rubber

processing.

Determination of select polyphenols in latex and effluent from rubber processing We assessed select polyphenols in the serum latex and effluent of rubber processing using a predetermined method according to optimization based on Mradu et al. (2012). Methanol and acetic acid were used as mobile phases and running time was 22 minutes. We detected three major peaks with two of the peaks having similar retention times as authentic gallic acid and naphtanoic acid standards. Figure 4 and 5 show the chromatogram of serum latex and effluent, respectively. There are unidentified peak could not be matched with any available standards that were tested in both samples, but can be further identified on liquid chromatography mass spectrometry (LCMS). We also detected gallic acid and naphtanoic acid in the effluent, with unknown peak as found in the latex.

Fig 3: Concentration of total polyphenol content in latex C-serum and effluent

Fig 4: HPLC chromatogram of latex from H. brasiliensis

Fig 5: HPLC chromatogram of rubber processing effluent

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The presence of polyphenols in the serum of latex and effluent from the rubber-processing was successfully determined by spectrophotometry, infrared and HPLC analysis. Obviously, the effluent contained lower concentration of polyphenols as compared to serum latex sample. This condition may contributed by the degradation polyphenols of thermal exposure, oxidation and light exposure4 during pooled in trap pond, while the polyphenols in serum latex was obtained as high as possible due to controlled condition. Polyphenols may play roles in rubber plant defence as it is protects the tapping wound from pathogens4. Polyphenol oxidase (PPO) was also found in higher concentration in wounded leaves of tomatoes, as well as systemin which was reported responsible to induce PPO in wounded leaves as insecticide mechanism, indicating an enzymic level of phenolic activity in plant defense6. Profiling of polyphenol in many crops provides a valuable knowledge to suggest potential markers for breeding in horticultural sectors. This has mainly been an effort to produce polyphenol-enriched plants due to interest in pharmacological benefits of polyphenols. However, the knowledge obtained from the profiling can also be useful for producing elite lines of Hevea species that may have profitable potential such as pest resistance and others. Conclusions Polyphenol presence were successfully determined in both latex serum and rubber-processing effluent via spectrometry and quantified spectrophtometrically. An optimized HPLC method also potentially can be used in effort to profile polyphenols in the sample. References

1. Boudet, A.M. 2007. Evolution and current status of research in phenolic compounds. Phytochemistry 68, 2722–2735.

2. Arimoro, F.O., Iwegbue, C.M.A. and Osiobe, O. 2008. Effects of industrial waste water on the physical and chemical characteristics of a tropical coastal river. Research Journal of Environmental Sciences, 2: 209-220.

3. Mradu, G., Saumyakanti, S., Sohini, M. And Arup, M. 2012. HPLC profiles of standard phenolic compounds present in medicinal plants. International Journal of Pharmacognosy and Phytochemical Research 4 (3). 162-167.

4. Trifunschi, S., Munteanu, M. F., Agotici, V., Ardelean, S. P., & Gligor, R. 2015. 5. Pavia, H., Cervin, G., Lindgren, A. and Åberg, P. 1997. Effects of UV-B radiation and

simulated herbivory on phlorotannins in the brown alga Ascophyllum nodosum. Marine Ecology Progress Series 157. 139-146.

6. Constabel, C. P., & Ryan, C. A. (1998). A survey of wound- and methyl jasmonate-induced leaf polyphenol oxidase in crop plants. Phytochemistry, 47(4), 507–511.