The Potential of Malaysian Seaweeds as the Source of Antifungal Compounds for Application in the Oil Palm Industry Zetty Norhana Balia Yusof, PhD Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, UPM, Serdang, Selangor, MALAYSIA

Oil Palm 39% palm oil production and 44% export in the world Kingdom: Plantae Family: Arecaceae Subfamily: Arecoideae Tribe: Cocoeae Genus: Elaies Species: E. guineensis Originated from Africa. Introduced to Malaysia by the British in 1870 and commercial planting started in 1917 G. boninense Daily uses Fusarium wilt

Oil Palm Estates in Malaysia

Basal stem rot disease  Oil palm productivity in Malaysia is threatened by basal stem rot disease caused by Ganoderma boninense  To date, no study is conducted on the use of antifungal properties from seaweeds for the disease control  A more environmental-friendly disease control is needed to replace the current heavy use of fungicides and harmful chemicals  Malaysia is blessed with the abundance of seaweeds which are underutilised Problem Statement

Seaweed as a Source of Natural Compounds  Several reports have shown that compounds deduced from macroalgae are highly bioactive, with properties that have antifungal and antibacterial activities (Oranday MA, MJ Verde, SJ Martínez- Lozano 2004; Freile-Pelegrín & Morales 2004).  Bioactive compounds extracted from seaweeds are capable of protecting human and plants from various serious diseases (Paul & Puglisi 2004; Bhadury & Wright 2004).

Antifungal Activity of Seaweeds Seaweed speciesPlant pathogen Effective extract References Acanthaphora spiciferaMicrosporum gypseumMethanol(Pandian et al., 2011) Rhodomela confervoides and Padina pavonica Candida albicans, Mucor ramaniannusMethanol(Saidani et al., 2012) Alaria esculenta, Fucus vesiculosus, Fucus sp., Spirulina platensis, and Ecklonia maxima Fusarium roseum, F. oxysporum, Alternaria alternata, A. dauci, A. longipes, Trichoderma viride, Botrytis cinerea, Aspergillus niger, Penicillium expansum Ethanol(Cosoveanu et al., 2010) Stypopodium zonale, Laurencia dendroidea, Ascophyllum nodosum, Sargassum muticum, Pelvetia canaliculata and Fucus spiralis. Colletotrichum lagenariumEthanol(Peres et al., 2012) Sargassum vulgare, Cystoseira barbata, Dictyopteris membranacea, Dictyota dichotoma, and Colpomenia sinuosa Alternaria alternata, Fusarium oxysporumCyclohexane(Am and Im, 2015) Sargassum myricocystum Gracilaria edulis Colletotrichum falcatumEthanol(Ambika and Sujatha, 2015)

Project Aims 1.To screen selected Malaysian seaweeds for antifungal activity 2.To investigate the potential of bioactive compounds with inhibitory activity against disease-causing fungi in oil palm 3.To identify the bioactive compounds possessing the antifungal activity against G. boninense

Collection of seaweeds Bioactive extract preparation - Soxhlet extractor and rotary evaporator Antifungal assay- Poisoned food technique (Bussaman et al., 2012; Schmitz, 1930) Phytochemical analysis- GC-MS & HPLC Methodolo gy

Collection of Seaweeds Location: Teluk Kemang, Port Dickson, Negeri Sembilan The beach of Teluk Kemang at Port Dickson (2°26 ′ N,101°51 ′ E) is located at the West Coast of Peninsular Malaysia, facing the Straits of Malacca. The study sites has vast variety of seaweed species can be found growing on the reefs. SamplingDateTimeLevelTide 1 st Thursday (10 th March 2016) 2.00 p.m.0.03mLow tide 2 nd Thursday (2 nd June 2016) a.m. 0.63mLow tide Tide schedule of Teluk Kemang, Port Dickson during sampling. Source :

Collected seaweeds and their potentials Seaweeds / BioactivityAntifungalAntibacterialAntioxidant Red seaweeds (Rhodophyta) Kappaphycus alvarezii√√√ Brown seaweeds (Phaeophyta) Sargassum duplicatumNI √ Green seaweeds (Chlorophyta) Halimeda opuntia√NI√ Caulerpa racemosa√√√ Caulerpa microphysa√√NI

Extract preparation MethanolWater Dichloromethane & Chloroform  Volatile  Dissolve wide range of organic compounds  Efficient for lipid extraction  Inexpensive  Recent publications reported a higher extraction yield in water for seaweeds identified with high levels of water- soluble components, such as soluble polysaccharides, protein and peptides, revealing the hydrophilic nature of the majority of the seaweed components (Farvin and Jacobsen 2013; Tierney et al. 2013).  Inexpensive  Volatile  Can dissolve many compounds  Low boiling point

Extraction Yield SeaweedsPowder weight (g) Extraction yield (g) MethanolDichloromethaneChloroformAqueous Sargassum duplicatum Caulerpa microphysa 5.0 NA Caulerpa racemosa Halimeda opuntia 5.0 NA

Antifungal Assay - Poisoned Food Technique (Bussaman et al., 2012; Schmitz, 1930)

Ganoderma boninense  Basidiomycetes  Reproduce through spores and mycelia  Spreads in the soil through roots and also through the air  G. boninense causes basal stem rot disease in oil palm Fusarium oxysporum  Ascomycetes  Soilborne pathogen  Produces macroconidia, microconidia, and chlamydospores  Chlamydospores allow survival in plant debris and soil  F. oxysporum causes fusarium wilt in oil palm  Attack oil palm at all ages from seedling to mature palms (Prendergast, 1957)

Ganoderma boninense Culture of G. boninense Microscopic identification of G. boninense with Lactophenol cotton blue staining. Right :Image observed under 40x100 magnification, Left: Image adapted from Rahamah et al., 2012

Fusarium oxysporum Culture of F. oxysporum Microscopic identification of F. oxysporum with Lactophenol cotton blue staining. Right :Image observed under 40x100 magnification, Left: Image adapted from oxysporum.html

Antifungal Assay (Bussaman et al., 2012; Schmitz, 1930) Fungi will be collected and inoculated on potato dextrose agar (PDA) plates. The plates will be incubated for 7 days at 25 ºc. All seaweed extracts were reconstituted to have the concentration of 5% and 10% by dilution with distilled water (Ambika and Sujatha, 2015). 1ml of each extract will be used for mixing with 19ml of warm PDA and poured into 9 cm sterile petri dish. The plates will be inoculated with 6mm agar piece of the one week old fungus mycelia. All treatments were duplicated and repeated three times and the averages of the experimental results determined. The inoculated plates will be incubated at 30˚c and the diameter of fungal colonies will be measured every day for 5 days.A plate consists of sterile distilled water will act as negative controls.

Antifungal Assay G.b.= Ganoderma boninense, F.o.= Fusarium oxysporum, X=contaminated Seaweed Extract concentration (µl/ml) Mycelial growth (mm) % reduction over control Control (mm) G.b.F.o.G.b.F.o. G.b.F.o. Sargassum duplicatum Caulerpa microphysa 5 X X

Summary  Methanol solvent gave the most extract yield compared to other solvents  A significant amount of reduction in the mycelial growth were observed in extract-added PDA  The seaweed extracts showed high levels of activities against the tested fungus  G. boninense is more sensitive towards the seaweeds extracts compared to F. oxysporum GC-MS,HPLC – for identification of compounds (phenolics and fatty acids) Testing of seaweed extracts on fungi-infected oil palm seedlings Compounds pathway analysis for overexpression studies Future Work

Project Benefit  This project will pave the way for the production of a more natural and environmental friendly method to control the disease caused by G. boninense in oil palm  The utilization of the readily abundant and naturally occurring seaweeds as a source of potential bioactive compounds with antifungal activity  This project will be crucial for the sustainability of palm oil production in the oil palm industry of Malaysia

Acknowledgements Syamimi Diyana Abdul Aziz Nur Sakinah Ghazali Atiqah Subki Aisamuddin Ardi Zainal Abidin Lee Li Fern Nur Husna Azim Lab 230, Department of Biochemistry, FBSB, UPM Geran Putra IPM Universiti Putra Malaysia ( )

Thank you Zetty Norhana Balia Yusof, PhD (Cambridge, UK) Senior Lecturer Department of Biochemistry, Faculty of Biotechnology & Biomolecular Sciences, Universiti Putra Malaysia, UPM, Serdang, Selangor, MALAYSIA / /