WHITE ROT FUNGI IN THE AREA OF DTI-UNIMAS TEST SITE, UNIMAS

ARBORETUM AND TIMBER RESEARCH AND TECHNICAL TRAINING

CENTRE (TRTTC)

Kan Yung Hao

Bachelor of Science with Honours

(Plant Resource Science and Management)

2015

WHITE ROT FUNGI IN THE AREA OF DTI-UNIMAS TEST SITE, UNIMAS

ARBORETUM AND TIMBER RESEARCH AND TECHNICAL TRAINING

CENTRE (TRTTC)

KAN YUNG HAO

This project report is submitted in partial fulfilment of the requirements for degree of

Bachelor of Science with Honors

(Plant Resource Science and Management Programme)

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2015

APROVAL SHEET

Name of Candidate : Kan Yung Hao

Tittle of Dissertation : White Rot Fungi in the Area of DTI-Unimas Test Site, Unimas A

rboretum and Timber Research and Technical Training Centre

(TRTTC)

……………………………….. ………………………………

(Associate. Prof. Dr. Andrew Wong) (Prof. Dr. Sepiah Muid)

Supervisor Co- Supervisor

………………………………..

(Dr. Rebicca Edward)

Coordinator of Plant Resource Science and Management Programme

Faculty of Resource Science and Technology

DECLARATION

I declare that no portion of the work referred to in this dissertation has been submitted in

support of an application for another degree of qualification of this or any other university or

institution of higher learning.

…………………………….

(KAN YUNG HAO)

Programme of Plant Resource Science and Management

Department of Plant Science and Environment Ecology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

i

ACKNOWLEDGEMENTS

This project was made possible with all the guidance and support by a lot of

people and agencies, to whom I owed so much and would like to express my deepest

gratitude and appreciation.

First of all, I would like to express my greatest gratitude to my supervisor, Assoc.

Prof. Dr. Andrew Wong for his guidance and encouragement, healthy criticism, brilliant

suggestions and motivation throughout the path of completing this study. My warmest

thanks also go to my co-supervisor, Prof. Dr. Sepiah Muid for her guidelines. I would like

to thank to the faculty members and fellows, lab assistant and other staff for their

cooperation, supply for materials in carrying out this project.

My gratefulness also goes to my fellow graduating student and master students,

Nor Fatin Natasha, Tan Wei Khong, and Carlson Tawi for their support, patience,

encouragement, suggestion and wonderful friendship throughout this study.

Finally, my deepest thanks go to my beloved family and my siblings for their

endless support, devotion and understanding which has always been the source of my

inspiration.

ii

TABLE OF CONTENTS

Page

Acknowledgement i

Tables of contents ii

List of Tables v

List of Figures v

List of Plates vi

Abstract viii

CHAPTER ONE

INTRODUCTION

1.1 General Background 1

1.2 Problem Statement 3

1.3 Objectives 4

CHAPTER TWO

LITERATURE REVIEW

2.1 The requirement of fungi colonization on wood 5

2.2 Wood decaying fungi features 7

2.2.1 Life cycle of decay fungi 8

2.3 White rot fungi 8

iii

CHAPTER THREE

MATERIAL AND METHODS

3.1 Preparation of Fungal Inoculums 10

3.1.1 Preparation of Media 10

3.1.2 Collected Fungi from DTI-Unimas Test Site 10

3.1.3 Identification of Collected Fungi 11

3.1.4 Preparation of Isolation and Pure Culture 11

3.2 Preparation of Agar Block Test 12

3.2.1 Preparation of Wood Samples 12

3.2.2 Agar Block Test 12

3.3 Statistical Analysis 14

CHAPTER FOUR

RESULTS

4.1 Identification of Fungi 15

4.2 Description of Collected Fungi 16

4.2.1 Ganoderma sp. 16

4.2.2 Trametes sp. 16

4.2.3 Microporus xanthopus 17

4.2.4 Lentinus conatus 18

4.2.5 Lentinus / Pleurotus sp. 18

4.2.6 Pycnoporus sanguineus 19

4.3 Distribution of Collected Fungi 22

4.4 Agar Block Test 23

iv

CHAPTER FIVE

DICUSSION 27

CHAPTER SIX

CONCLUSION AND RECOMMENDATION 31

REFERENCES 32

APPENDICES

APPENDIX A 40

APPENDIX B 41

APPENDIX C 42

v

LIST OF TABLES

Table Page

1 The fungi decay ability correlation with the mean of weight loss 13

2 Determination of white rot fungi as basidiomycete through 20

observing the presence of clamp connection

3 Average weight loss of blocks of Jelutong wood obtained

from individual fungi species when exposed in agar block test 24

4 Correlation between Jelutong wood and fungi species based

on weight loss. 25

5 Significance of mean for all fungi species on the weight loss

of wood blocks from One-way ANOVA. 26

6 Weight loss of individual of block samples when exposed in agar

block test in five weeks. 42

LIST OF FIGURES

Figure Page

1 DTI-UNIMAS Test Site 22

vi

LIST OF PLATES

Plate Page

1 Agar Block Test 13

2 Ganoderma sp. 16

3 Trametes sp. 17

4 Microporus xanthopus 17

5 Lentinus conatus 18

6 Lentinus / Pleurotus sp. 18

7 Pycnoporus sanguineus 19

8 Mycelium of Ganoderma sp. 20

9 Mycelium of Trametes sp. 20

10 Mycelium of Lentinus / Pleutotus sp. 20

11 Mycelium of Pycnoporus sanguineus 20

12 Mycelium of Microporus xanthopus 21

13 Mycelium of Lentinus conatus 21

14 Agar block test of Jelutong wood against Ganoderma sp.

after exposed in five weeks 23

15 Agar block test of Jelutong wood against Trametes sp.

after exposed in five weeks 23

16 Agar block test of Jelutong wood against Microporus xanthopus

after exposed in five weeks 23

vii

17 Agar block test of Jelutong wood against Lentinus/Pleurotus sp.

after exposed in five weeks 23

18 Agar block test of Jelutong wood against Lentinus conatus

after exposed in four weeks 24

19 Agar block test of Jelutong wood against Pycnoporus sanguineus

after exposed in four weeks 24

20 UNIMAS-DANISH Technological Institute (DTI) Test Site 40

21 Jelutong blocks were decayed by Ganoderma sp. 41

22 Jelutong blocks were decayed by Trametes sp. 41

23 Jelutong blocks were decayed by Microporus xanthopus 41

24 Jelutong blocks were decayed by Lentinus conatus 41

25 Jelutong blocks were decayed by Lentius / Pleurotus sp. 41

26 Jelutong blocks were decayed by Pycnoporus sanguineus 41

viii

White Rot Fungi in the Area of the DTI-Unimas Test Site, Unimas Arboretum and

Timber Research and Technical Training Centre (TRTTC)

Kan Yung Hao

ABSTRACT

Woods become valuable resources to human nowadays due to their multiple uses.

Meanwhile, most of them can be easily destroyed or bio-deteriorated by wood decay

fungi. The purpose of this study was to identify the collected fungi from DTI-UNIMAS

field test site, Unimas arboretum and Timber Research and Technical Training Centre

(TRTTC) which were Garnoderma sp., Trametes sp., Microporus xanthopus, Lentinus

conatus, Lentinus / Pleurotus sp., and Pycnoporus sanguineus. Most of the white rot

fungi composed essentially of lignin peroxidase enzyme, cellulase and laccase. The clamp

connection of certain fungi was identified. These respective fungi species were isolated

and then underwent the examination of wood decay test on Jelutong wood based on

weight loss. All fungi species were tested on agar block test (2% MEA agar). The average

percentage of weight losses were obtained and calculated after five weeks. The results

showed that Pynoporus sanguineus (17.14 %) had highest wood decay ability than

Ganoderma sp. (15.53 %), Trametes sp. (10.55 %), Microporus xanthopus (12.60 %),

Lentinus conatus (14.91 %) and Lentinus / Pleurotus sp. (15.29 %). Pcynoporus

sanguineus had no significantly different with Ganoderma sp., Lentinus connatus , and

Lentinus / Pleurotus sp., whereas both Trametes sp. and Microporus xanthopus showed

significantly different based on weight loss.

Key word: Wood decay fungi, wood decay test, agar block test.

ABSTRAK

Kayu menjadi sumber yang berharga untuk manusia pada masa kini oleh kerana

pelbagaian kegunaannya. Manakala, kebanyakan jenis kayu mudah dimusnahkan atau

dirosotkan melalui makro-kulat, terutamanya kulat pereput kayu. Tujuan kajian ini

adalah untuk mengenal pasti kulat-kulat yang diambil dari ujian lapangan DTI-UNIMAS,

Unimas arboretum dan pusat Penyelidikan Kayu (TRTTC) seperti Garnoderma sp.,

Trametes sp., Microporus xanthopus, Lentinus conatus, Lentinus / Pleurotus sp. dan

Pycnoporus sanguineus. Kebanyakan kulat perebut kayu mempunyai enzim lignin

peroxida, selulase dan lacase. Penyambungnan clamp telah terdapat dalam kulat tersebut.

Spesies kulat masing -masing ini telah dikurturkan dan kemudian menjalani ujian pereput

kayu pada kayu jelutong berdasarkan kehilangan berat kayu. Semua species kulat telah

diuji ke pada ujian blok agar (2% agar MEA). Peratusan purata tentang kehilangan

berat kayu telah diperolehi serta dikira selepas lima minggu. Hasil kajian menunjukkan

bahawa Pynoporus sanguineus (17.14%), mempunyai keupayaan pereput kayu yang

paling tinggi berbanding daripada Ganoderma sp. (15.53%), Trametes sp. (10.55%),

Microporus xanthopus (12,60%), Lentinus conatus (14.91%) dan Lentinus / Pleurotus sp.

(15.29%). Pcynoporus sanguineus menunjukkan persamanan yang ketara dengan

Ganoderma sp., Lentinus connatus dan Lentinus / Pleurotus sp., manakala hanya

Trametes sp. dan Microporus xanthopus mempunyai perbezaan yang ketara berdasarkan

kehilangan berat kayu.

Kata kunci: Kulat perebut kayu, ujian perebut kayu, ujian blok agar.

1

CHAPTER ONE

INTRODUCTION

1.1 General Background

Wood production plays significant roles in human activity since the beginning

of the human civilizations. Wood also serves as various purposes in our daily routine

such as cooking or heating material, furnitures, tools, as raw material for building

construction materials and other purposes. Nowadays, wood becomes more and more

valuable and dramatically in demand by humans due to the increasing human

population. Wood is considered the most abundant sources on earth. There are two

types of woods have been classified; softwoods (mostly conifers) and hardwoods

(flowering plants) (Rowell, 2005). Both of them have distinct physical appearances

strictly based on different formations of macrostructures and microstructures (Bowyer,

Shmulsky, & Haygreen, 2007).

Internal wood structure has consists of several extractives which also known

as natural products. These chemical products can be easily extracted by using organic

solvent or water (Lewin & Goldstein, 1991; Rowell, 2005). Wood extractives are

comprised of volatile oil, terpenes (turpentines, resin acids, sterols), fatty acids and

esters, waxes, polyhydric alcohols, mono-and polysaccharides, and some minor

organic compounds. Meanwhile, those particular organic extractives are also known

as fungal nutrition and intensively consumed by fungi as a source of energy,

especially carbon element (Eaton & Hale, 1993). Therefore, the degradation of wood

2

species can be resulted from wood decaying fungi and essentially prolong fungi life

cycle (Bodig & Jayne, 1982).

According to Wong and Pearce (1997, 1998), there are two common variety of

wood decaying fungi which attack on wood such as white rot (Basidiomycetes) and

soft rot (Ascomycetes & Deuteromycetes). Most of the white rot fungi have ability of

decaying carbohydrates and lignin, secondarily cellulose and hemicellulose (Luley,

2005). They are able to degrade the strength properties of the wood as the wood

undergoes decay process (Rowell, 2005). According to the book Wood Engineering

Handbook (1990), toughness of wood will decrease from 6 percent to more than 50

percent, nevertheless every 1 percent mass loss in the wood is related usually to

fungal deterioration. Furthermore, wood decaying process is also aggressively

accelerated by the external environmental stresses to induce the colonization of wood-

rotting fungi on the injured surface of bark or dead trunk (Lonsdale, 1999).

In ecosystem, majority of fungi are saprobes and also play an important role

as primary biotic decomposers of plants including plant debris and contribute to

carbon dioxide to provide requirement of photosynthesis in plants. In contrast, these

fungi are also known as decay agents of wood. Fungal hyphae do penetrate the wood,

absorb and digest external wood sources and metabolize the organic material from the

host (Zabel & Morrell, 1992). Generally, there are four groups of fungi that can be

found on or inside wood; mold, stain, soft-rot, and wood-rooting basidiomycetes fungi

(Lacasse & Vanier, 1999). There are two principle methods for studying fungal

biodegradation of lignocelluloic plant tissues; Soil block and agar block (Schilling &

Jacobson, 2011).

3

1.2 Problem Statement

Many wood products have been intensively deteriorated by wood decaying

agents such as decay fungi. As such, deteriorations of wood quality become one of

human concerns, at the same time to reduce the severe decay caused by the wood

decaying agents. In fact, white rot fungi are among the most active wood degraders in

nature in the tropics. There is several wood decay fungi present at the Universiti

Malaysia Sarawak (UNIMAS) test site, Unimas arboretum and Timber Research and

Technical Training Centre (TRTTC) field site, whereby causing treated and untreated

wood samples to decay naturally. However, inadequate information about the species

of these tropical fungi is available for reference. Thus, collection and identifications

of wood decaying fungi including their wood degrading ability are crucial to provide

a wealth of knowledge about these fungi and to provide solutions to reduce wood

decay of wood products.

4

1.3 Objectives

The objectives of this study are:

To identify various species of white-rot fungi collected from Danish

Technology Institute University Malaysia Sarawak (DTI-UNIMAS) Field test

site, Unimas arboretum and Timber Research and Technical Training Centre

(TRTTC).

To investigate the decaying ability of different fungi on Jelutong wood sp.

(Dyera costulata)

To compare the decay rate of the interspecies of collected fungi

5

CHAPTER TWO

LITERATURE REVIEW

Wood plant biomass constitutes about approximately 40 to 60 % cellulose, and

20 to 30% lignin (Hudson, 1980). Both compounds are very essential for fungal

growth due to the enrichment of carbon sources and also provide significant to

ecological cycles like carbon cycle.

2.1 The requirement of fungi colonization on wood

According to Wong and Pearce (1997, 1998), Malaysia is tropical rainforest

country which consist of high humidity and optimal ambient, attracting numerous of

decay fungal growth and development on local timbers, such as common white rot

(Basidiomycetes) and soft rot (Ascomycetes and Deuteromycetes). Decay fungi often

grow at optimal abiotic environment such as sufficiency of oxygen (air), relative

humidity which above the fiber saturation point; average 30% of moisture content,

and range of temperature within 10 to 35°C; aggressive stage is around 24 to 32°C

(Rowell, 2005). Microclimate surrounding the wood also extremely influences the

fungal colonization and interferes metabolic activities of fungi through alteration of

gaseous and moisture content interactions (Rayner & Boddy, 1988). When the

optimal ambient condition is achieved, fungi are capable to convert those complex

polymeric compounds from wood into simple compounds such as water and carbon

dioxide.

6

All wood decaying fungi need oxygen for growth and survival, similar with

other living organisms. Oxygen required by fungi to oxidise sugar that serve as an

energy which is essentially for fungal growth and hyphae elongation purposes. Simple

compounds like water and carbon dioxide are produced from carbohydrates while the

respiration process occurs (Baker, 1969). When the environmental oxygen becomes

low or absent, fungi are able to directly utilize the chemical substances to gain energy

through redox reactions, this process known as “Fermentation” (Zabel & Morrell,

1992).

1) C6H12O6 + 6O2 6CO2 + 6H2O + energy ---Aerobic respiration process

2) C6H12O6 2C2H5OH + 2CO2 + energy --- Anaerobic respiration process

In perspective of relative humidity, the growth of fungi is strongly influenced

or manipulated by moisture content. When the humidity of surrounding atmosphere

becomes low, water source is limited to access through hyphae thereby strictly

affecting hyphal growth. Therefore, the humidity content which is less than 20%

might not induce any fungal growth and decay process instantly (Baker, 1969).

However, high moisture level will cause unequal balance of gaseous-water

exchangeable system by creating a physical barrier inside water-filled void space

(Rayner & Boddy, 1988).

There are different requirement of temperature demand for various fungi

growing in wood. It is also one of the main factors to lead the growth of fungi, by

affecting metabolic activities such as digestion, translocation, respiration, assimilation

and enzyme synthesis (Zabel & Morrell, 1992). Most of the fungi may not survive or

grow at low temperature, but wood decaying fungi will not be executed by

temperature which is below the freezing point. Fungal growth will slow down

7

gradually when the temperature rises until 35°C or above, and then the metabolic

reaction process will completely cease when the temperature reach about 38°C (Baker,

1969).

2.2 Wood decaying fungi features

A study based on Schwarze, et al. (2000) reported that majority of wood decay

fungi which reduced the properties and sustainability of wood can be classified to the

polypores (Polyporales) or the gill fungi (Agaricales). Wong and Pearce (1997, 1998)

stated that they do absorb the nutrients from host and decompose the debris of plants,

thus also known as “decomposer”. Literally, those three types of fungi compose of

different significant pattern variations and development of the interaction processes

(abiotic and biotic factors) which are associated with degradation purpose (Raymer &

Boddy, 1988). They are concerned with cellulose, hemicellulose and/or lignin

compounds of wood and cause decay. Hence, they are frequently called as cellulolytic

or ligno-cellulolytic fungi that invade woody tissues to obtain nitrogen and

predominantly improve their carbohydrate intake (Barron, 2003).

The nitrogen element within wood is very limited whereby the ratio of Carbon

to Nitrogen (C: N) is particularly high that could cause limitation of fungi growth.

Moreover, Odum (1975) stated that 90% of energy flow in forest ecosystem belongs

to the decomposers and other 10% will be consumed by other living organisms such

as insect, termites, bacteria and so on.

8

2.2.1 Life cycle of decay fungi

In wood decaying fungi life cycle, they initially grow in or on the injured

surface of host by germinating spores, seem behave like other type of fungi as well.

The spores then keep on growing until the occurrence of long slender threads

widespread called “hyphae”. The length of individual hyphae is about approximately

0.5 to 20µm or even more in diameter, but mostly the range will be within 2 to 10µm

in diameter (Zabel & Morrell, 1992). The hyphae grow slowly along the surface,

follow by penetrating into the inner part of wood. The hyphae become predominantly

active in prolonging themselves from cell to adjacent cell via pits or holes formed in

the cell wall. Numerous of hyphae formed are basically called as “mycelium”.

Fruiting bodies (sporocarps) are formed producing large numbers of spores

which later on proliferated by wind, water and so on. For more information, fruiting

body can also be wisely used to identifying the species of fungi by in term of

characteristic (Johnson, 1990). Most wood decaying fungi utilize their hyphae to

undergo biochemical control by producing catalytic action of metabolites (Bowyer,

Shmulsky & Haygreen, 2007). After hyphae colonization has been stabilized, fungi

will further grow or constitute a tubular structure, known as “rhizomorph”. It

contributes water movement rapidly flow into wood and also obtain nutrient mineral

from host, by the time, increase the rate of wood decay.

2.3 White rot fungi

They are known as predominant agent of wood-fungi degradation. They

literally obtain sufficient enzymatic sources needed from wood host and strengthen

9

the fungi colonization on wood at any stages of decomposition (Rayner & Boddy,

1988).White-rot fungi basically enable to degrade on three essential cell wall

components of wood; lignin as primary source, cellulose and hemicellulose, as stated

before. According to Wilcox (1978), some strength and mechanical properties of

wood can be interfered by white-rot fungi before weight loss has been detected. The

result of decay rate is highly depending on distinct types of fungi. There are some

differences between white-rot fungi and other wood decaying fungi (brown-rot fungi),

whereas white-rot fungi will produce phenol-oxidases (Eaton & Hale in cited as

Davidson et al., 1938). It also called as ligninase or lignin peroxidase which is

significant enzymes in lignin degradation process. The purposed of lignified

degradation of white-rot fungi can promote bioconversion process on wood tissues,

such as biopulping and bioleaching (Istek, Sivrikaya, Eroglu, & Gulsoy, 2005).

White-rot fungi are formed in unique physical appearances such as pale colour,

fragile, soft, and fibrous texture while the wood materials have been colonized or

decomposed by fungi (Eriksson, Blanchette, Ander, 1990; Schwarze, 2007).

.

10

CHAPTER THREE

MATERIALS AND METHODS

3.1 Preparation of Fungal Inoculums

3.1.1 Preparation of Media

According to Smith and Onion (1994), Malt Extract Agar (MEA) was being

prepared using 20 gram for 1 litter of distilled water (2% MEA) with 1 gram of

streptomycin. MEA powder was then dissolved in boiled water and then autoclaved it

at 121°C for 45 minutes and 103 kPa (15 psi). The purpose of using streptomycin is

to inhibit bacterial growth or contaminations by unwanted species. This media was

very important to being used to isolate some selective fungal species. After that, MEA

was poured into petri dishes and sealed them tightly with parafilm tape. As a

precaution, it was necessary to prepare more MEA media for the pure culture and agar

block test in the next section.

3.1.2 Collecting Fungi at DTI-Unimas Test Site

Numerous of different white-rot fungi species had been collected. Those fungi

which contained fruiting bodies were significant to identification purpose. The

location of collecting fungi was located at DTI-UNIMAS test site, UNIMAS

arboretum and Timber Research and Technical Training Centre (TRTTC).

11

3.1.3 Identification of Collected Fungi

The morphological characteristics of collected fungi were observed by looking

at their physical appearance; macro-structure such as the length and colour of the cap,

height and colour of the fruiting body, type of gills and other aspects. For more details

and precise information of identification, microstructure of the fungi was being

assessed and observed under the microscope such as colour and shape of spores and

hypha (Seshikala & Charya as cited in Kriger, 1967; Suhirman, 2005). Observing the

presence of clamp connection of fungi was also conducted. The goal of accomplishing

method is predominantly to prove that that those significant species are

basidiomycetes. Every special characteristic of fungi had been recorded and searched

for further details from some relevant references.

3.1.4 Preparation of Isolation and Pure Culture

The fresh fruiting bodies of fungi were cleaned with distilled water to wash

away the dust and insect. Then, the fruiting bodies of fungi were being sterilized using

surface sterilization by dipping them onto 10% of Clorox solution for 5 minutes.

Clorox is a chemical agent that contains sodium hypochlorite which enables to avoid

contamination occurred after isolation was done. Fruiting body of fungi had been

broken up by using sterile scalpel to obtain inner tissues. After that, the tissues were

then transferred into sterile MEA media. Each fungal tissue taken from each fungus

were inoculated into five MEA media and incubated at room temperature to initiate

fungal growth. This method was repeated many times until the pure fungal cultures

were definitely obtained. The pure cultures were prepared with at least six replicates

12

and three stock cultures for agar block test. Each fungal isolate had at least

standardized six replicated agar plates for the agar block test.

3.2 Preparation of Agar Block Test

3.2.1 Preparation of Wood Samples

Wood sample used for agar block test was Jelutong wood (Dyera costulata).

The wood blocks were then cut into sized of 2 cm length x 2cm height x 0.5 cm

widths. About 200 pieces of sample blocks were prepared. They were being cleaned

off from unwanted material or dust on the block surface. The blocks were weighed

(W1) oven dry (105°C). After that, all wood blocks were autoclaved 2 times before

being exposed to the fungi to examine wood-decay ability of fungi.

3.2.2 Agar Block Test

This method is normally used for the decay resistance testing which can be

determined according to weight loss rate of the sample blocks (Cao, et. al., 2011).

Weight loss is dominantly applied to measure biological durability of wood, fungus

decay capability and the rate of decay. After the sample blocks had been autoclaved,

they were placed in sterile MEA media of pure culture and exposed onto fungal

hyphae on agar plates. Sterile blocks were incubated with fungus at 25°C for 4 - 6

weeks and observed every week to ensure that there is no plate contamination. After

that, the blocks were taken out and the mycelia were then cleaned off before the

blocks were oven dried again to constant weight at 105°C for 2 days. The wood

blocks were weighted (W2), therefore the rate of weight loss was eventually