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Endophytic Fungi Associated with Cinnamomum iners Leaves in UNIMAS, Malaysia.

YAP SHOO YUEN

39392

Bachelor of Science with Honours

(Plant Resource Science and Management)

2015

Faculty of Resource Science and Technology

Endophytic Fungi Associated with Cinnamomum iners Leaves in UNIMAS, Malaysia.

Yap Shoo Yuen

39392

This final project report is submitted in fulfilment of the requirement for the

Degree of Bachelor of Science with Honours

(Plant Resource Science and Management)

Supervisor: Prof Dr Sepiah Muid

Plant Resource Science and Management

Department of Plant Science and Environmental Ecology


University Malaysia Sarawak

2015

DECLARATION

I hereby declare that the thesis is based on my original work. All the quotations and citations

have been duly acknowledge. No portion of the work referred to this dissertation has been

previously or concurrently submitted for any other degree programs in UNIMAS or other

institutions of higher learning.

_____________________

Yap Shoo Yuen



Universiti Malaysia Sarawak (UNIMAS)

APPROVAL SHEET

Name of Candidate: Yap Shoo Yuen

Title of Dissertation: Endophytic Fungi Associated with Cinnamomum iners Leaves in

UNIMAS, Malaysia.

__________________________

Prof Dr Sepiah Muid

Supervisor




__________________________

Dr Rebicca Edward

Programme Coordinator




I

Acknowledgement

First and foremost, I gratefully acknowledge my wholehearted appreciation to Prof. Dr.

Sepiah Muid for her supervision, guidance and giving the opportunity to pursue my final

year project. She showed me different ways to approach a research problem. Her

enthusiasm and abilities are contagious and developed my interest and understanding of the

project. Without her everlasting encouragement, constant guidance and genuine care, I

could not have finished my final year project. This experience has been extremely valuable

to me and I will treasure it for the rest of my life.

Not forgotten, a special thanks also I proceed to the master students in Mycology

Laboratory for the guidance in using the equipment and their kindness in sharing ideas and

opinions when the project was carried out. I also would like to express my sincere gratitude

to the lectures and staffs of Faculty of Resource Science and Technology, Universiti

Malaysia Sarawak (UNIMAS) for their valuable cooperation, information, suggestion and

guidance in the compilation and preparation this final year project.

Last but not least, I would like to express my thanks to my family and friends, who

encouraged and supported me always in good and bad times.

II

Table of Content

Acknowledgement……...…………………………………………………………….. I

Table of Content…………………………………………………………………….. II

List of Abbreviations…………………………………………………………………. IV

List of Figures…………………………..……………………………………………. V

List of Tables………………………..……………………………………………….. VI

Abstract………………………………………………………………………………. 1

1.0 Introduction………………………………………………………………………

1.1 Problem statement……………………………………………………..…

1.2 Objective………………………………………………………………….

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4

2.0 Literature Review…………………………………………………………………

2.1 Description of Cinnamomum iners Reinw. ex Blume…………………....

2.2 Importance of cinnamon…………………………………………………..

2.2.1 Cinnamon in food production…………………………………

2.2.2 Cinnamon as fungicide………………………………………..

2.2.3 Cinnamon as anti-microbial agent…………………………….

2.2.4 Cinnamon as anti-cancer agent………………………………..

2.2.5 Cinnamon as analgesia………………………………………..

2.3 Description of endophytic fungi………………………………………….

2.4 Importance of endophytic fungi………………………………………….

2.4.1 Endophytic fungi in secondary metabolites production………

2.4.2 Endophytic fungi as antioxidants……………………………..

2.4.3 Endophytic fungi in enzymes production…………………….

2.4.4 Endophytic fungi as biocatalyst agent………………………..

2.4.5 Endophytic fungi as biocontrol agent…………………………

2.5 Relationship of endophytic fungi and other factors………………………

2.5.1 Fungal succession related to enzyme production…………….

2.5.2 Endophytic fungi related to decomposition process………….

2.5.3 Relating endophytic and parasitic fungi from Cinnamomum

sp. as pharmaceutical products..………………………………

2.6 Endophytic fungi associated with Cinnamomum spp. …………………..

2.7 Colonization and distribution of endophytic fungi………………………

2.7.1 Colonization of endophytic fungi on age of leaves………….

2.7.2 Distribution of endophytic fungi on environmental variations.

2.8 Physiological studies of endophytic fungi………………………………..

2.8.1 Effect of pH on growth of fungi………………………………

2.8.2 Effect of temperature on growth of fungi…………………….

2.8.3 Effect of light condition on growth of fungi………………….

2.9 Molecular Studies of endophytic fungi…………………………………...

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3.0 Materials and Method…………………………………………………………….

3.1 Sample collection…………………………………………………………

3.2 Isolation and pure culture of endophytic fungi…………………………...

3.3 Identification of fungi……………………………………………………..

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III

3.3.1 Morphological study of fungi…………………………………

3.3.2 Molecular study of fungi……………………………………...

3.4 Physiological study of fungi………………………………………………

3.4.1 Growth of fungi at different pH……………………………….

3.4.2 Growth of fungi at different temperature……………………..

3.4.3 Growth of fungi under light and in dark conditions…………..

3.5 Data Analysis……………………………………………………………

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4.0 Results.………………………………………….………………………………...

4.1 Occurrence of endophytic fungi…………….…………………...………..

4.1.1 Distribution patterns……………….………………………….

4.1.2 Abundance of fungi species…………………………………...

4.2 Morphological study of fungi.…………………………………………….

4.3 Molecular study of fungi………………………………………………….

4.4 Physiological study of fungi.……………………………………………...

4.4.1 Growth of fungi at different pH……………………………….

4.4.2 Growth of fungi at different temperature……………………..

4.4.3 Growth of fungi under light and in dark conditions…………..

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5.0 Discussion…………….…………………………………………………………..

5.1 Occurrence of endophytic fungi…………….…………………...………..

5.1.1 Distribution patterns……………….………………………….

5.1.2 Abundance of fungi species…………………………………...

5.2 Morphological study of fungi.…………………………………………….

5.3 Molecular study of fungi………………………………………………….

5.4 Physiological study of fungi.……………………………………………...

5.4.1 Effect of pH on fungi………………………………………….

5.4.2 Effect of temperature on fungi………………………………..

5.4.3 Effect of under light and in dark conditions on fungi...……....

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6.0 Conclusion..…………...……………………………………….…………………. 53

7.0 References……………………………………………….……………………….. 54

8.0 Appendices……………………………………………….………………………. 60

IV

List of Abbreviations

BenA β-tubulin

% Percent

°C Degree Celsius

bp Base pairs

C. iners Cinnamomum iners

CaM Calmodulin

cm Centimeter

Dec December

DNA Deoxyribonucleic acid

dNTP Deoxynucleoside-5’-triphosphate

et al. et alia (and other)

g gram

HCl Hydrochloric acid

hupA Huperzine A

ITS Internal transcribed spacer

MgCl2 Magnesium chloride

NaOH Sodium hydroxide

Nov November

Oct October

PCR Polymerase chain reaction

PDA Potato dextrose agar

PDB Potato dextrose broth

rpm Rotation per minute

sp. (spp.) Species (more than one species)

Taq Thermus Aquaticus

UV Ultra-violet

w/v Weight over volume

μl Micro liter

V

List of Figures

Figures Descriptions Page

Figure 1 The percentage of occurrence (%) of endophytic fungi isolated from

young, mature and senescence leaves of Cinnamomum iners with

respect to their times of isolation.

27

Figure 2 Aspergillus aculeatus (a) Mycelia and spores on water agar. (b) Colony

on PDA. (c) Reversed plate. (d)Mycelia and spores on PDA. (e) Spores

and hyphae.

35

Figure 3 Basidiomycetes sp. (a,b) Mycelia on water agar. (c) Colony on PDA.

(d) Reversed plate.

35

Figure 4 Beltrania sp. (a) Spores on water agar. (b) Spores and stipe. 36

Figure 5 Colletotrichum sp.1 (a) Mycelia and spores on water agar. (b) Colony

on PDA. (c) Reversed plate. (d) Spores and hyphae.

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Figure 6 Colletotrichum sp.2 (a) Mycelia and spores on water agar. (b) Colony

on PDA. (c) Reversed plate. (d) Spores.

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Figure 7 Colletotrichum sp.3 (a) Mycelia and spore on water agar. (b) Colony

on PDA. (c) Reversed plate. (d) Spores and hyphae.

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Figure 8 Penicillium rolfsii (a) Spore on water agar. (b) Colony on PDA. (c)

Reversed plate. (d) Spores and hyphae.

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Figure 9 Penicillium sp. (a) Spores on water agar. (b) Colony on PDA. (c)

Reversed plate. (d) Spores and hyphae.

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Figure 10 Species 3 (a) Mycelia and spores on water agar. (b) Colony on PDA.

(c) Reversed plate. (d) Spores and hyphae.

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(c) Reversed plate. (d) Spores and hyphae.

39

Figure 12 Species 8 (a,b) Mycelia on water agar. (c) Colony on PDA. (d)

Reversed plate.

40


(c) Reversed plate. (d,e) Spores and hyphae.

40

Figure 14 Species 13 (a) Mycelia on water agar. (b,c) Spores and hyphae. 41

Figure 15 Talaromyces aculeatus (a) Pale yellow colony. (b) Red colony. (c)

White colony. (d) Green colony on PDA. (d) Macro and microscopic

of spore.

41

Figure 16 Gel electrophoresis after PCR 42

Figure 17 The dried weight of mycelia (g) at different pH after one week in PDB

for selected fungi for seven days.

43

Figure 18 The average growth rates (cm day-1

) of the selected fungi in PDA at

different temperature (˚C) for seven days.

44

Figure 19 The average growth rates (cm day-1

) of the selected fungi in PDA

under light and in dark condition for seven days.

45

VI

List of Tables

Tables Descriptions Page

Table 1 The number of endophytic fungi isolates obtained from the young

mature and senescence leaves of Cinnamomum iners, with respect to

their percentage of occurrence (%) at different times of isolation.

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Table 2 The percentage of occurrence (%) of endophytic fungi isolated from

young leaf segments of Cinnamomum iners with respect to their times

of isolation.

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mature leaf segments of Cinnamomum iners with respect to their times

of isolation.

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senescence leaf segments of Cinnamomum iners with respect to their

times of isolation.

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Table 5 BLAST output of the finding. 42

1

Endophytic Fungi Associated with Cinnamomum iners Leaves in UNIMAS, Malaysia

Yap Shoo Yuen

Plant Resource and Management Programme

Faculty of Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Endophytic fungi are fungi that colonize within living plant tissues without causing any symptoms of disease,

but they might provide a novel, alternative and eco-friendly compound. The present study was to observe the

diversity, distribution and community structure of endophytic fungi of Cinnamomum iners in UNIMAS,

Malaysia. The leaf segments were immersed in 75% ethanol for about one minute, in 65% Clorox for ten

minutes, in 75% ethanol about 30 seconds and washed in sterilized distilled water for three times before

plated on water agar and obtained their pure culture. A total of 287 isolates belonging to 14 species of

endophytic fungi were isolated from 75 segments of young, mature and senescence leaves of C. iners. The

percentage of occurrence of the fungi on senescence leaf (112 isolates with 39.02%) was higher than on

mature (94 isolates with 32.75%) and young leaves (81 isolates with 28.22%). Of these 14 species, seven

species, 12 species and nine species were isolated from young, mature and senescence leaf segments of C.

iners, respectively. The percentage of occurrence of endophytic fungi isolated from young leaves was more in

November, mature leaves in December and senescence leaves in October as compared to other sampling

months. Species 3, Penicillium sp., Colletotrichum sp.2 and Colletotrichum sp.3 occurred in all the three leaf

maturity stages of C. iners. Species 3 was the most abundant endophytic fungi in the senescence leaf of C.

iners. Colletotrichum sp.3 occurred in all the three stages of leaf maturity but occurs at low percentages.

Aspergillus aculeatus, Penicillium rolfsii and Talaromyces aculeatus were identified by PCR and DNA

sequencing of ITS 4 and ITS 5. The optimum pH for fungus species growth was tends to be in slightly acidic

condition. All the fungi were growing well when under incubation temperature range of 20˚C to 30˚C. The

light and dark conditions did not influence much on the growth of fungi except of Aspergillus aculeatus and

Species 4.

Key words: Cinnamomum iners, endophytic fungi, leaf maturity, isolation time.

ABSTRAK

Kulat endofitik adalah kulat yang menjajah di dalam tisu tumbuhan hidup tanpa menyebabkan apa-apa

gejala penyakit, tetapi mereka mungkin menyediakan novel, alternatif dan mesra alam kompaun. Kajian ini

adalah untuk melihat kepelbagaian, taburan dan struktur masyarakat kulat endofitik daripada Cinnamomum

iners di UNIMAS, Malaysia. Segmen daun telah direndam dalam etanol 75% selama kira-kira satu minit,

dalam 65% Clorox selama sepuluh minit, dalam 75% etanol kira-kira 30 saat dan dibasuh di dalam air

suling steril selama tiga kali sebelum bersalut pada agar air dan diperolehi budaya murni mereka. Seramai

287 pencilan milik 14 spesies kulat endofitik telah diasingkan daripada 75 segmen muda, matang dan

penuaan daun C. iners. Peratusan berlakunya kulat pada hal menjadi tua daun (112 diasingkan dengan

39.02%) adalah lebih tinggi daripada di matang (94 mengasingkan dengan 32.75%) dan daun muda (81

mengasingkan dengan 28.22%). Daripada jumlah ini 14 spesies, tujuh spesies, 12 spesies dan sembilan

spesies telah diasingkan daripada segmen daun muda, matang dan penuaan C. iners, masing-masing.

Peratusan berlakunya kulat endofitik diasingkan daripada daun muda adalah lebih pada bulan November,

daun matang pada bulan Disember dan hal menjadi tua daun pada bulan Oktober berbanding bulan

persampelan lain. Spesies 3, Penicillium sp., Colletotrichum sp.2 dan Colletotrichum sp.3 berlaku di semua

peringkat kematangan tiga daun C. iners. Spesies 3 adalah kulat endofitik yang paling banyak terdapat

dalam daun hal menjadi tua di C. iners. Colletotrichum sp.3 berlaku dalam ketiga-tiga peringkat

kematangan daun tetapi berlaku pada peratusan yang rendah. Aspergillusa aculeatus, Penicillium rolfsii dan

Talaromyces aculeatus telah dikenal pasti oleh PCR dan penjujukan DNA daripada 4 ITS dan 5. ITS pH

optimum bagi setiap pertumbuhan spesies kulat yang menggembalakannya menjadi syarat dalam sedikit

berasid. Semua kulat telah berkembang dengan baik apabila di bawah pengeraman suhu antara 20˚C

kepada 30c. Keadaan cahaya dan gelap tidak mempengaruhi banyak kepada pertumbuhan kulat kecuali

Aspergillus aculeatus dan Spesis 4.

Kata kunci: Cinnamomum iners, kulat endofitik, kematangan daun, masa pengasingan.

2

1.0 Introduction

Endophyte literally derived from Greek, ‘endon’ means within and ‘phyton’ means plants

(Schulz & Boyle, 2005). Endophytes mean any organisms especially fungi and bacterial

that lives within plant tissues such as leaves, stems and roots for all or part of their life

cycle and without affecting the host plant (Schulz & Boyle, 2005; Maheshwari, 2006). In

the past 400 million years, the diversity of endophyte is the occurrence of development and

evolution continuously of endophytes (Schulz & Boyle, 2005). There are about 1 million

endophytes and about 300,000 plant species on Earth (Schulz et al., 2002; Tiwari &

Chittora, 2013). It showed that each individual plant hosts an average of four endophytes.

Endophytes are mostly found on tropical plant hosts in temperate areas (Azevedo et al.,

2000).

Endophytic fungi are fungi that colonize within living plant tissues without causing any

symptoms of disease (Aas, 2010). About 6,500 endophytic fungi were isolated from

herbaceous plants and trees, investigated their biological activities and identified their

bioactive compounds such as antimicrobial, antiviral and anticancer agents (Schulz et al.,

2002). The endophytic fungi may mimic the bioactive compounds as produced by the host

plant (Kaul et al., 2013). Hence, endophytic fungi are providing a novel, alternative and

eco-friendly source of bioactive compounds in pharmaceutical industries to overcome the

demand of drug resources (Jalgaonwala, Moite & Mahaian, 2011). These bioactive

compounds include secondary metabolites, plant hormones, enzyme proteins and

biological control agents (Azevedo et al., 2000). Some example of secondary metabolites

produced naturally by endophytic fungi are alkaloids, steroids, lignans, phenol, lactones

and others (Zhang, Song & Tan, 2006). Thus, the use of endophytic fungi is important for

biotechnological uses which lead to the essential of isolation and cultivation of them.

3

Endophytes which able to synthesizing chemical toxins as a biological control agent can

protect host plants from insect herbivores and pathogenic fungi. The exoenzymes produced

by endophytic fungi are very useful for biological control (Wu, 2012). For biologically

control host plant towards parasites and pathogens by endophytic fungi, an interactions

between organisms must be occur that is ranging from mutualistic or symbiotic to

antagonistic or pathogenic. In mutualistic, endophytes play an important role in protecting

the host plant from parasites and pathogens as well as controlling the disease of plant from

bacteria and fungi (Azevedo et al., 2000). Symbiotic endophytes can increase the nutrition

values of host plant as well as overcome environmental stress of host plants such as

drought tolerance (Tintjer & Rudgers, 2006). Antagonistic endophytes can excrete

metabolites which may be toxic to the host plants (Schulz et al., 1999). Pathogenic

endophytes may produce toxic metabolites and exoenzymes to infect and colonize the host

plants (Schulz & Boyle, 2005). Hence, there must be a biochemistry reaction between the

endophytic fungi, pathogenic fungi and host plants to sustain the balanced antagonism

between endophytic fungi and host plants as well as without causing the development of

disease to each other (Schulz et al., 1999). Endophytic fungi may impose certain

characteristics of fungal biodiversity on the evolution of plants community, diversity and

structure (Krings et al., 2006). Hence, endophytic continuum is occurred.

The taxa of endophytic fungi can be classified based on vegetative and reproductive

structures as well as resolved within genus and species based on molecular analysis

(Photita et al., 2005). Besides that, environmental factors such soil pH, climate and light

intensity will affect the growth and distribution of fungi (Rodrigues et al., 2009).

4

1.1 Problem Statement

The colonization and distribution of endophytic fungi associated with Cinnamomum spp.

leaves will be variable. Various abiotic and biotic factors such as pH, temperature, light

intensity and tissue colonization will be influencing the occurrence of endophytic fungi in

its host plants. There was no study reported that the diversity of endopytic fungi associated

with Cinnamomum iners in Malaysia. Traditional identification of fungi by morphological

study may be confusing in naming; hence, molecular study is important.

1.2 Objectives

The objectives of this study are to isolate the endophytic fungi in leaves of Cinnamomum

iners at different stages of leaf maturity, to identify and classify the isolated endophytic

fungi based on morphological and molecular analysis, and to investigate the growth of the

selected endophytic fungi found in leaves of C. iners based on a range of physiological

characteristics such as pH, temperature and light condition.

5

2.0 Literature Reviews

2.1 Description of Cinnamomum iners Reinw. ex Blume

Synonyms of C. iners are C. paraneuron Miq, C. iners var. agustifolium. Cinnamon, wild

cinnamon, medang teja and kayu manis hutan are the common name of C. iners. It is

grouped under family Lauraceae. There are naturally distributed along roadside in

Malaysia, Thailand, Indonesia, India, Laos, Myanmar, Vietnam, Cambodia and Sri Lanka

(Grieve, 2014). It grows at tropical rain forest at altitude 1200m (Grieve, 2014). It is an

evergreen aromatic tree. It has thick scabrous bark and cinnamon aroma with camphor

taste when distillation it (Li, Li & Werff, 2008). The leaves are alternately arranged,

glabrous abaxial and adaxial, trinerved venation (Li, Li & Werff, 2008). The mature leaves

are dark green whereas the young leaves are reddish brown (Li, Li & Werff, 2008). The

fruit is small, greenish when unripen and black when ripen with long stalked panicles (Li,

Li & Werff, 2008). Mustaffa et al. (2013) enumerated a review on phytochemical and

pharmacological activities of C.iners.

2.2 Importance of Cinnamon

2.2.1 Cinnamon in Food Production

Cinnamon is an important spices and culinary herbs. The dried inner bark of the shoot of

Cinnamon species is commercially used throughout the world. In 2001, 111,642 tons and

worth USD 91 millions of cinnamon were exported to Mexico and India from Sri Lanka,

China and Indonesia (FAO Corporate Document Repository, 2003). Since cinnamon is

aromatic, it is commonly used in preparing foods and beverages such as buns and coffee. It

adds sweet and savoury to the dishes. However, some people are not suitable to consume

cinnamon because it contains coumarin which is harmful to them. Coumarin could cause

6

liver damage (American Chemical Society, 2013).

2.2.2 Cinnamon as Fungicide

Singh et al. (2008) studied that Aspergillus sp. which grew abundantly in herbal raw

materials was treated with the essential oil of Cinnamomum camphora. They found that the

growth of Aspergillus flavus was decreased with increasing concentration of the essential

oil of C. camphora. They stated that there was no fungal mycelia growth when the

essential oil of C. camphora increased to 1,000 ppm. Thus, they concluded that the

essential oil of C. camphora could have the potential to prevent microbial and fungal

contamination of raw materials. It acts as herbal fungi toxicant.

Mishra et al. (2009) found that extraction of Cinnamomum zeylanicum leaf and bark were

inhibited the growth of pathogenis dematiaceous moulds such as Alternaria solani and

Curvularia lunata. They revealed that there was no spore germination of A. solani when

tested with both the extract of C. zeylanicum bark prepared in chloroform and acetone at

50μg/ml and the extract of C. zeylanicum leaves prepared in petroleum ether at 50μg/ml.

Besides, they investigated that there was no spore germination of C. lunata when tested

with both the extract of C. zeylanicum bark prepared in acetone at 100μg/ml the extract of

C. zeylanicum leaves prepared in petroleum ether at 100μg/ml. Hence, they concluded that

the bark and leaf extracts of C. zeylanicum could be act as fungicidal agents which

potentially prevent the activity of dematiaceous moulds.

2.2.3 Cinnamon as Anti-microbial Agent

Mustaffa et al. (2011) investigated that the methanolic extract and fraction of

Cinnamomum iners had the potential to inhibit microbial activity. They found that acetate

fraction of C. iners was the most efficient active compound to against methicillin-resistant

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Staphylococcus aureus (MRSA) and Escherichia coli, with minimum inhibitory

concentration (MIC) values of 100μg/ml and 200μg/ml respectively. They revealed that

thin-layer chromatography (TLC) using ethyl acetate fraction and chromatogram were used

to isolate the active compound and identified it as xanthorrhizol with MIC value of

25μg/ml to against MRSA and over 200μg/ml to against E. coli. Hence, they concluded

that the extract of xanthorrhizol in C. iners leaves could have antibacterial properties.

2.2.4 Cinnamon as Anti-cancer Agent

Pang, Thong and How (2009) revealed that cinnamon extracts had the potential to prevent

the proliferation of vary cell lines which can cause cancer. They found that the methanol

and acetone extracts Cinnamomum iners leaves had significant anti-kinase properties to

against mitogen-activated protein kinase kinase (MKK1). They mentioned that the MKK1

was an enzyme that activated the cancer cell to develop. They reported that the acetone

extract of C. iners leaves had higher polyphenolic content than methanolic extraction of C.

iners leaves. They indicated that the high content of bioactive compound such as

polyphenol and flavonoid with the anti-oxidation activity had the potential to inhibit the

activity of MKK1. Hence, they concluded that the extract of cinnamon could be an anti-

cancer drug.

2.2.5 Cinnamon as Analgesia

Annegowda et al. (2012) reported that the cinnamon extracts had the potential to anti-

inflammatory pain which caused by formalin. They found that the analgesic activity was

greater in the order of alkaloid, ethanolic and aqueous extract of Cinnamomum iners

leaves. They revealed that the presence of phenolic compounds at higher content could

inhibit the pain induced by formalin. Hence, they concluded that the extract of C. iners

bark could be used as a source of peripheral analgesia to relieve pain.

8

2.3 Description of Endophytic Fungi

Generally, there are two major groups of endophytic fungi namely the clavicipitaceous

endophytes (C-endophytes) and the non-clavicipitaceous endophytes (NC-endophytes).

The C-endophytes inhabit in some grasses species. The NC-endophytes inhabit wide range

of plant (Rodrigues et al., 2009). The NC-endophytes are sub-classified into three different

functional classes based on host range, type of tissues colonized, colonization in plant,

biodiversity level in plant, transmission patterns between host and ecological function

(Rodrigues et al., 2009).

2.4 Importance of Endophytic Fungi

2.4.1 Endophytic Fungi in Secondary Metabolites Production

Devi and Singh (2013) stated that six endophytic fungi were isolated from Phogacanthus

thyrsiflorus in Manipur. They stated that Colletotrichum gloeosporioides was chosen from

the six endophytic fungi to undergo gas chromatography–mass spectrometer (GD-MS)

analysis of secondary metabolites. They found that various metabolites such as phenol, 1,1-

dimethylethyl were detected by GD-MS spectrometer and had antioxidant properties.

Hence, they concluded that all the metabolites produced by C. gloeosporioides had the

potential to be used as an alternative source of human health in pharmaceutical aspects.

2.4.2 Endophytic Fungi as Antioxidants

Dhankhar et al. (2012) investigated that endophytic fungi isolated from Salvadora oleoides

in India were Aspergillus sp.JPY1, Aspergillus sp.JPY2, Penicillium chrysogenum, and

Phoma sp. and had antioxidant properties. They found that acetonic extract of fungi had

greater antioxidant potencies than methanolic and aqueous extract. They also indicated that

the acetonic extract of Aspergillus sp.JPY1 and Phoma sp. are safe to used and did not

9

shown any fatal effects on the animals despite consume up to 100mg/kg dosage.

2.4.3 Endophytic Fungi in Enzymes Production

Lumyong et al. (2002) revealed that isolated endophytic fungi such as Colletotrichum sp.,

Pestalotiopsis sp., Phoma sp., Phomopsis sp., and Xylaria sp. from six native tree species

which were Camellia sinensis var. assamensis, Cinnamomum iners, Garcinia cows, Litsea

salicifolia, Manglietia garrettii and Trichilla connaroides in Doi Suthep-Pui National Park,

Thailand had the ability to produce enzyme like cellulase, mannanase, proteinase and

xylanase. They reported that Colletotrichum sp. and Phomopsis sp. were isolated from C.

iners had the ability to produce these enzymes. However, mycelia sterilia isolated from C.

iners did not produce cellulase and xylanase but produced mannanase and protease.

Jeffrey, Son and Tosiah (2008) isolated 100 endophytic fungi from 19 species of medicinal

plants such as Acorus calamus, Alpinia conchigera, Andropraphis paniculata, Coleus

camosus, Curcuma domestica, Curcuma spp., Cymbopogon nardus, Gynura procumbens,

Hydrocotyle asiatica, Kaempferia galanga, Morinda citrifolia, Orthosiphon stamineus,

Panax spp., Plantago major, Polygonum minus, Vitex negundo, Zingiber cassumunar,

Zingiber minor, and Zingiber spp. at MARDI Sessang, Sarawak. They found that 15, 28

and 12 of the 100 isolates of endophytic fungi from 19 species of medicinal plants had the

potential to hydrolyse cellulose, xylan and mannan respectively. However, they revealed

that isolates of endophytic fungi from Z. cassumunar and A. paniculata did not secrete

those enzymes.

2.4.4 Endophytic Fungi as Biocatalyst Agent

Ying, Shan and Zhan (2014) investigated that among 49 isolates of endophytic fungi from

medicinal plant Huperzia serrata in Pan-An Country, Zhejiang Province, China, Ceriporia

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lacerata which has highest diversity in liquid potato dextrose medium was selected to

study the biotransformation of Huperzine A (hupA). They stated that different mycelium

fraction of C. lacerate was cultured with hupA as substrate and transformed to produce

huptermules. They mentioned that the huptremules were identified with nuclear magnetic

resonance (NMR) spectrometer. They reported that C. lacerate had the potential to

transform hupA to huptremules which were unique sesquiterpenoid-alkaloid hybrids

structures. Hence, they concluded that the endophytic fungi could act as biocatalyst agents

for biotransformation of natural products which are isolated from the host plant.

2.4.5 Endophytic Fungi as Biocontrol Agent

Wu (2012) studied the bioactivities of endophytic fungi in Cinnamomum kanehirai against

plant pathogens. They stated that 6 of 224 isolated endophytic fungi in C. kanehirai had

the potential to inhibit the growth of 8 fungal pathogens, 1 oomycete and 4 bacterial

pathogens. They found that Colletotrichum sp. isolate CKL005 had a significant inhibition

index against phytopathogens. They also revealed that 10mg/L of the mycelial extract from

Colletotrichum sp. isolate CKL005 had greater seed germination rate and seedling growth

of Chinese cabbage than the seed without treatment. They investigated that spraying

10mg/L of the mycelial extract from Colletotrichum sp. isolate CKL005 after pre-treatment

with the pathogen Colletotrichum higginsianum which causes anthracnose disease to

Chinese cabbage had greater inhibition of pathogen than the other treatments. Hence, they

concluded that Colletotrichum sp. isolate CKL005 could be as a biocontrol agent for the

anthracnose Chinese cabbage disease.

Kharwar et al. (2012) investigated that 162 endophytic fungi were isolated from more than

100 leaf segments of Cinnamomum camphora around the campus of Banaras Hindu

University, India. They revealed that isolated endophytic fungi such as Pestalotiopsis sp.

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was significantly inhibited the growth of Phytophthora cryptogea whereas Phomopsis sp.

was significantly inhibited the growth of Pythium aphanidermatum. Thus, they concluded

that endophytic fungi which had the inhibitory activity against those pathogens could have

the ability to produce bioactive metabolites.

2.5 Relationship of Endophytic Fungi and Other Factors

2.5.1 Fungal Succession Related to Enzyme Production

Promputtha et al. (2010) found that nine isolated endophytic fungi from Magnolia liliifera

leaves in Thailand were morphologically and phylogenetically similar to the isolated

saprobic fungi from M. liliifera leaves. They were Colletotrichum gloeosporioides,

Colletotrichum sp. 2, Corynespora cassiicola, Fusarium sp. 1, Guignardia mangiferae,

Leptosphaeri sp., Phomopsis sp. 2., Phomopsis sp. 6 and Phomopsis sp. 10 (Promputtha et

al., 2010). They mentioned that the fungal succession could be related to enzyme

production. They revealed that the productions of enzyme were related with the occurrence

of endophytic fungi and saprobic fungi in M. liliifera leaves.

They reported that xylanase and β-mannanase were produced by Phomopsis and Fusarium

which occur in both endophytic fungi and saprobic fungi at early stage of succession

whereas laccase was produced by Corynespora cassiicola which occur in both endophytic

fungi and saprobic fungi at middle to late stage of succession. Hence, they concluded that

endophytic fungi could be producing certain enzymes and colonising leaves as saprobic

fungi at different stages of leaves maturity during fungal succession. They also mentioned

the degrading enzymes were a factor influencing the process of endophytic fungi becoming

saprobic fungi.

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2.5.2 Endophytic Fungi Related to Decomposition Process

Pinruan et al. (2010) found that saprobic and endophytic basidiomycetes were collected

and isolated from the leaves, petioles and rachides of oil palm Elaeis guineensis in

Thailand. They reported that Schizophyllum commune and Pycnoporus sanguineus were

found in both saprobic and endophytic basidiomycetes. Hence, there is a possibility that

the endophytic fungi associated on leaves of oil palm may be important in decomposing

process as saprobic fungi.

He et al. (2012) reported that 39 endophytic fungi isolated from Cinnamomum camphora

leaves had the potential to degrade C. camphora leaf litter and some endophytic fungi had

lower decomposition rate of C. camphora leaf litter such as Basidiomycetes sp., Cerrena

sp., Cladosporium sp. 1 and Cladosporium tenuissimum after two months. They also found

that single endophytic fungi had lower decomposition rate of C. camphora leaf litter than

mixed microbial groups by comparing the mass loss of C. camphora leaf litter in

autoclaved soil and active soil. In pre-colonization, Chaetomium sp., Cladosporium sp. 1,

C. gloeosporioides, Colletotrichum sp. and Guignardia sp. were showed higher abundance

and caused greater mass loss of C. camphora leaf litter than other isolated endophytic

fungi (He et al., 2012). These endophytic fungi could have the ability to accelerate the

decomposition process. Thus, they concluded that decomposing endophytic fungi with

high abundance probably influence carbon and nutrient cycling in C. camphora

plantations.

2.5.3 Relating Endophytic and Parasitic Fungi from Cinnamomum sp. as

Pharmaceutical Products

Wang et al. (2011) investigated that the chemical components from endophytic fungus

Fusarium oxysporum which isolated from the bark of Cinnamomum kanehirai, a native

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plant in Taiwan. They found that a new oxysporidinone and 6-epi-oxysporidinone were

isolated from F. oxysporum had anti-fungal properties which against the growth of

Aspergillus fumigatus. They also revealed that beauvericin isolated from F. oxysporum had

the cytotoxic and antibacterial properties which against the growth of cancer cell lines (PC-

3, PANC-1 and A549), methicillin-resistant Staphylococcus aureus and Bacillus subtilise.

Liu et al. (2013) reported that Antrodia cinnamomea was isolated from C. kanehirai and

fermented with yeast extract and other substances to produce A. cinnamomea mycelial

fermentation broth (AC-MFB). A medicinal fungus A. cinnamomea is parasitise on the

inner cell of the Cinnamomum kanehirai which causes brown heart rot to the host plant

(Chang & Chou, 1995). They revealed that the cytotoxicity of AC-MFB was evaluated on

hepatocellular carcinoma cell (HCC) by tetrazolium-based colorimeter assay. They found

that AC-MFB was significantly inhibited the growth of HCC cell in the absence of hepatic

enzyme abnormality. They concluded that A. cinnamomea could have anticancer properties

and prevent the growth of cancer stem cell with its HCC characteristics.

From research works of Wang et al. (2011) and Liu et al. (2013), parasitic fungi Antrodia

cinnamomea and endophytic fungi F. oxysporum which isolated from Cinnamomum

kanehirai could have a relationship in the production of pharmaceutical products which

can be used as anticancer drug.

2.6 Endophytic Fungi Associated with Cinnamomum spp.

Suwannarach et al. (2012) reported that among 2,250 Cinnamomum bejolghota sample

collected in Doi Suthep Pui National Park, northern Thailand, 2,774 culturable endophytic

fungi were isolated. They found that 13 hyphomycetes, 9 ascomycetes and 6 coelomycetes

were isolated from the 2,774 endophytic fungi. They reported that Colletotrichum

gloeosporioides and Phomopsis spp. were grown abundantly in the leaves and stems of C.

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bejolghota and as endophytic fungi.

Kharwar et al. (2012) investigated that 162 endophytic fungi were isolated from more than

100 leaf segments of Cinnamomum camphora around the campus of Banaras Hindu

University, India. They found that 16 hyphomycetes, 4 coelomycetes and 1 ascomycete

were isolated from 162 endophytic fungi. They reported that Aspergillus niger grew

abundantly in stems of C. camphora. They also found that Cladosporium tennuissmum,

Nigrospora oryzae and Penicillium spp. grew abundantly in stems of C. camphora. They

revealed that Pestalotiopsis sp., Phyllosticta nobilis, and A. niger grew abundantly in

petioles of C. camphora.

He et al. (2012) found that among 69 Cinnamomum camphora leaves sample collected

from plantation forest site which near Jishou University, China, 2,861 culturable

endophytic fungi were isolated. They found that amount of 39 taxa including 36

ascomycetes and 3 basidiomycetes were isolated from the 2,861 endophytic fungi based on

phylogenetic analysis. They mentioned that Colletotrichum gloeosporioides grew

abundantly in the leaves of C. camphora with relative abundance of 68.79%. They stated

that C. gloeosporioides was distributed homogeneously across leaves of C. camphora with

colonization frequency of 95.65%. Hence, they concluded that endophytic fungi with high

colonisation frequency could also have high abundance.

2.7 Colonization and Distribution of Endophytic Fungi

2.7.1 Colonization of Endophytic Fungi on Age of Leaves

Mueller, Bills and Foster (2004) stated that age of leaves were influenced by fungal

colonization frequencies. They also mentioned that endophyte species diversity and

infection frequencies were increased with age of host organ and tissues. However, Selim et

yap shoo yuen - universiti malaysia sarawak fungi... · opinions when the project was carried out....

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