i

An investigation on the fungus associated with Aquilaria

malaccensis, their interaction and biological potentialities

By

DWRBI MOCHAHARI

DEPARTMENT OF LIFE SCIENCES

A THESIS

SUBMITTED

IN PARTIAL FULFILMENT OF THE REQUIREMENT

FOR THE DEGREE OF

MASTER OF SCIENCE

IN BIOTECHNOLOGY

TO

ASSAM DON BOSCO UNIVERSITY

TAPESIA CAMPUS

SONAPUR-782 402

ASSAM-INDIA

ii

DECLARATION

I, Dwrbi Mochahari, hereby declare that the subject matter of the thesis is the record of the

work done by me, that the content of this thesis did not form the basis of the award of any

previous degree to me or to the best of my knowledge to anybody else, and that the thesis

has not been submitted by me for any research degree in any other university/institute.

This is being submitted to the Assam Don Bosco University for the degree of Master of

Science in Biotechnology Department.

Signature

Dwrbi Mochahari

Research scholar

Signature

Dr.Shiny C.Thomas

Supervisor

Signature with date and seal

Dr. J.N.Vishwakarma

Head of the Department

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CERTIFICATE

This is to certify that the thesis entitled, ”An investigation on the fungus associated with

Aquilaria malaccensis, their interaction and biological potentialities,” being submitted by

Miss Dwrbi Mochahari, for the award of the degree of Master of Science in Biotechnology

department, of Assam Don Bosco University is the result of her investigation under my

supervision. She has fulfilled all the requirements for the submission of the thesis and is

worthy of being considered for the M.Sc. degree in Biotechnology department.

The work described in this thesis is original and has not been submitted for any other

degree or diploma in this or any other university.

Signature

Dr.Shiny C.Thomas

Supervisor

Signature with date and seal

Dr.J.N.Vishwakarma

Head of the Department

iv

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ACKNOWLEDGEMENT

I wish to express my sincere gratitude to Professor Dr. J.N.Vishwakarma, the head of the

department of School of Life Sciences, for providing me an opportunity to do my M.Sc. 4th

semester dissertation project work in the department of Biotechnology, Assam Don Bosco

University.

I sincerely thank my supervisor, Dr. Shiny C.Thomas for her guidance and encouragement

in carrying out this project work. I also wish to express my gratitude to Sir Ranjan, our

laboratory assistant, who rendered his help in the lab during the period of my project work.

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PREFACE

This thesis is a preliminary attempt to explore the biological interaction between the

vulnerable plant species Aquilaria malaccensis and the pathogens which infect them and

the endophytes present within them. The first phase of the study emphasises on the entire

literature review of this particular plant species, although very less research has been

carried out on this plant species around the world because of its less occurrence around the

globe. The ethnobotanical properties of the plant, its distribution and habitat around the

world and its endophytic relation with the fungi has been mentioned in the first chapter.

The second chapter includes the different materials and methods that had been undertaken

during the entire project work. Isolation of different fungal diversity and their biological

potentialities makes the work exciting. The third chapter reveals the results and also it

includes the discussion and conclusion at the end.

Finally I should say that this work has profited me tremendously with

the understanding of co-evolutionary relation between the endophytes and the plant

Aquilaria malaccensis and their biological potentialities.

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TABLE OF CONTENTS

1. Chapter 1 [INTRODUCTION AND LITERATURE REVIEW]………1

1.1 Introduction-plant and microbe interaction……………………………1

1.2 Endophytic fungus…………………………………………………….1

1.3 Aquilaria malaccensis...........................................................................2

1.3.1 Taxanomy…………………………………………………………...3

1.3.2 Vernaculars………………………………………………………….4

1.3.3 Distribution and Habitat…………………………………………….5

1.3.4 Ethnobotanical description……………………………………….....5

1.4 Economic importance of Aquilaria malaccensis……………………..6

1.5 Mystery of agarwood production- a hidden biological

process…………………………………………………………………….8

1.6 Objectives of the study………………………………………………...9

2. Chapter 2 [MATERIALS AND METHODS]…………………………10

2.1 Isolation of endophytic fungi from 1 year old Aquilaria malaccensis

plant stem…………………………………………………………………10

2.2 Isolation of fungi from the Agarwood chips………………………….15

2.3 Identification of the fungal isolates by lactophenol cotton blue……....19

2.4 Antibacterial activity of the leaves of 1 year old A. malaccensis........20

2.5 Antibacterial activity of the isolated fungi from different agarwood chips and

endophytic fungi from 1 year old A. malaccensis ………………………….23

2.6 The effect of 1 year old A.malaccensis stem pieces on fungal growth in PDA

plates………………………………………………………………………23

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3. Chapter 3 [RESULTS]……………………………………………………….....24

3.1 Isolation of endophytic fungi from 1 year old A. malaccensis………………...24

3.2 Isolation of fungi from the Agarwood chips…………………..........................25

3.3 Identification of the fungal isolates by lactophenol cotton blue……………....26

3.4 Antibacterial activity of 1 year old A.malaccensis leaves……………………..30

3.5 Antibacterial activity of the isolated fungi from different agarwood chips and

endophytic fungi from 1 year old A.malaccensis………………………………….34

3.6 Effect of A.malaccensis stem pieces on fungal growth in PDA

plates……………………………………………………………………………….37

4. Chapter 4 [DISCUSSION]……………………………………………………...40

5. Chapter 5 [CONCLUSION]…………………………………………………….42

6. References……………………………………………………………………….43

7. Appendix 1: Abbreviations used………………………………………………..48

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LIST OF FIGURES/TABLES

Figures:

Names Page no.

Figure 2.1 One year old A.malaccensis planted in

the greenhouse of the institution.

11

Figure 2.2 Culture of endophytic fungi in PDA

plates.

12

Figure 2.3 Subcultures of endophytic fungi. 13

Figure 2.4 Pure cultures of the endophytic fungal

isolates.

14

Figure 2.5 Agarwood chips of different forms. 15

Figure 2.6 Isolation of fungi from three types of

Agarwood chips.

16

Figure 2.7 Growth of fungus in PDA plates

isolated from Agarwood chips.

17

Figure 2.8 Sub-cultures of isolated fungi from

Agarwood chips.

18

Figure 2.9 Drying of Aquilaria malaccensis

leaves in the shade in laboratory.

20

Figure 2.10 Methanol leaves extract 21

Figure 2.11 Distillation process of the methanolic

leaves extract

21

Figure 3.1 Endophytic Fungi 24

Figure 3.2 Fungi isolated from infected agar

plant

25

Figure 3.3.1 Spores of Alternaria sp. under 45X. 26

Figure 3.3.2 Spores of Curvularia sp. under 45X 26

Figure 3.3.3 Spores of Curvularia sp. under 100X 27

Figure 3.3.4 Spores of Fusarium sp. under 100X 27

Figure 3.3.5 Mycelia of Sterilia sp. under 45X 28

Figure 3.3.6 Rhizopus sp. hyphae and spores under 28

x

45X.

Figure 3.3.7 Penicillium sp. under 45X 29

Figure 3.3.8 Fusarium sp. under 45X. 29

Figure 3.3.9 UNF(unidentified sp.) 29

Figure 3.4 Antibacterial activity of

A.malaccensis leaves on S.pneumonia,

B.subtillis, E.coli, S.dysentry, and

K.pneumonia

31

Figure 3.5 Antibacterial activity of UNF on

(1)E.coli and (2)B.subtilis.

34

Figure 3.6 Antibacterial activity of Penicillium

sp. on (1)E.coli and (2)B.subtilis.

35

Figure 3.7 Antibacterial activity of Rhizopus sp.

on (1)E.coli and (2)B.subtilis.

35

Figure 3.8 Antibacterial activity of Sterilia sp. on

(1)E.coli and (2)B.subtilis

35

Figure 3.9 Antibacterial activity of Curvularia

sp. on (1)E.coli and (2)B.subtilis

36

Figure 3.10 Antibacterial activity of Alternaria sp.

on (1)E.coli and (2)B.subtilis.

36

Figure 3.11 Effect of A.malaccensis stem pieces

on fungal growth in PDA plates

39

Tables:

Table 1.1 Vernaculars of Aquilaria malaccensis. 4

Table 2.1 Weight of the Agarwood chips 15

Table 3.1 Antibacterial activity of A.malaccensis

leaves on different bacteria.

30

Table 3.2 Antibacterial activity of fungal

isolates

34

Table 3.3 Effect of A.malaccensis stem pieces

on the growth of fungus

39

1

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction- plant and microbe interaction

Plants house several microorganisms like fungi and bacteria which live in a symbiotic or

mutualistic association. Mostly the fungi contribute more in the interaction. The fungal

species may be pathogenic or endophytic and the plant acts as the host mechanism. They

both interact with each other in different ways which produces many plant metabolites or

the secondary products as a outcome of the biochemical process that takes place at the time

of their interaction. The genetically coded possibilities for secondary metabolite production,

the stimuli of the production, and the special phytotoxins basically determine the

microscopic fungi-host plant interactions and the pathogenic lifestyle of fungi (Pusztahelyi

et al., 2015). When a microbe challenges a plant, co-evolutionary, multilayer defense

strategies are engaged in the plant as well as in the microbe (Joensen, 2011). These

roles play a great significance in the plant‟s life as well as in the fungi‟s life as they

produce magnificent valuable products which may be useful to the human kind.

1.2 Endophytic fungi

Endophytic fungi are fungi that colonize living plant tissue without causing any immediate,

overt negative effects. Most endophytes are capable of synthesizing bioactive compounds

that may provide plants with a defence against pathogens, and some of these compounds

have proven useful for novel drug discovery (Cui et al., 2011). Some species of endophytic

fungi have been identified as sources of anticancer, antidiabetic, insecticidal,

immunosuppressive and biocontrol compounds (Shoeb et al., 2010). The Aquilaria species

or plants belonging to Gyrinops family are known to house many endophytic fungi. The

main endophytic fungi found to have association with the Agarwood formation by natural

infection are Aspergillus, Lasiodiploidia, Chaetomium, Fusarium and Penicillium species

(Nagajothi et al., 2016). Fungi are very resistant microbes and thus they can undergo

mutations with time and with their changing environment. Thus, though many endophytic

2

fungi have been found associated with agarwood formation there can always be new fungi

evolving out of the Agar plant every time.

1.3 Aquilaria malaccensis

Agarwood or Agar plant is one of the most precious woods known for its

pharmacognostical (Satyanathanan et al., 2012), resinous oil producing (Chong et al.,

2015), essential agronomy based plants (Chowdhury et al., 2016) in the world. In India,

agar plant occurs mostly in foothills of North Eastern region as well as West Bengal.

Assam region of Northeast India is believed to be the origin of agarwood and spread

throughout Southeast Asia in to its current range (Burkill et al., 1966). Agarwood is an

aromatic resin product formed in the plants belonging to the genera of Gyrinops and

Aquilaria of the family Thymelaeaceae. Agarwood has different common names in

different places like Agar, Aloeswood, Eaglewood, Karas, Sasi, Gaharu and Kalamabak

etc. Agarwood is called „the wood of the Gods‟, because of its uses range from incense for

religious ceremonies, perfume for the Arabic world, medicinal wine in Korea and

ornamental functions in China (Persoon, 2007). Agarwood is one of the most priced non-

timber forest product traded in the international market. Agarwood plantation is also

known as green gold mine of the future.

Agarwood is mainly traded in three forms-wood chips, wood dust

or powder and Agar-oil (Abdin, 2014).

The main compounds in agarwood essential oil

have been revealed to be sesquiterpenoid (Liu et al., 2017). The agarwood producing

plants are so important now-a-days in the economic sector that people grow these plants in

a large scale due to its high value of resin production and has become a multidisciplinary

business mostly in the Muslim countries like Malaysia, Saudi Arabia, Bangladesh and in

the Middle East. International market price of agarwood chips is US $ 20 to US $ 6000 per

kilo based on its quality. Distilled agar oil valued as high as US$ 30,000 per kilogram and

the wood itself worth up to US$ 10,000 per kilogram (Akter et al.,2013). In Asia, 15

species of agarwood are known to produce essential oils (Sulaiman et.al., 2015). Besides

A. malaccensis, other potential species listed for domestication were A. crassna, A.

subintegra, A. hirta, A.rostrata, A. beccariana, A. filaria, A. khasiana, A.microcarpa, A.

grandiflora and A.sinensis (Zuhaidi, 2016) . Because of its multifarious uses and high

market demand, mature agarwood trees have been harvested indiscriminately from wild

3

resulting in rapid decline of the species in natural habitat (Beniwal et al., 1989). The

negative impact of such decline is felt not only in terms of biodiversity loss but also in

terms of the reduced availability of a highly valued and valuable forest resource. Due to

heavy exploitation of the trees producing agarwood in the countries where it is endemic,

the International Union for Conservation of Nature and Natural Resources (IUCN) has

listed the Aquilaria species in the red list of threatened species (The IUCN Red List of

Threatened Species: Aquilaria malaccensis – published in 1998). Moreover, it is also

found that the plant has low reproductive potential with seeds showing very short viability

period which offers a major limitation for its natural growth (Tasso et al., 2014). Very few

researches on agarwood have been carried out in the North East India inspite of the fact

that this plant is endemic only to this region of India, mainly the species Aquilaria

malacccensis. Therefore, it is important to find more and more interesting information on

this plant and thus, the present study focuses on the investigation of the fungus associated

with Aquilaria malaccensis, their interaction and biological potentialities.

1.3.1 Taxonomy-

The taxonomy of Aquilaria malaccensis is as follows-

Kingdom- Plantae

Phylum- Tracheophyta

Class- Magnoliopsida

Order- Myrtales

Family- Thymelaeaceae

Genus- Aquilaria

Species- malaccensis

Synonym- Aquilaria allagocha

(The IUCN Red list of threatened species –Aquilaria malaccensis, 1998).

4

1.3.2 Vernaculars-

The plant Aquilaria is known differently in different countries and regions.

Language Vernaculars

Arabic agare-hindi, ajmod, aud, aude-hindi, ud,

ude-hindi

Hindi Agar

Kannada agaru, agaru gandha, agilu gandha,

krishnagaru

Malayalam akil, karakil, kayagahru

Marathi agar, agara

Persian agar, agre-hindi

Sanskrit agaru, agnikashtha, aguru, aguruh, akil,

anaryaka, asara, bhringaja, jishvarupa,

jongaka, jongakah, jongakaichhila, jonk,

kalaguru, kalaloha, kashthaka,

krimigandha, krimija, krimijagandha,

krishna, krsnaguruh, laghu, loha,

lohakhya, maliyaka, pataka, pravara,

rajarahkalijya, rajarha, vanshika,

varnaprasadana, yogaja

Tamil agalicandana, agar, aggalichandana, agil,

akar, akaru, akil kattai, akilmaram,

akircilai, akirkattai, akkakalam, akkil,

akku, akkuru, akuru, aracakam, arakkam,

arukam,

Telugu agaru, agaru chettu, agru, krishna agaru,

krishnagaru

Tibetan a ga ru, a ka ru nag po

Urdu agar, agar hindi, agar hindi saida, agar

nim kofta, agar pisa hua, ood, ood hindi,

ood hindi (agar), ood hindi saida, roghan

ood, udghargi

Indonesia gaharu

Malaysia karas

Assamese sanchi, sasi-pat

Bengali agaru

(GBIF-The Plant List)

Table 1.1: Vernaculars of Aquilaria malaccensis.

5

1.3.3 Distribution and Habitat-

Agarwood is the heartwood produced by a number of Aquilaria species in Southeast Asia,

with Indonesia, India, Malaysia, Vietnam, Cambodia, Thailand, Laos and Papua New

Guinea as the main producing countries and Singapore being the main trade centre as

mentioned by Persoon published in a journal in IIAS Newsletter, 2007. In India, mainly

two Aquilaria species viz., Aquilaria malaccensis and Aquilaria khasiana are found.

Aquilaria khasiana is mainly found in Meghalaya, whereas, Aquilaria malaccensis is

native to the eight states of North-East India (Borah et al., 2012). As reviewed by Akter

and Neelim in 2008, A. malaccensis is found in 10 countries Bangladesh, Bhutan, India,

Indonesia, Iran, Malaysia, Myanmar, Philippines, Singapore and Thailand. As reported by

Kesler and Sidiyasa in their book “Trees of the Balikpapan-Samarinda Area, East

Kalimantan, Indonesia- A manual to 280 selected species”, the Aquilaria species remains

scattered on ridges and slopes, and on well-drained lands. Agar plant grows well in high

humid, sub-tropical climate with rainfall 1800-3500 mm per annum. It grows 500 metres

above the sea level. It requires a good amount of sunlight in order to grow well. The

traditional agarwood growing areas show that it prefers acidic soil (Hand book of

Medicinal and Aromatic plants).

1.3.4 Ethnobotanical description-

Trees of agarwood grow up to 20-40 metres tall and 60 centimeters in diameter. Bark is

smooth and whitish in color. Inner bark is creamy white, fibrous, homogeneous, tearing off

in long and strong strips. Sapwood is white in color. Twigs are slender. Leaves are spiral,

elliptic-oblong to oblong-lanceolate, . 5 - 1 2 cm long, 2.5-5.5 cm wide, base acute, apex

acute to acuminate, parchment-like, fibrous, not brittle, without translucent dots, secondary

veins 1 2 - 1 6 pairs, rather irregular, often branched, distinct beneath, obscure above.

Petioles are 3 -6 mm long, inflorescences terminal, axillary, or above the axils, branched

with 10- flowered umbels. The tree starts flowering and fruiting at the age of 5-6 years and

medium sized trees are reported to produce about 1.5 kg of seed during good seed years.

Flowers are green or dirty yellow, bell-shaped, 5-6 mm long, petaloid appendages twice as

many as the lobes, free, stamens twice as many as lobes, ovary ovoid, 1-1 .5 mm long,

stigma sessile. Fruits capsules are obovoid or obovoid-oblong, 3-4 cm long, 2.5 cm in

6

diameter, base wedge shaped, apex rounded. Seeds are ovoidal, 10 mm long and 6 mm in

diameter and are densely covered with red hairs (Adelina et al., 2004).

1.4 Economic importance of Aquilaria malaccensis

Agarwood is used for many different purposes. The following uses are given as follows-

1.4.1 Incense sticks

Agarwood is used in making several different varieties of incense sticks because of its high

quality of aroma. These incense sticks are useful for different occasions in different

religions and they are burnt in temples, mosques and also in the Buddhist monasteries.

Agarwood was used widely in Kodo, the art of incense ceremony in China and Japan

because the scent or aroma of agarwood is complex and pleasing, with few or no similar

analogues (Liu et al., 2017). As mentioned by Barden et al., in their paper “Heart of the

matter-Agarwood use and trade and cities implementation for Aquilaria malaccensis”,

agarwood powder and dust cannot be burnt directly in incense holders, but can be used to

make incense sticks or coils for indoor fragrance. Agarwood incense sticks are used in

many countries like India, Malaysia, China, Japan and in the Middle East. It is reported

that the Taiwanese people purchase agarwood raw material to make incense sticks for

their religious ceremonies to bring safety and good luck.

1.4.2 Perfumes

More excitingly, perfumes are the products formed after fermentation of the agarwood

chips. The resinous agarwood chips are fermented and then distilled to obtain the essential

oils which is the secondary product of agar plant for the production of perfumes of

different aromas. In the Old Testament, the term „Aloes‟ is mentioned for several time.

This marks the importance of agarwood perfumes since the olden times. Both agarwood

smoke and oil are customarily used as perfume in the Middle East (Chakrabarty et al.,

1994). The fragrance of agarwood is recognised as for its fine olfactory nuances, and its

consumption has positive effects on the body and the mind (Jung, 2016). Agarwood is said

to have been highly prized by European perfumers in the mid-1990s (Chakrabarty et al.,

1994)

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1.4.3 Medicine

Agarwood is not only important for its aromatic properties, but its medicinal properties are

as important as they produce aroma. Many researchers have found out its

pharmacognostical properties and with great values and applications. In the studies by

Sathyanathan et al, published at Industrial Journal of Pharmacy and Industrial Research,

they found out the phytochemical and physico-chemical properties of Aquilaria

malaccensis , the common species that produces agarwood. In the phytochemical screening

process they found proteins, sugar, alkaloids, tannins and essential oils which are essential

for medicinal values. Other secondary metabolites like lignin, saponin, flavonoid, quinine,

terpenoid, sterol, alkaloids were also found out. The leaves, stems and bark extracts of A.

malaccensis, A. sinensis, A.subintegra and A. crassna with different solvents showed

antioxidant, anticancer, anti-hyperglycemic, anti-inflammatory, antibacterial and antifungal

properties as reviewed by Jok et al., 2013. The seeds of Aquilaria malaccensis are found to

have anti-allergic properties (Korinek. et al., 2016).

1.4.4 Other uses

Agar wood pieces in natural shapes and sculptures are the highest value items in the

consumer market. Agar wood chips are mainly used as raw materials in fragrance industry.

A very small potion is using to make tea. Different types of agar wood fragrances (Agar

wood oil, Dihn al oudh, Arabian perfumes, Scented chips, Sprays, Bakhoor etc.) are used

as body fragrance, clothes fragrance, house fragrance and to receive honored guest. Agar

wood pieces are also made into beads for religious purpose. People use this oil fragrance

for personal devotion, applied on the hair, behind the ears, neck, nostrils, and clothes.

Traditionally the Buddhist masters used rosary beads made from agar wood. Agar wood is

used to manufacture several types of crude medicine, prepared medicine. Several types of

liquor products are producing with agarwood (Antonopoulou et al., 2010 TRAFFIC

research).

8

1.5 Mystery of Agarwood production- a hidden biological process

It has been studied that the Agar plant produces resinous constituent by many factors, the

endophytic fungi or fungus infection being one of the most important factors. Three

hypotheses exist regarding agarwood formation, namely that it is the result of pathological,

wounding/pathological and/or non-pathological processes (Ng et al., 1997). According to

Ng et al. 1997, studies have not provided conclusive evidence for any of these hypotheses.

Oldfield et al.,1998 states that resin production is a kind of response to fungal infection.

Although the understanding of the link between fungal infection and agarwood production

is discrete, fragrant oleoresin production in the heartwood of the trees is a kind of

biological response of the plant to wounding or infection of ascomycetes fungus. When

the Agar plant becomes naturally or artificially wounded, they become infected with the

pathogenic fungi and it is thought that this infection triggers the endophytic fungus present

in the plant to produce some metabolites or some biochemical reactions take place in

response to the pathogenic fungi and this metabolites production ultimately results in the

production of agarwood or resinous product. The resinous product is called the Oleoresin

(Nagajothi. et al., 2016). The actual mechanism that takes place behind the resin

production is still a mystery. Keeping this in mind the present research study aims to

isolate new fungal species from the one year old Agar plant and to compare them with the

ones isolated from the infected agarwood chips and to identify their morphology and to

investigate on the connection between fungal infection and oleoresin production, and

evaluate biological potentialities of agarwood extract.

9

1.6 Objectives of study-

Isolation of fungi from one year old Aquilaria malccensis plant stems.

Isolation of fungi from old infected agarwood chips, uninfected and infected fresh

agarwood chips.

Identification of the isolated fungi by lactophenol cotton blue.

Antibacterial activity of methanol extract of Aquilaria malaccensis leaves.

Antibacterial activity of endophytic fungi isolated from one year old Aquilaria

malaccensis plant.

Effect of Aquilaria malaccensis stem pieces on the growth of six different fungi in

PDA medium.

10

CHAPTER 2

MATERIALS AND METHODS

Source of materials

In the experiments chemicals and reagents and agarwood samples were used which are

given below:

Chemical and reagents

Potato dextrose agar (PDA) (infusions from potato 200 g, dextrose 20 g and 15 g in

1 litre of purified water) ; potato dextrose broth (PDB) (infusions from potato 200 g

and dextrose 20 g in 1 litre of purified water) (Hi Media laboratories Pvt. Ltd,

India).

Luria Agar(LA); Luria Broth(LB). (Hi Media laboratories Pvt. Ltd, India).

Agarwood sample materials

Agarwood sample materials were obtained from Harisinga of Udalguri district, Assam.

They were labelled and transported to the laboratory.

2.1 Isolation of fungi from one year old Aquilaria malaccensis plant

stems.

2.1.1 Collection of one year old Aquilaria malccensis plants

The plant sample was collected from Harisinga of Udalguri district, Assam on 11th

February 2017. Ten healthy plants with strong stem and leaves were selected and

transported to the green house of Assam Don Bosco University.

11

2.1.2 Plantation of the collected sample in the green house

The plant samples were brought to the green house in good condition and replanted in 7-8

kg of soil in plastic sacks. The plants were kept under observation till they became

acclimatized with the new environment inside the green house. The plants were watered

once daily in the morning for 1 week and after a week they were watered on alternative

days.

Figure 2.1:One year old A.malaccensis planted in the greenhouse of the institution.

2.2.2 Isolation

For the isolation of endophytic fungus from 1 year old agarwood plant, first the small

stems from the plant were taken and cut into small pieces. They were than surface

sterilized by dipping in 70% ethanol for 2 minutes which was followed by rinsing with

distilled water for 3-4 times and then dried for sometime. The stem pieces were then

ground by using mortar and pestle. 15 ml of distilled water was added to it and then filtered

using a clean filter paper. The filtrate was used as fungal source, and it was cultured in

PDA plates by pour plate technique. 200 μg/ mL of Ampicillin was added into the media

prior to pouring into the plates in order to prevent any bacterial contamination or to inhibit

the growth of bacteria. Then the plates were kept under incubation at room temperature for

5-6 days till the fungal growth was obtained. Each colony that was grown in the plates was

sub-cultured on new PDA plates until a pure isolate was obtained. For each sample type,

12

two plates were prepared to grow in replicates to maximize the growth of different possible

endophytes and the isolates were incubated again at room temperature for seven days.

Figure 2.2: Culture of endophytic fungi in PDA plates.

13

Figure 2.3: Sub-cultures of endophytic fungi to obtain the pure cultures.

14

Figure 2.4: Pure cultures of the endophytic fungal isolates.

15

2.2 Isolation of fungi from old infected agarwood chips, uninfected and

infected fresh agarwood chips.

2.2.1 Collection of Aquilaria malaccensis wood chips

The sample was collected from Harisinga of Udalguri district, Assam on 11th

February

2017. Three types of agarwood chips were collected which were- dried infected chips,

fresh infected chips and fresh uninfected chips.

Figure 2.5: Agarwood chips of different forms-dried infected, fresh infected and fresh

uninfected.

2.2.2 Preliminary observations of the agarwood chips sample

The weight of the three different wood chips were recorded separately which is given

below-

Dried infected wood chip Fresh infected wood chip Fresh uninfected wood

chip

1.22g 5.88g 4.12g

Table 2.1: Weight of the three Agarwood chips.

16

2.2.3. Isolation

For the isolation of fungi from the agarwood chips, the standard protocol for isolation was

followed. The three different agarwood chips were first surface sterilized by dipping in

70% ethanol for 2 minutes all separately and followed by rinsing with distilled water for 3-

4 times and then dried for some time. The wood chips were then crushed by mortar and

pestle till powdered form. Then 15 ml of distilled water was added to it and then filtered by

using a filter paper all separately. The filtrate obtained after filtration was then used in the

culturing of endophytic fungus in the PDA medium. Three replica plates of each different

agarwood chips were made which were- the dried infected, fresh infected and the fresh

uninfected chips. The filtrate of fungal source was cultured in the plates by pour plate

technique to obtain the isolates. 200 μg/mL of Ampicillin was added into the media prior

to pouring into the plates in order to prevent any bacterial contamination or to inhibit the

growth of bacteria. Then the plates were kept under incubation at room temperature for 5-6

days till the fungal growth was obtained. Each colony that was grown in the plates from

the wood chips was sub-cultured in new PDA plates until a pure isolate was obtained. For

each sample type, two plates were prepared to grow in replicates to maximize the growth

of different possible endophytes and the isolates were incubated again at room temperature

for seven days.

Figure 2.6: Isolation process of fungi from three types of agarwood chips.

17

Figure 2.7: Growth of fungus in PDA plates isolated from Agarwood chips.

18

Figure 2.8: Sub-cultures of isolated fungi from Agarwood chips.

19

2.3. Identification of the fungal isolates from the agarwood chips and the

endophytic fungus from 1 year old plant by lactophenol cotton blue.

For the identification of the fungal isolates from the samples a standard protocol of

morphological identification was followed using the lactophenol cotton blue. Here in this

mounting method, lactophenol cotton blue stain is used where lactophenol acts as a

cleaning agent as phenol is fungicidal in nature so it kills the organism. Lactic acid acts as

a preservative and it preserves the structure of fungus where as cotton blue stains the

cytoplasm in light blue colour so here we can observe the fungal structure colourless with

light blue background. First a clean grease free slide was taken which was sterilized by

using 70% ethanol , then with the forcep a mycelia mat was taken from the fungal culture

and was placed on the slide. The mycelia was teased very carefully in order to spread the

hyphae and to make the spores and other structures of the fungus visible. Then a drop of

the mounting fluid which was the lactophenol cotton blue stain was added and was covered

by a cover slip very carefully. Then each slides were observed under a simple microscope

for morphological identification at 10X, 45X and 100X magnification.

20

2.4 Antibacterial activity of methanol leaves extract of one year old

Aquilaria malaccensis.

2.4.1 Collection and drying of leaves from 1 year old planted Aquilaria malaccensis

from the green house

The leaves were collected from the planted 1 year old A. malaccensis from the green

house of the institute. Then the leaves were washed carefully in the running water tap.

Then they were dried in the shade for 7 days at room temperature (28 ±2°C) and ground to

coarse powder. The powder samples were kept at room temperature in a covered glass

containers to protect them from humidity and light prior to extraction.

Figure 2.9: Drying of Aquilaria malaccensis leaves in the shade in laboratory.

2.4.2 Extraction of leaves of 1 year old Aquilaria malaccensis

27 grams dried powder leaves of 1 year old A. malaccensis plant was exhaustively

extracted by maceration in 300 ml absolute methanol solvent for 24 hours in the incubator

shaker at 35°C. The solvent-containing extract was then decanted and filtered by Whatman

filter process. The extraction of the ground leaves was further repeated once again with

absolute methanol (300 ml). The filtrate from the extraction was combined and the excess

solvent was evaporated by performing distillation process to give concentrated crude

21

methanolic extract. The weight of the extract was measured after distillation and then kept

into an eppendorf tube prior to use.

Figure 2.10: Extract of A.malaccensis leaves in methanol.

Figure 2.11: Distillation process of the methanolic leaves extract.

22

2.4.3. Disc diffusion method

Five bacterial strains Streptococcus pneumonia, Escherichia coli, Shigella dysentery,

Klebsiella pneumonia and Bacillus subtilis cultured in broth media were obtained from the

Department of Life Sciences, Assam Don Bosco University. Agar diffusion test was

carried out to establish the antibacterial activity of the methanol extract against the test

pathogens. Nutrients agar (NA) plates were prepared as per manufacturer instructions.

Then the five bacterial cultures were inoculated in 3 plates each for triplicate experiment.

They were then spread all over the plate by spread plate technique. Small disc made of

filter paper measuring 5mm in diameter were then dipped in the leaves extract till it was

wet. These discs were then placed at 2 positions in the plate, 1 disc was dipped in the

methanol alone to serve as the control. The plates were incubated at 37°C for 24 h. The

zone of the clearance around each well after the incubation period, confirms the

antimicrobial activity of the respective extract.

23

2.5 Antibacterial activity of the isolated fungi from different agarwood

chips and endophytic fungi from 1 year old A. malaccensis

Antibacterial activity of the isolated fungi from Agarwood chips and endophytic fungi

from 1 year old Agar plant was carried out by direct method of antibacterial assay. The

activity was checked against two bacterial strains, E.coli and B. subtilis respectively. The

experiment was done in triplicate. First the bacteria were inoculated in LA plates and then

spread all over by spread plate method. Then a small amount of the mycelia mat from the

different fungi was placed at 3 positions on the plates. One plate each of E.coli and

B.subtilis were kept as control plates without fungal inoculation. Then all the plates were

kept overnight for incubation at 37°C. The zone of the clearance around each fungal mat

after the incubation period, confirms the antimicrobial activity of the respective extract.

2.6 The effect of 1 year old A.malaccensis stems pieces on fungal growth

in PDA plates.

The effect of Agarwood chips in different fungi were checked by simply adding the agar

chips in PDA plates to enrich the media and compare the growth of different fungal

species. 2 different non-endophytic fungal species which were ordered from MTCC

laboratory, Chandigarh were used to compare the growth with that of the isolated fungi.

Total 6 fungi ( Sterilia sp., Rhizopus sp., Curvularia sp., Ustilago maydis and

Cladosporium herbarum) were used for this test. The PDA plates were prepared as per the

manufacturer‟s instructions and already autoclaved agar chips were added on to the plates

in different concentrations ( 200 mg and 300 mg) while the media was poured on the

plates. Then the mycelia mat of each fungus was inoculated in the plate. The plates were

incubated at room temperature for some days until the fungal growth occurred.

24

CHAPTER 3

RESULTS

3.1 Isolation of endophytic fungi from 1 year old Aquilaria malaccensis

Four different fungi were isolated as endophytic fungi from the 1 year old Aquilaria

malaccensis plants. The figures are given below:

Figure 3.1: Endophytic fungi

25

3.2 Isolation of fungi from dried infected and uninfected and infected

fresh Agarwood chips

Three different fungi were isolated from the infected agarwood chips. The figures are

given below:

Figure 3.2: Fungi from infected wood chips

26

3.3 Identification of the fungal isolates by lactophenol cotton blue stain.

The four isolates from 1 year old Aquilaria malaccensis were identified to be Alternaria

sp. , Curvularia sp. , Rhizopus sp., and Sterilia sp. , by looking into their hyphae and

spores structures.

Figure 3.3.1: Spores of Alternaria sp. under 45X

Figure 3.3.2: Spores of Curvularia sp. under 45X.

27

Figure 3.3.3: Spores of Curvularia sp. under 100X.

Figure 3.3.4: Spores of Fusarium sp. under 100X

28

Figure 3.3.5: Mycelia of Sterilia sp. under 45X.

Figure 3.3.6: Rhizopus sp. hyphae and spores under 45X

29

Three different fungi were isolated from the three different agarwood chips out of which

only two were identified. The unidentified one is termed as the UNF but it was used for all

the other experiments along with the identified ones.

Figure 3.3.7: Penicillium sp. under 45X Figure 3.3.8: Fusarium sp. under 45X

Figure 3.3.9: UNF (Unidentified species)

30

3.4 Antibacterial activity of the leaves extract of 1 year old Aquilaria

malaccensis

The methanolic extract of 1 year old Aquilaria malaccensis leaves showed antibacterial

activity against 3 strains of bacteria out of 5. The strains which showed clear inhibition

zones were S.pneumonia, E.coli, and S. dysentery respectively. While, K.pneumonia and

B.subtilis did not form distinct clear zones.

Bacterial strain Presence or Absence of

inhibition

Streptococcus pneumonia + +

Escherichia coli + +

Shigella dysentry + +

Klebsiella pneumonia ─ ─

Bacillus subtilis ─ ─

Table 3.1: Antibacterial activity of A.malaccensis leaves on different bacteria.

31

The pictures given below indicates the antibacterial activity of the methanolic leaves

extract of Aquilaria malaccensis by showing the zone of inhibition in the LA plates of

different bacterial strains.

(1)

(2)

32

(3)

(4)

33

(5)

Figure 3.4: Antibacterial activity of A.malaccensis leaves on S.pneumonia,

B.subtillis, E.coli, S.dysentry, and K.pneumonia

34

3.5 Antibacterial activity of the isolated fungi from different agarwood

chips and endophytic fungi from 1 year old Aquilaria malaccensis

Out of the 7 fungi (3 from agarwood chips and 4 endophytic fungi), only one showed

antibacterial effect on E.coli and B.subtilis. All the others showed negative results as

shown in the table below-

Fungi Bacillus subtilis Escherichia coli

UNF + + + + + +

Penicillium sp. ─ ─ ─ ─ ─ ─

Fusarium sp. ─ ─ ─ ─ ─ ─

Alternaria sp. ─ ─ ─ ─ ─ ─

Curvularia sp. ─ ─ ─ ─ ─ ─

Sterilia sp. ─ ─ ─ ─ ─ ─

Rhizopus sp. ─ ─ ─ ─ ─ ─

Table 3.2: Antibacterial activity of the fungi isolated from 1 year old A.malaccensis and

Agarwood chips.

The pictures given below shows the antibacterial activity of different fungal isolates in

PDA plates of two bacterial strains which is E.coli (gram negative) and B.subtilis (gram

positive).

Figure 3.5: Antibacterial activity of Unknown fungus(UNF) on (1)E.coli and (2)B.subtilis

35

Figure 3.6: Antibacterial activity of Penicllium sp. on (1)E.coli and (2)B.subtilis

Figure 3.7: Antibacterial activity of Rhizopus sp. on (1)E.coli and (2)B.subtilis

Figure 3.8:Antibacterial activity of Sterilia sp. on (1)E.coli and (2)B.subtilis

36

Figure 3.9:Antibacterial activity of Curvularia sp. on (1)E.coli and (2)B.subtilis

Figure 3.10:Antibacterial activity of Alternaria sp. on (1)E.coli and (2)B.subtilis

37

3.6 The effect of 1 year old A.malaccensis stems pieces on fungal growth

in PDA plates.

The addition of 1 year old A. malaccensis stem pieces was used to enrich the culture media

(PDA). All the 6 different fungi showed good growth and faster growth than the control

plates where the stem pieces were not added.

38

39

Figure 3.11: Effect of A.malaccensis stem pieces on fungal growth in PDA plates.

Fungal species Control(PDA) PDA+Agarwood

chips

UNF(unidentified) + + (fast) + +(fast)

Sterilia sp. + + (slow) + +(fast)

Mucor sp. + + (moderate) + +(fast)

Curvularia sp. + + (fast) + +(fast)

Ustilago maydis + +(slow) + +(fast)

Cladosporium herbarum + +(fast) + +(fast)

Table 3.3: Effect of A.malaccensis stem pieces on the growth of fungus

40

CHAPTER 4

DISCUSSION

The Aquilaria malaccensis and its interaction with the fungi were studied by many

researchers (Mohamed et al.,2014; Nagajothi et al.,2016; Than, 2013; Chong et al., 2014).

The present study was focused on the isolation of the endophytic fungi from one year old

Aquilaria malaccensis plant. It is believed that the Agarwood production or the oleoresin

production mainly takes place by infection of the plant by either pathogenic fungi after

wounding or cutting of trees or by other environmental factors (Ng et al., 1997; Oldfield et

al., 1998). But in this study four different endophytic fungi were isolated from one year old

Aquilaria malaccensis plant. They were also identified by its morphological structures

under the microscope. Thus, it was confirmed that the Agarwood formation or the

oleoresin production not only takes place due to the pathogenic fungi that infects the plant

after the wounding or cutting or by several environmental factors, but also the endophytic

fungi contributes its role in the resin production.

Moreover, the pathogenic fungi were also isolated from the infected agar plant

and their growth in PDA medium alone were tested and found that the pathogenic fungi

grew normally as the other fungi grow in normal conditions.

Though the plant has been known for its agarwood production, the methanol

extract of the leaves showed visible inhibition zones in LA plates (Figure 3.4) which

indicates antibacterial activity against S.pneumonia, E.coli and S.dysentry. Thus, the

leaves also possess antibacterial activity, but rigorous research is needed to confirm this

finding. It was thought that if the leaf shows biological properties that would be an

additional benefit over the Aquilaria malaccensis. Therefore, the antibacterial activity of

the methanol leaf extract has been evaluated in this study.

In continuation with the antibacterial evaluation of leaf extract, the endophytic

fungi isolated (Alternaria sp., Curvularia sp., Rhizopus sp., and Sterilia sp.) from one year

old Aquilaria malaccensis and pathogenic fungi (Penicillium sp., Fusarium sp. and UNF-

Unidentified) isolated from the infected Agarwood chips were also screened for their

antibacterial property. Out of the 7 fungi used in this screening only one fungi which was

41

unidentified fungus (UNF) from the infected agarwood chips has shown promising

antibacterial effect (Figure-3.5).

Since the plant-microbe interaction in the agarwood production is still a

mystery, it was assumed that the addition of one year old agar plant stem pieces in the

fungal culture medium (PDA) may give a pilot clue about their pathophysiology. The

result of this experiment showed that the plates which were added with agar chips did not

inhibit the growth of the fungus as compared to the control (Figure-3.11). The same result

has been shown by other fungi which were isolated from the one year old Aquilaria

malaccensis which is growing in the university campus. The study gives a clue that the

resin production takes place in Aquilaria malaccensis is due to many biological processes.

The initiation and regulation of agar resin production is the combined biochemical

processes beginning from the early stages of growth of the plant and the resin production is

visible only in the mature plant.

42

CHAPTER 5

CONCLUSION

The present study reveals that Aquilaria malaccensis is an expensive multipurpose

non-timber plant that produces many valuable compounds and thus by determining the

interaction and influence of the Agar plant chips on the fungal growth it may be

concluded that there is a mysterious bonding between the Agar plant and the fungus in

and around its environment . This interaction can be studied in a broader way in the

future perspectives and by determining the biological potentialities of the other parts of

the plant like leaves and stems, more sustainable way of conserving this vulnerable

species can be introduced mostly in the places where it is endemic and utilized and also

where the exploitation of this species is high. This study will help the future

researchers to concentrated on the relationship between the endophytic and non-

endophytic fungi and the Aquilaria malaccensis plant. The metabolomic and

metabonomic studies can be the future perspectives which can be taken up in the

studies of agarwood formation.

43

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48

Appendix 1: Abbreviations Used

mg Milligram

μg Microgram

μg/mL Microgram per milli litre

g Gram

kgs Kilograms

°C Degree celcius

mm Milli meter

cm Centimeters

LA Luria Agar

PDA Potato Dextrose Agar

UNF Unidentified fungus