Isolation of Chitinolytic Bacteria Used as Biological Control of Suspected Pathogenic Fungi on Oil palm Seedlings

ISOLATION OF CHITINOLYTIC BACTERIA USED AS
BIOLOGICAL CONTROL OF SUSPECTED PATHOGENIC
FUNGI ON OIL PALM SEEDLINGS

AGUSTINUS HARYANTO

DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCE
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2013

STATEMENT ABOUT UNDERGRADUATE THESIS,
INFORMATION SOURCES, AND ACT OF SPILLING OVER
COPYRIGHTS
By this writing I clarify that the undergraduate thesis Isolation of
Chitinolytic Bacteria Used as Biological Control of Suspected Pathogenic Fungi
on Oil Palm Seedlings is my own work under the supervisions of the advising
committee and hasn’t been proposed for any institution. Copied information
source of published and unpublished writing of other author has been mentioned
in the text and incorporated in the references at the last part of this thesis.

By this writing I hand over the copyright of my undergraduate thesis to
Bogor Agricultural University.
Bogor, November 2013
Agustinus Haryanto
NIM G34090116

ABSTRACT
AGUSTINUS HARYANTO. Isolation of Chitinolytic Bacteria Used as Biological
Control of Suspected Pathogenic Fungi on Oil Palm Seedlings. Supervised by
NISA RACHMANIA MUBARIK and SRI LISTIYOWATI.
Oil palm (Elaeis guineensis Jacq.) is one of the main plantation
commodities in Indonesia. Production of oil palm is influenced by several
environmental condition such as rainfall, soil, climate, and pathogen outbreak.
Pathogen outbreak such as anthracnose and leaf blight cause rotting on leaves of
oil palm seedling which in the end will kill the plant. Prevention on pathogen
outbreaks on oil palm which was caused by pathogenic fungi can be done by
using chitinolytic bacteria which can produce chitinase enzyme as biological
control. Chitin is one of the element in cell wall of the fungi. The aims of this
experiments were to isolate chitinolytic bacteria and to investigate under in vitro
test of their potential as biological control of suspected pathogenic fungi on oil

palm. Results showed isolate SAHA12.08, SAHA12.10, and SAHA12.13 had
antagonistic activity to the growth of Curvularia sp. and Colletotrichum sp.
suspected pathogenic whereas isolate KAHN15.12 only had antagonistic activity
to Curvularia suspected pathogenic.
Key words: antagonistic activity, chitinolytic, pathogenic fungi, oil palm

ABSTRAK
AGUSTINUS HARYANTO. Isolasi Bakteri Kitinolitik yang Berpotensi sebagai
Biokontrol terhadap Cendawan yang Diduga Patogen pada Bibit Kelapa Sawit.
Dibimbing oleh NISA RACHMANIA MUBARIK dan SRI LISTIYOWATI.
Kelapa sawit (Elaeis guineensis Jacq.) merupakan salah satu komoditas
perkebunan utama di Indonesia. Produksi kelapa sawit sangat dipengaruhi oleh
berbagai faktor lingkungan seperti curah hujan, tanah, iklim, dan serangan
penyakit. Serangan penyakit seperti antraknosa dan bercak daun menyebabkan
pembusukan pada daun kelapa sawit saat pembibitan yang pada akhirnya akan
membunuh tanaman kelapa sawit tersebut. Upaya pencegahan serangan penyakit
pada kelapa sawit yang disebabkan oleh cendawan patogen dapat dilakukan
dengan penggunakan bakteri kitinolitik yang mampu menghasilkan enzim kitinase
sebagai pengendali biologi. Kitin merupakan salah satu komponen penyusun
dinding sel dari cendawan. Penelitian ini bertujuan untuk mengisolasi bakteri

kitinolitik dan menguji potensinya sebagai pengendali hayati dalam keadaan in
vitro terhadap cendawan patogen pada kelapa sawit. Hasil menunjukkan isolat
SAHA12.08, SAHA12.10, dan SAHA12.13 memiliki aktivitas antagonis terhadap
pertumbuhan cendawan yang diduga patogen Curvularia sp. dan Colletotrichum
sp., sedangkan isolat KAHN15.12 hanya memiliki aktivitas antagonis terhadap
pertumbuhan Curvularia sp. yang diduga patogen.
Kata kunci: aktivitas antagonis, cendawan patogen, kelapa sawit, kitinolitik

ISOLATION OF CHITINOLYTIC BACTERIA USED AS
BIOLOGICAL CONTROL OF SUSPECTED PATHOGENIC
FUNGI ON OIL PALM SEEDLINGS

AGUSTINUS HARYANTO

An Undergraduate Thesis
Intended to Acquire Bachelor Degree
In Faculty of Mathematics and Natural Science

DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCE

BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2013

Thesis Title: Isolation of Chitinolytic Bacteria Used as Biological Control of
Suspected Pathogenic Fungi on Oil palm Seedlings
Agustinus Haryanto
Name
NIM
G34090116

Approved by

ｾ＠

Dr Nisa Rachmania Mubarik, MSi
Supervisor I

Dr Sri Listiyowati, MSi
Supervisor II

Head of Department Biology

Pass Date: : i 9

CJ 2913

Thesis Title: Isolation of Chitinolytic Bacteria Used as Biological Control of
Suspected Pathogenic Fungi on Oil palm Seedlings
Name
: Agustinus Haryanto
NIM
: G34090116

Approved by

Dr Nisa Rachmania Mubarik, MSi
Supervisor I

Dr Sri Listiyowati, MSi

Supervisor II

Acknowledged by

Dr Ir Iman Rusmana, MSi
Head of Department Biology

Pass Date:

FOREWORDS
First of all, I would like to give thanks to Jesus Christ for all blessing I’ve
got. This research is made through an experiment entitled Isolation of Chitinolytic
Bacteria Used as Biological Control of Suspected Pathogenic Fungi on Oil Palm
Seedlings which was conducted from January until July 2013 on IPB. This study
was funded by Start Up Funding Collaborative Research Centre (CRC) 990 in
2012 and Directorate of Higher Education, Ministry of National Education
Republic of Indonesia through Penelitian Unggulan Perguruan Tinggi Institut
Pertanian Bogor in 2013 to Nisa Rachmania Mubarik.
Biggest acknowledgment is given to Dr Nisa Rachmania Mubarik, MSi and
Dr Sri Listiyowati, MSi as my supervisor for the advice and supervisions.

Acknowledgment is also given to name as Dr Achmad Farajallah, MSi for the
advice and discussion. Special acknowledgment is given to my families and my
friends for all their love and support.
At last, I hope this research will be helpful for all the readers.

Bogor, September 2013
Agustinus Haryanto

TABLE OF CONTENTS
LIST OF TABLES

vi

LIST OF FIGURES

vi

LIST OF APPENDIXES

vi

INTRODUCTION

1

Background

1

Aims

1

MATERIALS AND METHOD

1

Time and Place

1

Materials

2

Isolation and Identification of Chitinolytic Bacteria

2

Isolation and Identification of Leaf Spot Fungi from Oil Palm

2

In vitro Screening of Chitinolytic Bacteria against Suspected Pathogenic
Fungi

2

Identification of Chitinolytic Bacteria which has Antagonistic Activity

3

Bacterial Growth Curve from Selected Isolates and Antagonistic Activity

3

RESULTS

3

Isolation and Identification of Chitinolytic Bacteria

3

Isolation and Identification of Leaf Spot Fungi from Oil Palm

4

In vitro Screening of Chitinolytic Bacteria against Suspected Pathogenic
Fungi

5

Identification of Chitinolytic Bacteria which has Antagonistic Activity

6

Bacterial Growth Curve from Selected Isolates and Antagonistic Activity

7

DISCUSSION
CONCLUSION AND SUGGESTION

8
11

Conclusion

11

Suggestion

11

REFERENCES

11

APPENDIXES

16

CURRICULUM VITAE

17

LIST OF TABLES
1 Identification results of chitinolytic isolates
2 Antagonistic activity of ten highest chitinolytic index (CI) isolates
3 Antagonistic properties on suspected pathogenic fungi

4
6
6

LIST OF FIGURES
1
2
3
4
5

Symptoms of sick leaves caused by leaf spot fungi of oil palm
Cell morphology of suspected pathogenic fungi from oil palm. (a)
Curvularia sp. (b) Colletotrichum sp.
Colony morphology of suspected pathogenic fungi from oil palm. (a)
Curvularia sp. (b) Colletotrichum sp.
Growth curve of isolate SAHA12.08 and its antagonistic activity
toward Curvularia sp.
Growth curve of isolate SAHA12.08 and its antagonistic activity
toward Colletotrichum sp.

5
5
5
7
8

LIST OF APPENDIXES
1 Soil samples data from Taman Nasional Bukit Dua Belas (Jambi)
2 Isolates origin from soil samples data
3 Biochemical characteristics of isolates SAHA12.08 and SAHA12.13
using API 50 CH

14
15
16

1

INTRODUCTION
Background
Production of oil palm products increased approximately from 167.000
tones in 1967 to 18 million tones in 2009, or almost 107 times within 42 years.
Areas of oil palms have increased from 105.000 hectares in 1967 to 7.8 million
hectares (Ditjenbun 2009). Some of pathogens Botryodiplodia palmarum,
Melanconium sp., Glomerella cingulata and Curvularia eragrostidis which are
commonly found at Southeast Asia greatly damaged oil palm seedling
(Aderungboye 1977). Therefore, by controlling the pathogen outbreaks, it will
lead to increase in oil palm production itself.
Biological control using microorganism has been studied intensively since it
means as an available environmental friendly alternative. Introduction of
chitinolytic bacteria as antagonist agents is to control pathogenic fungi. As stated
in many previous reports, the production of chitinase enzyme was related to
fungal growth inhibition and the biological control of fungal pathogen was
possible because of the ability of the chitinolytic bacteria to degrade fungal cell
walls (Kamil et al. 2007; Suryanto et al. 2010; Gomaa 2012). Chitinolytic bacteria
is capable to inhibit fungal activity because it can produce chitinase to degrade
chitin which is one of the element in cell wall in the fungi (Peter 2005).
Application of chitinolytic products from bacteria is one of environmental friendly
alternatives which is safer to control pathogenic fungi than using synthetic
fungicide. While using chitin colloidal as common substrate for chitinase enzyme
inducer to screen chitinolytic bacteria, it probably lead to degradation of chitin
structures of other chitin substrate in its application which has the same chitin
structures with chitin colloidal (Haran and Chet 1995). Therefore, this study need
further investigation in its application on the field.

Aims
The aims of this research were to isolate chitinolytic bacteria and to
investigate under in vitro test of their potential as biology control of suspected
pathogenic fungi on oil palm seedlings.

MATERIALS AND METHOD
Time and Place
The research was held on January until July 2013 in Microbiology
Laboratory, Department of Biology, Faculty of Mathematics and Natural Science,
Bogor Agricultural University, Bogor and Indonesia Biotechnology Research
Institute for Estate Crops, Bogor.

2

Materials
Soil samples were obtained from Taman Nasional Bukit Dua Belas, Jambi
(Appendix 1). Leaf spot fungi were obtained from infected leaves of oil palm
from Indonesia Biotechnology Research Institute for Estate Crops, Bogor.
Isolation and Identification of Chitinolytic Bacteria
In total of five plots were used in chitinolytic bacteria screening with twice
repetition from each plot. Every 3.0 g of soil sample is diluted in 30 mL of
nutrient broth (NB) with 1% chitin colloidal and it was incubated at room
temperature for 24 hours. Then, all cultures were done with serial dilution from
10-6 to 10-8 in NaCl 0.85 %. Suspension was spreaded on chitin agar (1% chitin
colloidal, 0.1% MgSO4·7H2O, 0.02% K2HPO4, 0.1% yeast extract, and 1.5%
agar) which was incubated at 37oC for 48 hours. Each colony of different bacteria
is streaked on the new agar chitin until single colony of bacteria was found.
Cultures were incubated at 37oC for 48 hours. Isolates were observed based on
chitinolytic index (CI) which was hinted by clear zone. Every chitinolytic was
identified by using Gram staining.
Isolation and Identification of Leaf Spot Fungi from Oil Palm
Isolation was began with preparation of infected leaves. Every three spots
on infected leaves was cut into 1x1 cm covering half healthy leaf and half sick
leaf. The leaves were rinsed with flowing water and then it was soaked into
sodium hypochlorite (NaClO) 1% for a minute. The leaves then were rinsed with
sterilized water and it was dried with sterilized tissue. Finally, the leaves were put
on potato dextrose agar (PDA) containing chloramphenicol 0.5 %. Fungal
cultures were incubated at room temperature for a week. Every hyphae were
reinoculated into new PDA to get pure cultures. Cultures were identified by its
morphological characteristic.
In vitro Screening of Chitinolytic Bacteria against Suspected Pathogenic
Fungi
The bacterial isolates were screened for their antagonistic activity against
suspected pathogenic fungi in vitro based on percentage inhibition of radial
growth (Fokkema 1973). To evaluate the antagonistic activity of the chitinolytic
bacteria isolates, a loopful of the bacterial isolates was streaked 2 cm from the
margin of PDA plates (9 cm diameter) and 4 cm long. Opposite the bacterial
isolates, at a distance of at least 3 cm, suspected pathogenic fungi was placed.
After incubation for 6 days at room temperature, inhibition of the pathogen’s
development was assessed by the percentage of inhibition of radial growth [100%
x (r1-r2)/r1). R1 is length of radial growth towards plate margin (4 cm) and R2 is
length of radial growth towards antagonistic (length of inhibited hyphae). This
procedure was repeated two times.

3

Identification of Chitinolytic Bacteria which has Antagonistic Activity
Isolates which had antagonistic activity to all suspected pathogenic fungi
were identified by using kit BioMérieux, USA. Isolates with antagonistic activity
were streaked on chitin agar. Cultures were incubated at 37oC for 24 hours.
Cultures then were stained with Gram staining for verification. Three loopful of
the cultures of each isolates were diluted into kit API 50 CHB/E medium. In
amount of 200 µl kit API 50 CHB/E medium was filled into the tube. In addition,
the first tube was filled by kit API 50 CHB/E medium which had not been
inoculated by isolates as control. Result which was showed was interpretated by
using the apiweb identification software with the database (V4.0).
Bacterial Growth Curve from Selected Isolates and Antagonistic Activity
Inoculation of 2-4 loopful of selected isolates into 50 ml NB with 1% chitin
colloidal as enzyme production medium which was incubated at 37oC for 15 hours.
In amount of 1 ml culture was inoculated into 100 ml NB with 1% chitin colloidal.
Enzyme production medium was incubated on incubator shaker at 120 rpm at
37oC. Optical density (λ = 600 nm) and antagonistic activity of the culture were
measured every 12 hours. Antagonistic activity was tested by using culture and
crude chitinase of the isolate with agar well diffusion method. Supernatant (crude
chitinase) which contains extracellular metabolites was obtained by centrifugation
on 12.000 rpm (Centrifuge MiniSpin with rotor F-45-12-11) for five minutes.
Well which was containing 100 µl culture or crude chitinase was made 2 cm from
margin of PDA plate. Opposite the well, at a distance of at least 3 cm, suspected
pathogenic fungi was inoculated. After incubation for 6 days at room temperature,
inhibition of the pathogen’s growth was assessed by the percentage of inhibition
of radial growth [100% x (r1-r2)/r1). R1 is length of radial growth towards plate
margin (4 cm) and R2 is length of radial growth towards antagonistic (Fokkema
1983).

RESULTS
Isolation and Identification of Chitinolytic Bacteria
Among soil samples from five plots, there were 28 isolates which had been
successfully isolated and identified. Those isolates were only obtained from four
plots, despites of five plots were used in chitinolytic bacteria screening (Appendix
2). There were 14 isolates which were isolated from soil around oil palms
whereas the other 14 isolates were isolated from soil around rubber tree. Isolates
were identified with Gram staining (Table 1). Results showed that chitinolytic
index was ranged from 0.04 to 3.40. The isolate SAHA12.08 had the highest
chitinolytic index (CI) in amount 3.40 while isolate KAHN10.05 had the lowest
chitinolytic index in amount 0.04 (Table 1).

4

Plantation

Oil Palms

Rubber
Tree

Table 1 Identification results of chitinolytic isolates
Total
Chitinolytic
Isolates Code
Gram
Isolate
Index
SAHA3.01
+
0.33
SAHA3.02
+
0.15
SAHA12.04
+
0.53
SAHA12.05
+
0.25
SAHA12.06
+
1.27
SAHA12.07
+
0.29
SAHA12.08
+
3.40
14
SAHA12.09
+
0.25
SAHA12.10
+
0.40
SAHA12.11
+
1.00
SAHA12.12
+
0.29
SAHA12.13
+
0.71
SAHA12.14
0.20
SAHA12.15
+
0.50
KAHA7.01
+
0.33
KAHA7.02
+
0.21
KAHA7.03
+
0.22
KAHN10.01
0.08
KAHN10.02
+
0.16
KAHN10.03
+
0.20
KAHN10.04
+
0.11
14
KAHN10.05
+
0.04
KAHN10.06
+
0.07
+
0.25
KAHN10.07
KAHN10.08
+
0.18
KAHN13.09
+
0.13
KAHN13.10
+
1.67
KAHN15.12
+
0.76

Isolation and Identification of Leaf Spot Fungi from Oil Palm
Two suspected pathogenic fungi were obtained from sick leaves of oil palm
seedlings at Indonesia Biotechnology Research Institute for Estate Crops, Bogor.
The symptom for infected leaves include small lesions which turn to varying
shades of yellow, gray, reddish-brown, brown, or black (Figure 1). Identification
results through morphology characteristics showed that the isolates are most likely
belonging to Curvularia sp. and Colletotrichum sp. (Figure 2). The morphology of
Curvularia sp. colony was white and turned to black as the colony mature. While
the morphology of Colletotrichum sp. colony was snow white colored which
turned to grey eventually (Figure 3).

5

Figure 1 Symptoms of sick leaves caused by leaf spot fungi of oil palm

a

b

Figure 2 Cell morphology of suspected pathogenic fungi from oil palm (a)
Curvularia sp. (b) Colletotrichum sp.

a

b

Figure 3 Colony morphology of suspected pathogenic fungi from oil palm (a)
Curvularia sp. (b) Colletotrichum sp.

In vitro Screening of Chitinolytic Bacteria against Suspected Pathogenic
Fungi
Ten chitinolytic bacteria with highest chitinolytic index were antagonistic
tested against two isolates suspected pathogenic fungi. The result showed isolate
SAHA12.08, SAHA12.10, and SAHA12.13 were able to inhibit the growth of two
suspected pathogenic fungus at once (Colletotrichum sp. and Curvularia sp.)
whereas KAHN15.12 was only able to inhibit the growth of Curvularia sp.
suspected pathogenic (Table 2).
Isolate SAHA12.13 showed highest inhibition to Curvularia sp. in 58.75%
and Colletotrichum sp. in 52.50% while isolate SAHA12.08 showed the lowest
inhibition to Curvularia sp. in 36.25% and Colletotrichum sp. in 16.82% (Table 3).

6

The result showed that isolate SAHA12.08 which had the highest chitinolytic
index did not show stronger inhibition to all suspected pathogenic fungi than
isolate SAHA12.13 which had chitinolytic index lower than SAHA12.08.
Table 2 Antagonistic activity of ten highest chitinolytic index (CI) isolates
Antagonistic Activity
Isolates Code

CI
Curvularia sp.

Colletotrichum sp.

SAHA12.08

3.40

+

+

KAHN13.10

1.67

-

-

SAHA12.06

1.27

-

-

SAHA12.11

1.00

-

-

KAHN15.12

0.76

+

-

SAHA12.13

0.71

+

+

SAHA12.04

0.53

-

-

SAHA12.15

0.50

-

-

SAHA12.10
SAHA3.01

0.40
0.33

+
-

+
-

+ antagonistic activity

- no antagonistic activity

Table 3 Antagonistic properties on suspected pathogenic fungi
Suspected
Inhibition on Radial
Isolates Code
Pathogenic Fungi
Growth (%)
SAHA12.08
36.25
SAHA12.10
57.50
Curvularia sp.
SAHA12.13
58.75
KAHN15.12
41.25
SAHA12.08
16.82
Colletotrichum sp. SAHA12.10
46.97
SAHA12.13
52.50
Identification of Chitinolytic Bacteria which has Antagonistic Activity
Two isolates of higher chitinolytic index which had antagonistic activity
towards two suspected pathogenic fungi were isolate SAHA12.08 and
SAHA12.13. The isolates were identified by using kit BioMérieux (API 50 CH,
USA) which identification was based on biochemical characteristics (Appendix 3).
Results showed that isolate SAHA12.08 had similarities to Geobacillus
thermoglocosidasius in amount of 99.9% while isolate SAHA12.13 had
similarities to Bacillus cereus in amount of 82.7%.

7

Bacterial Growth Curve from Selected Isolates and Antagonistic Activity

5
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0

45,00
40,00
35,00
30,00
25,00
20,00
15,00
10,00
5,00

Antagonistic Activity (%)

Log of Number of Bacteria

Two isolates of higher chitinolytic index with antagonistic activity to all
suspected pathogenic fungi were measured its growth as well as antagonistic
activity. Those isolates were SAHA12.08 and SAHA12.13. The results showed
that isolate SAHA12.08 probably ended exponential phase at 24 hours. Stationary
phase of isolated SAHA12.08 probably occurred within 24 hours to 60 hours
while death phase was probably reached after 60 hours. Antagonistic activity of
culture to Curvularia sp. showed that the highest inhibition of 38.75% occurred at
24 hours while decreasing occurred after 24 hours (Figure 4), whereas
antagonistic activity of the culture to Colletotrichum sp. showed that the highest
inhibition of 26.97% occurred at 12 hours while decreasing occurred after 12
hours (Figure 5). Antagonistic activity of crude chitinase to Curvularia sp.
showed that the highest inhibition of 33.75% occurred at 24 hours while
decreasing occurred after 24 hours, but on 48 hours highest inhibition occurred
again in 33.75% of inhibition and decreasing occurred past 48 hours.
Antagonistic activity of crude chitinase to Colletotrichum sp. showed that the
highest inhibition of 26.67% occurred at 12 hours while decreasing occurred past
12 hours. Results also showed that antagonistic test with culture had stronger
antagonistic activity than crude chitinase (Figure 4 and 5). Isolate SAHA12.13
was also observed its growth and antagonistic activity through culture and crude
chitinase, however this isolate showed no activity at all to inhibit suspected
pathogenic fungi.

0,00
0

12

24

36

48

60

72

Incubation Time (hour)
Logofofnumbers
numbersofofbacteria
bacteria
Log

Antagonistic activity
Culture/Curvularia
sp. of Culture

Crude
Chitinase/Curvularia
sp. Chitinase
Antagonistic
activity of Crude

Figure 4 Growth curve of isolate SAHA12.08 and its antagonistic activity toward
Curvularia sp.

8

5

30,00
25,00

4
3,5

20,00

3
2,5

15,00

2
10,00

1,5
1

5,00

Antagonistic Activity (%)

Log of Number of Bacteria

4,5

0,5
0

0,00
0

12

24

36

48

60

72

Incubation Time (hour)
Log
Logofofnumbers
numbersofofbacteria
bacteria

Culture/Botryodiplodia
sp.Culture
Antagonistic activity of

Crude
Chitinase/Botryodiplodia
Antagonistic
activity of Crude sp.
Chitinase

Figure 5 Growth curve of isolate SAHA12.08 and its antagonistic activity toward
Colletotrichum sp.

DISCUSSION
An amount of 28 isolates of chitinolytic bacteria were successfully isolated
by using chitin agar containing chitin colloidal. Chitinolytic isolates can be
detected through clear zone around bacterial colony. Low numbers of isolates
which were isolated were caused by using enrichment and dilution techniques.
Enrichment and dilution techniques were only capable in selecting those
microorganism that are numerically abundant or those microorganism that show
superior growth in a given medium but not the actual number of bacteria which
were found in the sample (Jackson et al 1998). From 28 isolates of chitinolytic
bacteria, isolate SAHA12.08 showed highest chitinolytic index in amount of 3.40.
Chitinolytic index of isolate SAHA12.08 (3.40) was higher than the average of
chitinolytic index of isolates from chili plant roots which was 1.00
(Nurdebyandaru et al. 2008). Isolate SAHA12.08 also showed higher chitinolytic
index than the highest chitinolytic index of isolates from rubber tree which was
0.52 (Muharni and Widjajanti 2011).
In amount of 10 highest chitinolytic index isolates were used in antagonistic
test against suspected pathogenic fungi. There were two suspected pathogenic
fungi which were successfully isolated. One of them was Curvularia sp. which
showed morphological characteristics including white to pinkish gray initial
colony which turns to olive brown or black as the colony matures, brown hyphae,
septate hyphae, and brown conidiophores which are simple or branched. Most
species of Curvularia are facultative pathogens of plants in tropical or subtropical

9

areas, while the remaining few are found in temperate zones (Larone 1995).
Another suspected pathogenic fungus which was successfully isolated was
Colletotrichum sp. It had sparse, cottony, white to pale gray colored colony.
Spores are formed in acervuli, erumpent, cushion like masses of hyphae bearing
conidiophores, hyaline, and oblong to fusoid conidia (Horst 2008). While
Colletotrichum species cause serious plant disease that devastate crop plants
worldwide, they also commonly isolated as endophytes from healthy plants and as
saprobes on dead plant material (Photita et al 2005). The majority of the fungi
including Curvularia sp. and Colletotrichum sp. have chitin but not cellulose in
their cell walls, therefore its cell wall could be degraded by chitinolytic bacteria
(Landecker 1996).
From 10 of highest chitinolytic index isolates, only 4 isolates showed
antagonistic activity. Those isolates were SAHA12.08, SAHA12.10, SAHA12.13,
and KAHN15.12. Isolate SAHA12.13 showed strongest inhibition towards
suspected pathogenic fungus which was indicated by 58.75% of inhibition of
radial growth to Curvularia sp. and 52.50% of inhibition of radial growth to
Colletotrichum sp. Isolate SAHA12.08 and SAHA12.10 were also able to inhibit
the growth of Curvularia sp. and Colletotrichum sp. however its antagonistic
activity were not as strong as isolate SAHA12.13. Isolate SAHA12.10 showed
inhibition of radial growth to Cuvularia sp. in amount of 57.50% and 46.97% of
inhibition to the growth of Colletotrichum sp. Inhibition of radial growth to
Curvularia sp. and Colletotrichum sp. by isolate SAHA12.10 was slightly lower
than SAHA12.13, however SAHA12.10 showed low chitinolytic index and
therefore this isolate was not tested any further. KAHN15.12 were only able to
inhibit the growth of Curvularia sp. Variation in inhibition maybe was caused by
specification enzyme to the species, differences chitinase activity, and chitin
composition in fungi cell wall, also existence of secondary metabolites. The cell
wall of fungi generally consist of not only chitin but also another type of sugars.
For example, β-1,3 glucan which was bound to chitin. In that case, there are more
than one enzyme which are responsible in degradation of cell wall (Anand and
Reddy 2009).
Isolate KAHN13.10 showed the second highest chitinolytic index but
surprisingly isolate KAHN13.10 was not able to inhibit suspected pathogenic
fungi. On the other hand isolate SAHA12.10 and SAHA12.13 showed lower
chitinolytic index than isolate KAHN13.10, but they were able to inhibit the
growth of suspected pathogenic fungi especially isolate SAHA12.13 which had
strongest inhibition to suspected pathogenic fungi.This results showed that
compatibility of enzyme to the substrate is very essential in playing antagonistic
activity as well as antifungal properties (Gohel et al. 2006).
Optimum phase of isolate SAHA12.08 to inhibit the growth of suspected
pathogenic fungi which occurred at 0 hour to 24 hours probably was caused by
high level of chitinase extracellular production on exponential phase. It has been
reported that extracellular chitinase production was significantly increased during
the exponential phase and dramatically declined when the cells entered the the
stationary phase (Prapagdee et al. 2008). Isolate SAHA12.08 was still able to
inhibit Curvularia sp. on stationary phase probably due to secondary metabolites
which were produce on stationary phase, although concentration of hydrolytic
enzyme had decreased. Somehow, SAHA12.08 was not able to inhibit

10

Colletotrichum sp. past 12 hours. This might be caused by the decreasing
production of hydrolytic enzymes and incapability of secondary metabolites
which were produced on stationary phase to inhibit Colletotrichum sp.
The antagonistic test by using supernatant (cell-free-filtrate) which contains
extracellular metabolites was aimed to observe whether hydrolytic enzyme such
as chitinase was responsible in inhibiting suspected pathogenic fungal growth.
The result showed that the filtrate was able to inhibit the growth of suspected
fungi, although the inhibition was lower than inhibition by culture. Differences in
ability to inhibit might be caused by differences in concentration of hydrolytic
enzyme or even secondary metabolites on the filtrate (Prapagdee et al. 2008).
Multiple chitinases as antagonistic properties also contribute important role in
antagonistic activity. It is believed that multiple chitinases within a single
organisms lead to a more efficient use of the respective substrate of chitin as a
result of synergistic enzyme interactions. Therefore, it leads to a more efficient
antagonistic activity (Svitil et al. 1997). There are many types of bacterial
chitinase which had been reported such chitinase A, chitinase A1, chitinase B,
chitinase C, chitinase D, exochitinase, etc. Those chitinase are grouped in two
different families which is families 18 and 19 of the classification of glycoside
hydrolases based on amino acid sequence similarities (Henrissat 1999). In
addition, another factor that affect the ability of the isolates to inhibit the growth
of pathogenic fungi probably caused by the other bioactive compounds which
were produced by the isolate other than extracellular chitinase. In order to prove
either extracellular chitinase or other bioactive compounds which were
responsible in antagonistic activity to pathogenic fungi, this study needs further
investigation.
Identification using kit API 50 CH (BioMérieux, USA) showed isolate
SAHA12.08 was most likely Geobacillus thermoglucosidasius, whereas isolate
SAHA12.13 was most likely Bacillus cereus. Isolate SAHA12.08 was Grampositive and rod shaped bacteria which was isolated from top soil around palm
tree. Geobacillus thermoglucosidasius which was identified from SAHA12.08 is
rod-shaped bacteria, occurring either singly or in short chains and motile by
means of peritrichous flagella. The cell wall structure is Gram-positive, but the
Gram-stain reaction may vary between positive and negative. It is aerobic or
facultatively anaerobic bacteria and obligately thermophilic (Nazina et al. 2001).
Identification using API 50 CHB system turned out to be inapplicable for isolate
SAHA12.08 because discrepancies in the reaction patterns resulted in differing
identifications. This misidentified was caused by limited database for the species,
therefore the use of genomic methods such as 16S rRNA sequencing may be
preferable (Boyd et al. 2005).
Identification through Gram staining showed isolate SAHA12.13 was
Gram-positive and rod shaped bacteria. Bacillus cereus is group of ubiquitous
facultative anaerobic, spore forming, and Gram-positive rods commonly found in
soil (Tallent et al. 2012). Identification using kit API 50 CH on isolate
SAHA12.13 and Gram-staining showed compatible results to the profile of
Bacillus cereus. According to Huang et al. (2005), Bacillus cereus which was
isolated from lily plant in Taiwan had the ability to produce chitinase as well as
the ability to inhibit the growth of several pathogenic fungi. This bacteria
produced at least two types of chitinases (ChiCW and ChiCH) which acted as

11

antifungal properties. Prokaryotes which were commonly found be able to
degrade chitin were Pseudomonas, Vibrio, Photobacterium, Actinomycetes,
Bacillus, Clostridium, and Enterobacter (Gooday 1990).

CONCLUSION AND SUGGESTION
Conclusion
An amount of 28 chitinolytic isolates was successfully isolated. Isolate
SAHA12.08, SAHA12.10, and SAHA12.13 were able to inhibit the growth of
Curvularia sp. and Colletotrichum sp. Whereas, isolate KAHN15.12 was only
able to inhibit Curvularia sp. Isolate SAHA12.08 showed optimally antagonistic
activity to all suspected pathogenic fungi at exponential phase. Identification of
isolate SAHA12.08 which was most likely Geobacillus thermoglucosidasius by
API 50 CHB turned out to be inapplicable. Identification of isolate SAHA12.13
by using API 50 CHB which was Bacillus cereus showed acceptable result.

Suggestion
There are some suggestions about this research. First, suspected pathogenic
fungi should be explored for its actual pathogenic ability on oil palm. Exploration
of the other abilities of all chitinolytic isolates also needs to be investigated. Last,
characterization of chitinase molecule and also molecular identification of
chitinolytic isolates and suspected pathogenic fungi isolates should be researched
in the future.

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14

Appendix 1 Soil samples data from Taman Nasional Bukit Dua Belas (Jambi)
Plot
Subplot
Soil Code
Plantation
A1
FG01
A2
HI01
A3
1
DE45
N1
Oil Palm
HI23
N2
AA01
N3
HI23
N4
JJ45
A4
JJ67
A5
B02
A6
2
Oil Palm
JJ89
N5
JJ67
N6
FG07
N7
BC67
A7
DE67
A8
BR26
A9
FG45
A10
FG67
A11
3
Rubber Tree
AA45
N8
BC45
N9
DE45
N10
HI23
N11
HI01
N12
BC23
A12
BO43
A13
4
Oil Palm
BC67
N13
FG23
N14
DE89
A14
BC89
A15
FG10
A16
5
Rubber Tree
BR48
N15
FG45
N16
JJ67
N17

15

Appendix 2 Isolates origin from soil samples data
Plot
Soil Code
Isolates Code
SAHA3.01
A3
1
SAHA3.02
N1
A6
2
N5
KAHA7.01
A7
KAHA7.02
KAHA7.03
KAHN10.01
KAHN10.02
3
KAHN10.03
KAHN10.04
N10
KAHN10.05
KAHN10.06
KAHN10.07
KAHN10.08
SAHA12.04
SAHA12.05
SAHA12.06
SAHA12.07
SAHA12.08
SAHA12.09
A12
SAHA12.10
4
SAHA12.11
SAHA12.12
SAHA12.13
SAHA12.14
SAHA12.15
KAHN13.09
N13
KAHN13.10
A14
5
N15
KAHN15.12

16

Appendix 3 Biochemical characteristics of isolates SAHA12.08 and SAHA12.13
using API 50 CH
Test
Glycerol
Erythritol
D-Arabinose
L-Arabinose
Ribose
D-Xylose
L-Xylose
Adonitol
Β-Methyl – D-Xyloside
Galactose
Glucose
Fructose
Mannose
Sorbose
Rhamnose
Dulcitol
Inositol
Mannitol
Sorbitol
a-Methyl-D-Mannoside
a-Methyl-D-Glucoside
N-Acetyl Glucosamine
Amygdalin
Arbutin
Esculin
Salicin
Cellobiose
Maltose
Lactose
Melibiose
Sucrose
Trehalose
Inulin
Melezitose
Raffinose
Starch
Glycogen
Xylitol
Gentiobiose
Turanose
D-Lyxose
D-Tagatose
D-Fucose
L-Fucosel
D-Arabitol
L-Arabitol
Gluconate
2-Keto Gluconate
5-Keto Gluconate

SAHA12.08
+
+
+
+
+
+
+
+
+
+
+
+
-

SAHA12.13
+
+
+
+
+
+
+
+
-

APPENDIXES

17

CURRICULUM VITAE
Author was born on 12th of August 1992 as the first of three from the
parents Dany Haryanto and Tjhi Djam Mi. In 2009, author graduated from SMA
Kristen Yusuf and entered the Department of Biology, Faculty of Mathematics
and Natural Science, Bogor Agricultural University by SNMPTN selection. In
2011, author conducted the Field Study with the research title Isolation and
Identification of Potential Bacteria From Soil Around the Roots of Legumes in
Gunung Walat University Forest and in 2012, author conducted the Field Work at
Kilang Minyak Kelapa Sejahtera with the title Management of Quality Control in
the Processing of Copra into Low Grade Coconut Oil. The author also became
laboratory assistance for Prokaryotes Physiology on year study 2011-2012. On
August 19th 2013, author has participated in poster session with poster title
Isolation of Chitinolytic Bacteria used as Biological Control of Pathogenic Fungi
on Oil Palm at IGN-TTRC The 2nd International Student Conference about
Biodiversity and Biotechnology for Human Life, Airlangga University, Surabaya.