Faculty of Resource Science and Technology

PROFILING OF MICROBES WITH PLANT-GROWTH PROMOTING TRAITS

FROM SAGO HUMUS

Ng Ping Ping

Bachelor of Science with Honours

(Resource Biotechnology)

Year 2009

ii

Profiling of Microbes with Plant-Growth Promoting Traits from Sago Humus

Ng Ping Ping

This project is submitted in partial fulfillment of the requirement for

the degree of Bachelor of Science with Honours

(Resource Biotechnology)

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2009

iii

Profiling of Microbes with Plant-Growth Promoting Traits from Sago Humus

Ng Ping Ping

Resource Biotechnology Program

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Sago hampas undergoes decompositions by microorganisms to become stable and simpler form

organic compounds called sago humus. In this study, screening tests for plant-growth promoting

traits were done on all the isolated bacteria from sago humus collected at Mukah. For bacteria

that show positive result were further identified by using biochemical test, morphology and

molecular methods. From the 43 isolates that had successfully isolated from 5 sago samples, 8

isolates demonstrated positive results for at least one of the 4 screening tests. Isolate 3 and 4 had

ability to produce indole-acetic acid (IAA) and ammonia. Isolate 6 was able to produce hydrogen

cyanide (HCN) and ammonia. Other 5 isolates showed only the ability in production of ammonia.

All the 8 isolates showed positive result for catalase test and there were 3 isolates demonstrated

positive result for oxidase test. Two of the isolates were capable to use citrate as sole source of

carbon. Most of the morphologies of the bacteria were in rod shape and belong to gram-negative.

Bacillus subtilis and Peseudomonas as PGPB had successfully been identified. In this study,

molecular method was considered as direct method in identifying bacteria.

Key words: Sago humus, plant-growth promoting traits, screening tests, PCR

ABSTRAK

Hampas sagu mengalami penguraian oleh mikroorganisma untuk menjadi bentuk campuran

organik yang stabil dan ringkas yang biasanya dipanggil humus sagu. Dalam kajian ini, ujian

penapisan dijalankan untuk memilih bakteria yang mempunyai ciri- ciri merangsang

pertumbuhan tumbuhan daripada humus sagu dipungut dari Mukah. Bakteria yang menunjukkan

keputusan positif kepada ujian tersebut selanjutnya dikenalpasti menggunakan ujian biokimia,

kaedah morfologi dan molekul. Daripada 43 bakteria yang berjaya diasingkan daripada 5

sampel sagu, 8 isolasi menunjukkan sekurang-kurangnya satu keputusan positif daripada 4 ujian

penapisan. Isolasi 3 dan 4 mempunyai keupayaan untuk menghasilkan asid asetik indol (IAA)

dan ammonia. Isolasi 6 berupaya menghasilkan hidrogen sianida dan ammonia. 5 isolasi yang

lain menunjukkan keupayaan dalam penghasilan ammonia sahaja. Kesemua 8 isolasi

menunjukkan keputusan positif untuk ujian katalasi dan 3 isolasi memberi keputusan positif

untuk ujian oksida. 2 isolasi berkemampuan menggunakan sitrat sebagai satu-satunya sumber

karbon. Kebanyakan bakteria adalah berbentuk rod dan bergram negatif. Bacillus subtilis dan

spesies Pseudomonas sebagai PGPB telah berjaya dikenalpasti. Dalam kajian ini, kaedah

molekul adalah sebagai kaedah yang terus untuk mengenal pasti bakteria.

Kata kunci: Humus sagu, ciri-ciri merangsang petumbuhan tumbuhan, ujian penapisan, asid

asetik indol, ammonia

iv

ACKNOWLEDGEMENT

This final year project was successfully produced on date. This is the time to say thank you to

those people who ever guide or help me in my project.

First of all, I would like to take this opportunity to express my sincere thanks to my

supervisor, Dr. Lesley Maurice for her guidance, assistance and encouragement throughout this

project. Besides, I would also like to thank my co-supervisor, Dr. Awang Ahmad Sallehin for his

advice and assistance in lab work.

Not forgetting to dedicate my appreciation to the lab assistance, Encik Azis for his

kindness to help me to search for reagents needed at other laboratories and providing laboratory

equipments for my lab work. I am very thankful for his sincere in answering my questions when

I had problems in something.

Apart from that, special gratitude goes to all the master students in Microbiology lab and

Genetic Engineering lab for their assistance and guidance. My sincere appreciation to my entire

course mate for their companionship and support.

Finally, I am grateful to my family members for their love, care, and morale support

throughout my studies in UNIMAS.

v

TABLE OF CONTENTS

Page

TITLE AND COVER PAGE i

TABLE OF CONTENTS ii

LIST OF TABLES iv

LIST OF FIGURES v

LIST OF ABBREVIATIONS vi

CHAPTER 1 INTRODUCTION

1.1 Introduction 1

1.2 Problem statement/Rationale 4

1.2 Research objectives 4

CHAPTER 2 LITERATURE REVIEW 6

2.1 Sago Palm (Metroxylon sagu) 6

2.2 Uses of sago palm 6

2.3 Sago cultivation in Sarawak 7

2.4 Use of sago waste 8

2.5 Microorganisms inside sago humus with plant-growth 9

promoting traits

2.5.1 Production of plant-growth hormones and nutrients 9

2.5.1.1 Indole-3-acetic acid (IAA) production 9

2.5.1.2 Phosphate-solubilizing bacteria 12

2.5.1.2.1 Mechanism for phosphate 13

solubilization

2.5.1.2.2 Isolation of phosphate 14

solubilizing microorganism

(PSM)

2.5.2 Biocontrol 16

2.5.2.1 Production of HCN 16

vi

CHAPTER 3 MATERIALS AND METHODS 19

3.1 Collection and prosessing of sample 19

3.2 Preparation of overnight bacterial culture 19

3.3 In vitro screening for plant-growth promoting traits in 20

bacterial isolates

3.3.1 Indole acetic-acid (IAA) production 20

3.3.2 Ammonia production 20

3.3.3 HCN production 20

3.3.4 P-solubilization 21

3.4 Conventional method 21

3.4.1 Biochemical test 21

3.4.1.1 Oxidase test 21

3.4.1.2 Catalase test 21

3.4.1.3 Motility test 22

3.4.1.4 Triple-sugar ions & H2O2 Test 22

3.4.1.5 Citrate utilization test 22

3.4.1.6 Methyl red-Voges Prokaeur (MR-VP) test 23

3.4.17 Pseudomonas isolation agar 23

3.5 Morphology of bacterial isolates 23

3.5.1 Gram staining 23

3.6 Molecular identification of bacterial isolates 24

3.6.1 DNA extraction procedures 24

3.6.2 Polymerase chain reaction (PCR) amplification 25

3.6.3 Agarose gel electrophoresis 27

3.6.4 DNA sequencing 27

CHAPTER 4 RESULTS 28

4.1 Screening tests

4.1.1 Production of indole-acetic acid 28

vii

4.1.2 Production of ammonia 28

4.1.3 Production of hydrogen cyanide 30

4.1.4 P-solubilizing 30

4.2 Conventional method 31

4.2.1 Biochemical test 31

4.2.1.1 Oxidase test 31

4.2.1.2 Catalase test 31

4.2.1.3 Motility test 32

4.2.1.4 Triple-sugar ions (TSI) & hydrogen peroxide (H2O2 ) 32

4.2.1.5 Citrate utilization test 33

4.2.1.6 MR-VP test 34

4.2.1.6.1 Methyl red test 34

4.2.1.6.2 Voges-Proskaeur (VP) test 34

4.2.1.7 Pseudomonas isolation agar 35

4.2.2 Morphology of bacterial isolates 35

4.3 DNA sequencing 38

CHAPTER 5 DISCUSSION 41

5.1 Screening test for plant-growth promoting traits 41

5.2 Biochemical test 47

5.3 Molecular characterization 50

CHAPTER 6 CONCLUSION AND RECOMMENDATIONS 53

REFERENCES 55

APPENDICES 62

viii

LIST OF TABLES

Table 2.1 Microbial strains producing organic acid 15

Table 2.2 Pikovskaya medium 16

Table 2.3 Morphological features and plant growth promotion traits of the non-

rhizorbial from Kudzu nodules 18

Table 3.1 Oligonucleotide primers used to target the 16S rRNA gene 26

Table 3.2 Specific PCR reaction of 25.0 μl volume reaction 26

Table 3.3 Specific PCR amplification parameter 26

Table 4.1 The percentage of plant-growth promoting traits showed by 43 isolates 37 Table 4.2 Summary of biochemical characteristics of the eight isolates 38

ix

LIST OF FIGURES

Figure 2.1 Microbial solubilization of phosphate ` 15

Figure 4.1 Positive control (P.aeruginosa and S.marcescens) 28

Figure 4.2 Isolate 3 (Left) and Isolate 5 (Right) 28

Figure 4.3 Positive control (Bacillus amyloliquefaciens) 29

Figure 4.4 Positive results for ammonia production 29

Figure 4.5 Isolate 8 showed a change of yellow on the filter paper soaked 30

with picrate solution

Figure 4.6 The concentrated blue spot 31

Figure 4.7 The bubbling or foaming formed 31

Figure 4.8 A clearly visible straight line shows non-motile bacteria (left). 32

Cloudy media formed shows motile bacteria (right)

Figure 4.9 Alkaline slant-acid butt (red/yellow) for all the three tubes 33

Figure 4.10 Blue slant medium 33

Figure 4.11 Green medium (negative result) 33

Figure 4.12 Left: Positive result (red) Right: Negative result (yellow) 34

Figure 4.13 Left: Red color (positive result) Right: Yellow colour (negative 35

result)

Figure 4.14 Left: Isolate 1 grew on NA Right: morphology isolate 1 36

(coccobacilli, gram negative)

Figure 4.15 Left: Isolate 2 grew on NA Right: morphology isolate 2 36

(coccobacilli, gram negative)

Figure 4.16 Left: Isolate 3 grew on NA Right: morphology of isolate 3 36

(diplobacilli, gram negative)

Figure 4.17 Left: Isolate 4 grew on NA Right: Morphology of isolate 4 36

(diplobacilli, gram negative)

x

Figure 4.18 Left: Isolate 5 grew on NA Right: Morphology of isolate 5 36

(bacilli, gram negative)

Figure 4.19 Left: Isolate 6 grew on NA Right: Morphology of isolate 36

(bacilli, gram negative)

Figure 4.20 Left: Isolate 7 grew on NA Right: Morphology of isolate 7 37

(bacilli, gram negative)

Figure 4.21 Left: Isolate 8 grew on NA Right: Morphology of isolate 8 37

(diplobacilli, gram negative)

Figure 4.22 The PCR product amplified using the forward and reverse (PA 39

and PH) primers separated on a 2% agaraose gel. LaneM: 1kb

ladder (Fermentas) L 1-4 : represent Isolate 3, 4, 6, and 7,

respectively

xi

LIST OF ABBREVIATIONS

ACC 1-amino cyclopropane carboxylic acid

bp Base pairs

CMC Carboxymethylcellulose

DNA Deoxyribonucleic acid

dNTPs Deoxyribonucleotide triphosphates

EtBr Ethidium bromide

ETOH Ethanol

EDTA Ethylenediamine tetra-acetic acid

G Gram

GC-MS Gas-chromatography mass spectrophotometry

h Hour

H2O2 Hydrogen peroxide

HCN Hydrogen cyanide

HPLC High- performance liquid chromatography

IAA Indole acetic acid

L Liter

LB Luria Bertani

kb Kilobase pairs

MR Methyl red

MRVP Methyl red-Voges Prokaeur

MgCl2 Magnesium chloride

M Molarity

Min Minute(s)

mg Miligram

ml Mililiter

mM MiliMolar

µ micro

µg Microgram

µl Microliter

xii

NA Nutrient Agar

NaCl Sodium chloride

Na-OAC Sodium Acetate

NCBI National Center for Biotechnology Information

PCR Polymerase Chain Reaction

PCI Phenol-Chloroform-Isoamyl

Psi Pound(s) per square inch (Ib/ in2)

PSM Phosphate solubilizing microorganisms

PGPB Plant-growth promoting bacteria

PGPR Plant-growth promoting rhizophere

KOH Potassium hydroxide

rDNA Ribosomal deoxyribonucleic acid

rpm Revolution per minute

SCA Simmon citrate agar

SDS Sodium Dodecyl Sulphate

SIM Sulphide ion motility

YEM Yeast Extract Mannitol Agar

sec Second(s)

spp. Species

Taq Thermus aquaticus DNA polymerase

TAE Tris-Acetic acid EDTA electrophoresis buffer

TE Tris-EDTA buffer

TSI Triple-sugar ions

UV Ultraviolet

V Volts

VP Voges-Proskaeur

w/v Weight per volume

% Percent

°C Degree Celcius

1

CHAPTER 1

INTRODUCTION

1.1 Introduction

The sago pith or bark residue disposed after the starch processing is popularly known as

sago hampas. Sago hampas is considered as an unfavorable environmental pollutant due

to its ligno-cellulosic materials and has no demand for other industries, except for the use

as animal feed supplement (Pongsapan et al., 1984). Sago hampas will undergo

decomposition by diverse types of microbes or fungi to convert it into sago humus. Sago

hampas is a starchy fibrous made up of ligno-cellulosic materials that is abundantly

available at the low price in Malaysia. It is one type of waste from sago debarking and

processing. The content of hampas is mainly composing of 66% of starch (Chew & Shim,

1993) and 14% of fibre on a dry weight basis. The decay of sago hampas into sago

humus is starting with the decomposition of starch into simpler sugar and followed by the

time-consuming breakdown mechanism of lignin and cellulose by white-rot fungi

(Wikipedia, 2008). Sago humus is the decomposed or rotten organic materials such as

bark or base that has achieved a stable stage where it has been converted from complex

organic compounds into simpler forms. Stable humus is highly insoluble and has ability

to withstand from further decomposition. The stable humus has excellent physical

structure to remains the humidity in the soil by increasing microporosity. Chemically, it

serve as excellent source of plant nutrient to increase the fertility of soil while it provides

a conducive environment for living organisms in soil to feed and reproduce biologically

(Burns, 1986). Starch as a renewable natural raw material is well-known to be extracted

2

from sago than potato, tapioca, rice, and wheat in Sarawak (Abd-Aziz, 2002). Sago is

preferable by consumer as food supplement as it is tasteless.

There are different kinds of microorganisms grow inside the sago humus. Some of

the microorganisms such as bacteria and fungi are able to use the nutrients inside the sago

humus to survive. They can even carry out certain biological reactions to produce useful

enzymes to degrade materials in sago humus. These bacteria can stimulate plant growth

indirectly and they are called as plant-growth promoting bacteria (PGPB). They have

ability to enrich soil and promote plant growth by carrying out a series of mechanism

(Bashan & de-Bashan, 2005). The continuous supply of inorganic substances such as

ammonia, water, carbon dioxide, and various compounds of nitrate, phosphate, and

calcium from the breakdown of organic matter can ensure the plants to grow (Csuros,

1998). For example, Pseudomonas spp. has ability to produce secondary metabolites such

as hydrogen cyanide which help in disease suppression (Blumer & Haas, 2000).

Plants need complete nutrients types such as vitamins, salts, minerals and proteins

to ensure their healthy growth. Generally, plants get their nutrients directly from soil or

from fertilizer. The lands with the problem of scarcity are usually difficult in supporting

development of healthy plants. Therefore, despite of chemical fertilizers or biofertilizers,

they usually will be supplied to these lands. Apart from that, some microorganisms that

reside in soil are able to produce essential nutrients to compensate the insufficient of

nutrients in soils. The inadequacy of phosphate available in soil for plant usage are due to

the low solubility of general phosphate like Ca3(PO4)2 hydroxyapatite and aluminium

3

phosphate. Thus, with the presence of bacteria that have ability to solubilize insoluble

phosphate, they may help plant growth (Rodriguez and Fraga, 1999).

Hydrogen cyanide found in soil is usually in the form of cyanide. Cyanide is an

alkaline metal salts or immobile metallocyanide complexes. It is more easily to volatilize

and very mobile if the soil condition is with pH less than 9.2 (Koren & Bisesi, n.d.). It

could be recognized as an organic compound as well as inorganic compound with high

toxicity. It can be found in wastewater form chemical producers, coking operations,

electroplating operations, and petrochemical operations (Michael et al., 2006). The

environmental cyanide is produced biologically by cyanogenic plants such as alfalfa,

almonds, peaches, and sorghum and cyanogenic bacteria and fungi under a particular

growth conditions. Besides that, a certain of organotrophic bacteria are capable to

undergo detoxification and assimilation mechanisms to remove cyanide from wastewater,

sludge, and soil (Michael et al., 2006). Ammonia in the form of ammonium ion is the

main necessary inorganic cation since it has a central role in nitrogen metabolism.

Meanwhile, phosphate is the most important inorganic anion as it is needed during

biomass formation such as DNA, RNA and phospholipids (El-Mansi & Bryce, n.d.).

Indole-3-acetic acid (IAA) helps to control the physiological processes of plant such as

enlargement and division, tissue differentiation, and response to light and gravity (Taiz &

Zeiger, 1998).

This project was carried out using four screening tests on plant-growth promoting

traits namely determination of plant-growth hormone production, indole acetic acid

4

(IAA); production of plant-promoting nutrients which are ammonia, and phosphate and

production of pathogen-resistance substances namely hydrogen cyanide (HCN). For

bacterial colonies selected that shown positive result for the screening tests; the bacterial

isolates will be further identified and characterized using conventional method and

molecular method. In conventional method, biochemical characterization and

morphologies of bacterial colonies are identified through gram staining reaction and cell

shape while in molecular method; the bacterial isolates will be characterized using PCR

method.

1.2 Problem Statement/ Rationale

In 2002, at Mukah, the first project targeting of 50,000 hectares of sago plantation can

contribute in many sago starch productions; sago pith residue waste and sago bark waste.

Both the ligno-cellulosic waste is rich in soil microorganisms. Large quantities of sago

waste from sago starch mill produced everyday have no commercial value. Improper

discharge of effluents resulting from sago debarking and processing to river nearby had

lead to river pollutions. If sago bark can used as biofertilizers, it will bring benefit in

sustainable development. During the stripping of the bark process to get sago starch from

the sago trunk, remaining sago bark contains a considerable amount of starch especially

in its inner part of bark.

1.3 Research objectives

This study was undertaken with the following objectives:

1. To in vitro screen for different plant-growth promoting traits in bacterial isolates

5

2. To identify and characterize bacterial isolates with plant-growth promoting traits

using conventional method and molecular method.

6

CHAPTER 2

LITERATURE REVIEW

2.1 Sago palm (Metroxylon sagu)

The true sago palm (M. sagu) is a pinnate-leaved palm which is found mostly in hot

humid Oceania and peatland delta or riverine areas of South-East Asia especially in

Papua New Guinea, Indonesia and Malaysia (McClatchey et al., 2006).

It belongs to the Kingdom Plantae, Division Magnoliophyta, Class Liliopsida,

Order Arecales , family Arecaceae, subfamily Calamoideae Griffth, tribe Calameae

Drude, subtribe Metroxylinae Blume and genus Metroxylon (Wikipedia, 2008; Flach,

1997).

The word “sago” is originally from Javanese which means the starch-containing

palm pith. The sago palm (Metroxylon sagu) where its scientific name, “metra” means

pith or parenchyma and “xylon” means xylems (Flach, 1997). Sago palm is

environmentally friendly, where it tolerates most soil conditions. It is called as extremely

durable plant as it is able to thrive in flooding area, high salinity and acidity soil where

only few crops can survive. It grows with a minimum care and usually with no use of

fertilizer and pesticide treatments (Flores, n.d.).

2.2 Uses of sago palm

Almost the whole part of sago palm is versatile. Starch obtained from the sago trunk is

use as a source of carbohydrates which has many functions in food industry example are

7

bee hoon making, high fructose syrup, glucose, maltose and dextrose as well as housing

construction such as sago-leaf roof thatching and wall-siding. In states of Kelantan,

Terengganu and some parts of Pahang, it can be made into delicious fish crackers where

fish is mixed in sago flour with other ingredients. Besides, it can be used to make cream-

type and fruit puddings and acts as thickener in other dishes. Recently, many researchers

are working on sago in producing biodegradable plastics, fuel alcohol and ethanol (Abd-

Aziz, 2002).

Sago bark is a waste from sago production factories, but if this natural material

resource is used, it can help in global environmental conservation and sustainable

development. The property of its hardness makes it possible to be used as timber fuel,

wall materials, ceilings and fences in local areas. Sago bark has potential to be processed

into more durable building materials such as sago plywood and particleboards through

the bio-composite method. A project done in UNIMAS working on the waste from sago

bark also has successfully produced numerous decorative products from the waste (Azlin,

2005).

2.3 Sago cultivation in Sarawak

There is about 19,720 hectares lands situated particularly in the Division of Sibu had

been planted with sago palm (Tie & Lim, 1991). Oya- Dalat, Mukah, Pusa- Saratok, Igan

and Balingan are among the main sago cultivation parts in Sarawak. Presence of

approximate 1.69 million hectares of peat land in Sarawak which suited for sago

plantation has made the future for sago plantation in Sarawak to be bright (Chew et al.,

8

n.d.). The state of Sarawak in Malaysia is the largest exporter of starch in the world.

Japanese buy about 20,000 tons of starch from Sarawak each year. Besides, Peninsular

Malaysia, Taiwan, Singapore and other countries also import sago from Sarawak. The

plantation of sago palm gives considerable revenue to Sarawak in which it becomes the

fifth largest earnings after oil palm, pepper, cocoa and rubber (Abd-Aziz, 2002). In 1993,

the exportation of sago of RM 23.15 million had successfully overtaken the earning from

exportation of rubber in that year (Chew et al., n.d.).

2.4 Use of sago waste

The processing of 600 logs of sago palms every day can produce three types of sago

waste which are 15.6 tons of woody bark, 237.6 tons of wastewater and 7.1 tons of

starchy fibrous sago pith residue or hampas (Khan & Sallehin Awang Husaini, 2006).

Previous study has reported that starch (41.7-65%), fiber (14.8%) and a fair amount of

minerals are the major components in sago pith waste or hampas (Wina et al., 1986). The

decomposition of sago waste using microorganisms through biotechnological approach is

known as an attractive and proficient ways. Food processing industry such as sago

debarking industry releases a substantial amount of effluents rich in substrates like starch,

cellulose, fats and proteins. This effluent is more likely for microbial degradation to be

taken in place to produce products with additional values (Carmelo et al., 2002). Bacteria

and fungi are well-known in composing cellulose and starch components (Coughlan,

1985).

9

2.5 Microorganisms in sago humus with plant-growth promoting traits

2.5.1 Production of plant-growth hormones and nutrients

There are two groups of plant growth-promoting bacteria (PGPB) namely PGPB or

biocontrol PGPB, depend on either they stimulate plant growth directly or indirectly,

respectively (Bashan and Holguin, 1998). PGPB can influence the uptake of nutrients,

growth and yield of a plant by carrying out their own processes. It affects plant-growth

directly by nitrogen fixation (Han et al., 2005), solubilization of nutrients (Rodriguez and

Fraga, 1999), production of growth hormones, and indirectly by antagonizing pathogenic

fungi by production of siderophores, chitinase, β-1, 3-glucanase, antibiotics, fluorescent

pigments, and cyanide (Renwick et al., 1991; Pal et al., 2001).

According to Mahafee and Kloepper (1997), Bacillus and Pseudomonas colonize

plant more frequently than other bacteria. They can usually be isolated from surface-

disinfected plant tissue or within the tissue. Bacillus species had been successfully be

identified in different plant tissues such as citrus (Araujo et al., 2005), oak, maple,

cauliflower, grape, corn, and sunflower (Kobayashi, 2000). Bacillus and Pseudomonas

had exhibited their ability in producing IAA and ammonia (Joseph et al., 2007) and they

are most efficient phosphate solubilizing microorganism (PSM) amongst bacteria and

fungi (Tilak et al., 2005).

2.5.1.1 Indole-3- acetic acid (IAA) production

IAA is one of the common plant growth regulator and among the most physiologically

active auxins in plants. It affects several plant growth processes such as cell enlargement

10

and division, and tissue differentiation of plant. Its action is usually inhibited by light

where it concentrates at the covered site of stem and promotes growth of plant towards

light. Microorganisms such as plant-growth promoting rhizobacteria (PGPR) use L-

tryptophan to produce IAA (Frankenberger & Brunner; 1983 & Lynch, 1985). Therefore,

L-tryptophan is known as a precursor for IAA production. Various types of soil

microorganisms including bacteria (Muller et al., 1989), fungi (Stein et al., 1990) and

algae (Finnie & Van Staden, 1985) have ability to exhibit obvious effect on plant growth

by produce physiologically active quantities of auxins (Muller et al., 1989). The viable of

IAA concentration influences the growth of seedling. Low concentration will stimulate

the development while high concentration will give inhibitory effect to plant (Arshad &

Frankenberger, 1991). However, different plants respond differently towards the

fluctuation of auxin concentrations (Sarwar & Frankenberger, 1994).

The production of IAA is increased when the formation of adventitious roots

increases with the presence of L- tryptohan. L- tryptohan is considered as the precursor

factors to induce the development of adventitious roots (Madmony et al., 2004). However,

plant growth would be affected if tryptophan supplied to plant is in high concentration as

this will toxic to plant. IAA is common as root elongation inducer which leads to better

water and nutrient absorption from soil (Hoflich et al., 1994). But, the availability of high

bacterial source of IAA that exceeds above a threshold of 10-6

to 10-9

not beneficial to

root elongation anymore (Loper & Schroth, 1986). Although tryptophan and other related

compounds have been known in root exudates (Loper & Schroth, 1986), their

concentrations and stabilities in soil environment still reveal unclear.

11

Bacterial IAA producers (BIPs) are likely to impede on the physiological

processes in plant by input of IAA into the plant’s auxin pool. The fluctuation of IAA

concentration can be easily detected by root organ with high sensitive to IAA changes.

The plant-associated BIPs are considered as the factor that affecting the true

measurement of amount of IAA presents in plant tissues itself. BIDs can found plentifully

related with plants. In addition, they are the source of all the symptoms associated with

diverse plant diseases like gypsophila gall (Manulis & Barash, 2003), knot disease of

olive and oleander (Silverstone et al., 1993), and russet of pear fruit (Libber & Risch,

1969). In the contrast, bacteria that have the converse characteristic of destroying IAA

are called as bacterial IAA degraders (BIDs).They are also known to contaminate the

source of IAA as they have ability to destroy IAA and subsequently obscured the exact

quantities of IAA in plant tissues (Nissl & Zenk, 1969; Tomaszewski & Thimann, 1966).

In the study by Selvakumar et al. (2007), Serratia marcences was able to produce IAA.

In the past, an auxin type compound has been detected in Azotobacter culture by

using biological methods (Raznicina, 1938). However, in the fifties, the detection of

indole-3-acetic acid was successfully detected by using the paper chromatography

(Bukatsh et al., 1956). After the discoveries, many studies on the formation of

phytohormones by Azotobacter culture have been carried out. The production of the

phytohormone was found to be depended on the strains of microorganisms and their age.

The maximum IAA production was observed in stationary phase and the IAA was

transformed into indole-3- carbonic acid during further aging of the culture (Vancura &

Macura, 1960).

12

2.5.1.2 Phosphate- solubilizing bacteria

Phosphorus usually exists in nature in the forms of organic and inorganic. Organic forms

of phosphorus are contributed by the decomposition of dead plant or animal whereas

inorganic forms of phosphorus is available in soil whenever soluble phosphorus are

precipitated with inorganic forms of compounds such as calcium, Ferum, and Aluminium.

The existence of phosphorus in either organic form or inorganic form is pH-dependent. It

is common for the large amount of phosphorus on earth to present in the apatites with the

formula of M10 (PO4)6 X2. The M represents calcium and the X refers to the anion

fluorine. However, X can also be choride ion (CI-), hydroxide ion (OH

-) or carbonate

(CO3) which means that phosphorus can exist as flour, Chloro, hydroxy and carbonate

apatites.

Phosphorus is a major essential macronutrient form for biological growth and

development (Pradhan & Sukla, 2005). It is the second important factor after nitrogen in

limiting the growth of plants and it occupy 0.2% of plant dry weight. Phosphorus must

be converted into orthophosphate (H3PO4, H2PO4-

, HPO42-

, PO43-

) form before it can be

used by microorganisms. Phosphate solution in soil as phosphate anions are acquired by

plants for development. However, in some type of soil, these anions are more likely to

form precipitation with other cations like Ca2+

, Mg2+

, Fe3+

and Al3+

, due to their known

extremely reactive properties. The reaction converts the phosphate into insoluble forms

and makes it unavailable to plants. Phosphate in fertilizers that apply to soil is easily

become insoluble forms by precipitation (Nasreen, et al., 2005).