PROFILING OF MICROBES WITH PLANT-GROWTH … of Microbes With Plant-Growth...Bacillus subtilis and...
Transcript of PROFILING OF MICROBES WITH PLANT-GROWTH … of Microbes With Plant-Growth...Bacillus subtilis and...
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
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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2. To identify and characterize bacterial isolates with plant-growth promoting traits
using conventional method and molecular method.
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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
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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.,
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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).
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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).
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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).