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UNIVERSITI PUTRA MALAYSIA
APPLICATION OF BACILLUS SPHAERICUS UPMB10 FOR GROWTH ENHANCEMENT OF BANANA (MUSA SPP. VAR. BERANGAN) AND
ITS EFFECT ON FUSARIUM WILT
ILLANI ZURAIHAH BINTI IBRAHIM
FP 2008 32
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APPLICATION OF BACILLUS SPHAERICUS UPMB10 FOR GROWTH
ENHANCEMENT OF BANANA (MUSA SPP. VAR. BERANGAN) AND ITS
EFFECT ON FUSARIUM WILT
ILLANI ZURAIHAH BINTI IBRAHIM
MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA
2008
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APPLICATION OF BACILLUS SPHAERICUS UPMB10 FOR GROWTH
ENHANCEMENT OF BANANA (MUSA SPP. VAR. BERANGAN) AND ITS
EFFECT ON FUSARIUM WILT
By
ILLANI ZURAIHAH BINTI IBRAHIM
Thesis submitted to the School of Graduate Studies, Universiti Putra
Malaysia, in Fulfillment of the Requirement for the Degree of Master of
Science
SEPTEMBER 2008
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DEDICATION
Special dedication to:
Zull and Danial
My lovely family…. For all the supports, endless loves and advices throughout
the long journey to complete my study…. Keeps me alive!
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Master of Science
APPLICATION OF BACILLUS SPHAERICUS UPMB10 FOR GROWTH
ENHANCEMENT OF BANANA (MUSA SPP. VAR. BERANGAN) AND ITS
EFFECT ON FUSARIUM WILT
By
ILLANI ZURAIHAH BINTI IBRAHIM
September 2008
Chairman : Professor Zulkifli Hj Shamsuddin, Ph.D. Faculty : Agriculture
A locally isolated strain Bacillus sphaericus UPMB10, categorized as plant
growth promoting rhizobacteria (PGPR), was reported to have beneficial
effects in stimulating growth and yield of many agricultural crops. This study
explores the indirect mechanism of B. sphaericus UPMB10 that would support
the hypothesis that this PGPR could act as a biocontrol agent. Through in-
vitro study, B. sphaericus UPMB10 was observed to inhibit the growth of
Fusarium oxysporum f.sp cubense race 4 (FOCR4) mycelium by 70% in
culture medium. Malformation of the fungal hyphae by vacuole formation,
swelling and thickening of the hyphal strands occurred with the presence of B.
sphaericus UPMB10. In addition, B. sphaericus UPMB10 showed positive
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results in the tests for production of cell wall-degrading enzymes and
phosphate solubilizing activity by producing clear zones or halos. It can also
produce a plant growth regulating hormone, indole acetic acid (IAA). An initial
glasshouse study showed that inoculation with B. sphaerius UPMB10
increased plant growth up to 30% in plant height and increased nutrient
uptake namely for N, P, K, and Ca, and also reduced the disease severity
through visual symptoms and vascular discoloration of the corm. In the last
glasshouse experiment, the appearance of the disease incidence was
observed. All of the treatments with FOCR4 application were infected by
FOCR4 including those treated with B. sphaericus UPMB10. Significantly, the
infection occurred at the late stage of the experiment indicating that B.
sphaericus UPMB10 could delay the infection process. Solid inoculum
inoculation with B. sphaericus UPMB10 showed a delayed onset of
symptoms, lower percentage in disease incidence and epidemic rate. As
liquid and solid substrate were selected for evaluation against the disease in
the glasshouse, it showed that soil application of B. sphaericus UPMB10 with
solid inoculum provided the best suppression of Fusarium wilt of banana. In
this study, an inoculum dose of 100 g of solid formulation per plant was
optimum, because it suppressed the disease to the same extent as higher
doses of the antagonist. This showed B. sphaericus UPMB10 has a high
potential for its use as a plant growth enhancer and biocontrol agent in the
disease management of Fusarium wilt in bananas.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan ijazah Master Sains
APLIKASI BACILLUS SPHAERICUS UPMB10 UNTUK MENINGKATKAN
PERTUMBUHAN PISANG (MUSA SPP. VAR. BERANGAN) DAN
KESANNYA KE ATAS PENYAKIT LAYU FUSARIUM
Oleh
ILLANI ZURAIHAH IBRAHIM
September 2008
Pengerusi : Professor Zulkifli Hj Shamsuddin, Ph.D.
Fakulti : Pertanian
Strain bakteria tempatan Bacillus sphaericus UPMB10 yang dikategorikan
sebagai rhizobakteria penggalak pertumbuhan pokok (PGPR) dilaporkan
mempunyai keupayaan dalam menggalakkan pertumbuhan dan hasil dalam
pelbagai tanaman pertanian. Kajian ini merupakan explorasi yang
menunjukkan secara tidak langsung mekanisme yang dapat menyokong
hipotesis PGPR ini untuk bertindak sebagai agen kawalan biologi. Melalui
kaijan in vitro, B. sphaericus UPMB10 telah diperhatikan dapat merencatkan
pertumbuhan Fusarium oxysorum f.sp.cubense race 4 (FOCR4) sebanyak
70% di dalam medium kultur. Ketidakseragaman pertumbuhan hifa fungi yang
disebabkan oleh pembentukan vakuol, pembengkakkan dan penebalan
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jaluran hifa telah berlaku dengan kehadiran B. sphaericus UPMB10.
Tambahan pula, B. sphaericus UPMB10 juga telah menunjukkan keputusan
yang positif di dalam ujikaji penghasilan enzim pelupus dinding sel dan ujikaji
aktiviti pelarutan fosfat dengan menunjukkan kesan zon jernih yang jelas atau
halo. Ia juga berupaya menghasilkan hormon pertumbuhan iaitu indole asetik
asid (IAA). Pada permulaan eksperimen rumah kaca menunjukkan yang
inokulasi dengan B. sphaericus UPMB10 telah meningkatkan pertumbuhan
pokok sebanyak 30% ketinggian pokok dan kadar pengambilan nutrient
terutamanya N, P, K dan Ca. Ia juga telah mengurangkan kadar keterukan
penyakit melalui pemerhatian simptom luaran pokok dan perwarnaan umbisi
pokok. Pada akhir eksperimen, kadar insiden penyakit ditunjukkan melalui
simptom luaran dengan menunjukkan kesemua rawatan yang melibatkan
Fusarium telah dijangkiti walaupun dirawat dengan B. sphaericus UPMB10.
Secara signifikannya, serangan hanya bermula lewat di akhir eksperiment
dan ini menunjukkan keupayaan B. sphaericus UPMB10 untuk melambatkan
serangan penyakit tersebut. Inokulum pepejal B. sphaericus UPMB10 telah
menunjukkan perlambatan kemunculan simptom, peratus insiden penyakit
dan kadar epidemik yang rendah. Aplikasi kepada tanah telah menunjukkan
B. sphaericus UPMB10 dengan cara pepejal telah memberikan ketahanan
terbaik kepada penyakit layu fusarium. Kombinasi samada menggunakan
aplikasi substrat cecair atau pepejal telah dipilih untuk penilaian terhadap
penyakit di eksperimen rumah kaca. Di dalam kajian ini, didapati bahawa
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inokulum dengan formulasi 100 g pepejal per pokok adalah optimum untuk
memberikan ketahanan terbaik pada tahap antagonis pada dos yang sama.
Kesimpulan menunjukkan B. sphaericus UPMB10 berpotensi tinggi untuk
digunakan sebagai penggalak pertumbuhan pokok dan agen kawalan biologi
dalam pengurusan penyakit layu Fusarium pada tanaman pisang.
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ACKNOWLEDGMENTS
All praises and thanks to Almighty Allah SWT, the beneficent and merciful for
the blessing and the strength in completion of my study. Firstly, I would like to
express my deepest and sincerest appreciations with a heartfelt gratitude to
Professor Dr. Zulkifli Hj Shamsuddin, the Chairman of Supervisory Committee
for his invaluable advice, constructive criticism, support, encouragement,
patience and understanding that made this study possible. Not forgetting to
Professor Dr. Sariah Meon and Associate Professor Dr. Zakaria Wahab as
members of the Supervisory Committee, for their guidance, suggestions and
invaluable advice and comments throughout the duration of this study and
preparation of this thesis.
Also with deep sense of honour, I wish to extend my sincere gratitude to all
staffs of the Department of Land Management especially in the Soil
Microbiology Laboratory and to Mrs Zarinah and Mr Dzulkifli Duaji for their
time and assistance in the preparation of materials. Heartfelt thanks are also
extended to my friends Wahida, Noraini, Maliza, Zakry, Prema, Negash, Ina,
Palie, Yani, Ziana, Siti and others.
I also would like to thank the Ministry of Science, Technology and Innovation
(MOSTI) for the research grant no. 01-02-04-PR0010/04-02 and financial
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support through PASCA Scheme that provided me the opportunity to pursue
my master degree.
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I certify that an Examination Committee met on 25th September 2008 to conduct the final examination of Illani Zuraihah Ibrahim on her thesis entitled “Application of Bacillus sphaericus UPMB10 for Growth Enhancement of Banana (Musa spp.var. Berangan) and Its Effects On Fusarium Wilt“ in accordance with Universities and University Colleges Act 1971 and the Constitution of the Univeristi Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded Master of Science. Members of the Thesis Examination Committee were as follows: Zaharah Abdul Rahman, Ph.D. Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Jugah Kadir, Ph.D. Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Halimi Mohd Saud, Ph.D. Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Nik Masdek Nik Hassan, Ph.D. Lecturer Horticultural Research Centre Malaysian Agricultural Research and Development Institute (External Examiner)
BUJANG KIM HUAT, PhD Professor and Deputy Dean, School of Graduate Studies, Universiti Putra Malaysia. Date: 19 February 2009
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This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows:
Zulkifli Hj. Shamsuddin, Ph.D. Professor, Faculty of Agriculture, Universiti Putra Malaysia. (Chairman) Sariah Meon, Ph.D. Professor, Faculty of Agriculture, Universiti Putra Malaysia. (Member) Zakaria Wahab, Ph.D. Associate Professor, Faculty of Agriculture, Universiti Putra Malaysia. (Member)
HASANAH MOHD GHAZALI, Ph.D.
Professor and Dean, School of Graduate Studies, Universiti Putra Malaysia. Date: 9 April 2009
.
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DECLARATION
I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.
___________________________
Candidate: ILLANI ZURAIHAH IBRAHIM Date:
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TABLE OF CONTENTS
PAGE iii iv vi ix xi xiii xvii xviii
DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST PLATES LIST OF FIGURES LIST ABBREVIATION
xx xxi
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 2.1 Overview of banana industry 2.2 Fusarium wilt
2.2.1 Systematics and Characterization of Fusarium oxysporum
2.2.2 Characterization of Fusarium oxysporum f.sp.cubense
2.2.3 Fusarium Wilt of Banana 2.2.4 Disease Distribution and Losses 2.2.5 Epidemiology 2.2.6 Current Management of Fusarium Wilt
2.3 Beneficial Microbes as An Alternatives for Biocontrol 2.3.1 Bacillus sphaericus
2.4 Mechanisms of Biocontrol by PGPR 2.4.1 Antibiosis 2.4.2 Antifungal Metabolites 2.4.3 Enzymes 2.4.4 Competition 2.4.5 Plant Ethylene Levels 2.4.6 Induced Systemic Resistance 2.4.7 Direct Stimulation of Plant Growth
and Development 2.5 Microbial Inoculum Performance
6 6 7 7 9
10 13 15 17 19 21 24 25 25 27 28 32 33 35
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3 4
MATERIALS AND METHODS 3.1 Morphological and Metabolite Production
Characteristics, and Biological Activity of B. sphaericus UPMB10 3.1.1 PGPR Strain 3.1.2 Fungal Strain 3.1.3 Culture, Media and Growth Condition 3.1.4 Dual Culture Technique 3.1.5 Hyphal Morphology 3.1.6 Production of Cell-wall Degrading Enzyme 3.1.7 Test for Phosphate Solubilizing Activity 3.1.8 Production of Indole Acetic Acid (IAA)
3.2 B. sphaericus UPMB10 Inoculation on Growth of Banana Seedlings and Pathogenecity Toward Fusarium oxysporum 3.2.1 Tissue-cultured Banana Plantlets 3.2.2 Preparation of Fusarium Inoculant 3.2.3 Plant Tissue Analysis 3.2.4 Plant Growth Measurement 3.2.5 Disease Assessment
3.3 Infectivity Studies and Effect of Different Rates of B. sphaericus UPMB10 Inoculation on the Disease Incidence Caused by Fusarium oxysporum 3.3.1 Inocula Preparation and Application Rate 3.3.2 Plant Growth Performance (based on height
increment) 3.3.3 Evaluation on Different Rates of Inoculum on
Disease Assessment 3.4 Statistical Analysis
RESULTS AND DISCUSSION 4.1 Morphological and Metabolite Production
Characteristics, and Biological Activity of B. Sphaericus UPMB10 4.1.1 Dual Cultural Studies 4.1.2 Hyphal Morphology 4.1.3 Secondary Metabolite Production of B.
sphaericus UPMB10 4.1.3.1 Test for Phosphate Solubilizing Activity 4.1.3.2 Production of Indole Acetic Acid (IAA) 4.1.3.3 Production of Cell Wall-degrading Enzymes
38
38 38 38 39 40 41 41 42 42
43 43 43 44 44 45
48 48
49
50 53
54
54 54 56
60 60 61 63
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4.2 Evaluation of B. sphaericus UPMB10 Inoculation on Plant Growth Performance 4.2.1 Evaluation on Nutrient Status of Different Plant
Components 4.2.1.1 Nitrogen (N) 4.2.1.2 Phosphorus (P) 4.2.1.3 Potassium (K) 4.2.1.4 Calsium (Ca)
4.2.1.5 Magnesium (Mg) 4.2.2 Evaluation on Plant Height Development
4.2.3 Evaluation on Disease Progress 4.3 Effect of Different Rates of B. sphaericus UPMB10
Inoculation on Disease Incidence of Fusarium oxysporum 4.3.1 Evaluation and Comparison Between Liquid vs.
Solid Inocula and Application Rate on Growth Performance
4.3.2 Evaluation on Disease Assessment (Disease Incidence and Symptom Development)
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73 73 76 77 78 79 79 83
89
92
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5 GENERAL DISCUSSION AND CONCLUSION 96
REFERENCES/BIBLIOGRAPHY APPENDICES BIODATA OF THE STUDENT
105 131 139
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LIST OF TABLES
No. Table
Page
1 S Some free-living rhizobacteria (PGPR) that are also plant growth-promotive. (Note: Not all strains of the microorganisms can act as PGPR)
20
2 Summary of beneficial and antagonistic characteristic of B. sphaericus UPMB10
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3 The nutrient status in root, pseudostem, corm and leaves of banana plantlets inoculated with B. sphaericus UPMB10 after 12 weeks. (Means having the same letter (s) in a column of a parameter do not differ significantly at 0.05% level by Tukey Test)
74
4 Total nutrient accumulation in banana plantlets inoculated with B. sphaericus UPMB10 after 12 weeks. (Means having same letter (s) in a column do not differ significantly at 0.05 levels by Tukey’s Test)
75
5 Disease severity (vascular discoloration of corm) index (DSI) of banana seedlings var. Berangan cv. Intan challenged with FOCR4 at 90 days. Each value is the mean of five replicates
86
6 Area Under Disease Progress Curve (AUDPC) and epidemic rate of Fusarium wilt under different treatments
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LIST OF PLATES
No. Plate
Page
1 Inoculated banana plants under evaluation for tolerance to Fusarium oxysporum in a glasshouse.
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2 B. sphaericus UPMB10 colony in a ring inhibiting the growth of FOCR4 on NA plate after 5 days of incubation
55
3 5mm agar disc of FOCR4 on NA plate as the control after 5 days’ incubation
55
4 A streak colony of B. sphaericus UPMB10 bacteria inhibiting the growth of FOCR4 on NA plate after 5 days of incubation.
56
5 (a) (a) Light microscopy shows abnormalities of FOCR4 hyphae with B. sphaericus UPMB10 in the culture medium; thickened and vacuolar hyphal strand.
57
5 (b) Normal fungal growth with smooth hyphal wall (no swelling and vacuoles) in absence of B. sphaericus UPMB10.
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5 (c) Photo from Chaurasia et al., 2005 showed (A) Normal hyphae macroconidia of F.oxysporum , (B) swelling of hyphal tip and deformation of conidia in F.oxysporum
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6 Clear zone around B. sphaericus UPMB10 cultured on chitin agar plate after 5 days of incubation
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7 Comparison of inoculation effect of B. sphaericus UPMB10 (T2) with control (T5) after 4 weeks of treatments.
68
8 Effect of B. sphaericus UPMB10 inoculation on plant height of banana tissue cultured plantlet at six weeks. Left B. sphaericus UPMB10 (T2) and right control (T5).
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9 Infected banana plant (left) showing foliar symptoms of Fusarium wilt caused by FOCR4 compared to healthy control plant (right).
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10 Infected banana plant showing the development of foliar associated symptoms of Fusarium wilt of banana on cultivar INTAN caused by Fusarium oxysporum f. sp. cubense. Healthy plant (A); chlorosis or yellowing of lower leaves (B); desiccation of lower leaf and chlorosis of upper leaves (C); advanced stage of infection with severe chlorosis and wilting (D).
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11 Disease severity index, (based on vascular discoloration of corm caused by FOCR4): (a) uninfected control, (b, c) less severe (inoculated with UPMB10) and (d) most severe (FOC4); of banana seedlings var. Berangan cv. Intan challenged with FOC4 after 60 days.
87
12 Culture of banana pseudostem on PCNB agar. New Fusarium colonies formed from tissue of an infected plant (a) and no fungal proliferation from tissue of a healthy plant (b).
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LIST OF FIGURES
No. Figure
Page
1 Effect of B.sphaericus UPMB10 inoculation on fresh weight of banana tissue culture plantlets (pseudostem and corm, roots, leaves) at 12 weeks of treatments. (Means having same letter (s) in a column of a parameter do not differ significantly at 0.05 level by Tukey’s Test)
67
2 Effect of B.sphaericus UPMB10 inoculation on dry weight of banana tissue culture plantlets (pseudostem and corm, roots, leaves) at 12 weeks of treatments. (Means having same letter (s) in a column of a parameter do not differ significantly at 0.05 level by Tukey’s Test)
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3 The effect of B.sphaericus UPMB10 inoculation on root volume of banana tissue cultured after 12 weeks of treatments. (Means having same letter (s) between treatment not differ significantly at 0.05 level by Tukey’s Test)
71
4 Effect of different treatments of inoculation in plant height of banana plantlets. (Each value represent mean of 5 replicates). Vertical bars on the top indicate LSD for treatment comparisons at a given week of treatment. Vertical bars on the right indicate LSD for comparisons of growth height over weeks within a treatment.
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5 Disease development on banana seedlings challenged with FOCR4 based on foliar symptoms. (Each value is the mean of 5 replicates).
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6 Figure 6: Effect of different rates of B. sphaericus UPMB10 inoculation to FOCR4 challenge on growth height of banana plants. (Each value represent mean of 12 replicates). Vertical bars on the right indicate LSD for comparisons of growth height over weeks within a treatment
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7 Effect of different treatments on the development of Fusarium wilt in banana based on foliar symptoms. Each value represent mean of 12 replicates.
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LIST OF ABBREVIATIONS
ANOVA Analysis of variance
AUDPC Area under Disease Progress Curve
cfu Colony forming units
DI Disease incidence
DS Disease severity
DSI Disease severity index
FOCR4 Fusarium oxysporum f.sp. cubense race 4
FSM Fusarium selective media
FW Fresh weight
GA Gibberelic acid
IAA Indole acetic acid
M monit
PDB Potato Dextrose Broth
PGPR Plant growth promoting rhizobacteria
PIRG Percentage of inhibition of radial growth
PSB Phosphate solubilizing bacteria
RCBD Randomised Complete Block Design
rm Epidemic rate
TSA Tryptic Soy Agar
TSB Tryptic Soy Broth
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CHAPTER 1
INTRODUCTION
Non-symbiotic rhizobacteria, associated with plant roots and able to
enhance plant growth, known as plant growth promoting rhizobacteria
(PGPR) has generated much interest in a wide range of research. It has
impacts in different fields of study because it provides enormous information
on different aspects of plant growth promotion and development while at the
same time promises possible biological control solutions to pytopathogens of
some plant diseases. At present, phytopathogens have caused a great loss in
crop productivity and had to depend largely on chemicals, fumigation, steam-
treatment and solarization of soils in order to control it (Gamliel and Katan,
1992). Earlier, it was believed that the advantages of chemical controls of
diseases could be obtained without any deleterious environmental effects.
Currently, researchers have realized that these chemical agents are both
hazardous to animals and humans because they can persist and accumulate
in natural ecosystems. Consequently, there is a strong desire to replace
chemicals with biological approaches that are potentially more “friendly” to the
environment in the long term. These biological approaches include the use of
transgenic plants, which are resistant to one or more plant pathogens
(Greenberg and Glick, 1993), or either to use of biocontrol plant growth-
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promoting bacteria which can suppress or prevent the plant damage
(O’Sullivan and O’Gara, 1992; Sivan and Chet 1992; Sutton and Pegg, 1993;
Cook, 1993). Biocontrol seems to be a reliable alternative compared to
chemical fungicides, which have raised serious concerns of food
contamination and environmental pollution. Some saprotrophic bacteria can
serve as excellent biocontrol agents against plant pathogens (Fernando et al.,
2005).
The introduction of beneficial microbes as biological control of soil
borne plant pathogen as well as enhancement of plant growth has a
considerable potential in agriculture. Biological control using antifungal
rhizobacteria to suppress plant diseases offers a powerful alternative to the
use of synthetic chemicals. Numerous studies have demonstrated the ability
of several bacteria to suppress diseases caused by soil-borne and seed-
borne plant pathogens (Weller, 1988; O’Sullivan and O’Gara, 1992; Whipps,
1997). The anti-fungal abilities of these beneficial microbes have been known
since a few decades ago and there have been extensive efforts to use them
for plant disease control and more recently on a commercialized scale.
The uniqueness of microorganisms has never been understood until
their beneficial functions were discovered. It is a paradox that bacteria can be
both beneficial and antagonistic towards a plant pathogen to maintain its life
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cycle. In this manner, it is possible to introduce the beneficial microorganism
as a biological control for soil borne plant pathogen as well as enhancement
of plant growth. This creates a great implication on plant growth development
through direct or indirect mechanisms (Glick et al., 1999). A direct promotion
of plant growth by PGPR is generally based on the ability of the bacteria to
synthesize and facilitate nutrient uptake from the environment. Meanwhile
indirect mechanism is through its effect on the phytopathogenic organism
(usually fungus) by one or several mechanisms such as production of
antibiotic and antifungal metabolite or enzyme that could lyse the fungal cell
wall. It was believed that this advantage would lead to the potential of PGPR
as a biocontrol agent besides stimulating plant growth. A local rhizobacterial
strain, isolated from oil palm roots and identified as Bacillus sphaericus
UPMB10 (B. sphaericus UPMB10), has shown great potential in promoting
plant growth development and has been characterized for its optimum growth
requirement, inoculum carrier (ground oil palm frond) and inoculum production
technique (Premalalatha, 2006). Bacillus sphaericus is categorized as a free-
living soil bacterium that can function as a plant growth promoting
rhizobacteria (Kloepper et al., 1989). A few studies have demonstrated that
inoculation with B. sphaericus UPMB10 enhanced plant growth and fixed
nitrogen with oil palm (Amir et al., 2001 and banana (Mia et al., 2000). The
current research interest is to assess the ability of B. sphaericus UPMB10 as