Title Page
ABSTRACT
Contents
Key to Abbreviation 13
INTRODUCTION 14
Chapter 1. Literature review 17
1.1. Plant growth regulators produced by PGPR 17
1.2. Improved nutrient availability by PGPR 19
1.3. Bioremediation by PGPR 20
1.4. Tolerance to abiotic stress conferred by PGPR 21
1.5. Control of plant disease 22
1.6. Bacillus subtilis 24
Chapter 2. Volatiles of Bacillus subtilis JS promote growth in plants (Nicotiana tabacum and Ipomoea batatas) 26
2.1. Introduction 26
2.2. Materials and methods 27
2.2.1. Identification of bacteria and bacterial culture 27
2.2.2. In vitro plant growth promotion 28
2.2.3. Analysis of hormone related gene expression level by RT-PCR 29
2.2.4. Growth promotion response of Arabidopsis thaliana mutants 30
2.2.5. Effect of the B. subtilis JS on the growth of sweet potato in field tests 30
2.3. Results and Discussion 31
2.3.1. Identification of bacteria 31
2.3.2. In vitro plant growth promotion 31
2.3.3. Analysis of hormone related gene expression levels by RT-PCR 32
2.3.4. Growth promotion response of Arabidopsis thaliana mutants 34
2.3.5. Effect of the B. subtilis JS on the growth of sweet potato in field tests 35
Chapter 3. Gene expression profile affected by volatiles of Bacillus subtilis JS in tobacco 48
3.1. Introduction 48
3.2. Materials and methods 49
3.2.1. Bacterial culture and treatment 49
3.2.2. Construction of SSH libraries 50
3.2.3. EST analysis 51
3.2.4. RT-PCR analysis 51
3.3. Results 52
3.3.1. SSH derived cDNA library and analysis 52
3.3.2. RT-PCR analysis 54
3.4. Discussion 56
Chapter 4. Volatiles of Bacillus subtilis JS induce the pathogenesis-related genes (PR genes) expression result in disease resistance in tobacco 72
4.1. Introduction 72
4.2. Materials and methods 73
4.2.1. Bacterial and fungal culture 73
4.2.2. Plant materials 74
4.2.3. Resistance assay of tobacco exposed to volatiles of B. subtilis JS 75
4.2.4. Gene expression analysis by RT-PCR 75
4.3. Results and Discussion 76
4.3.1. Resistance assay of tobacco exposed to volatiles of B. subtilis JS 76
4.3.2. PR genes expression analysis by RT-PCR 77
Chapter 5. General discussion 85
Literature Cited 89
국문요약 101
Table 2-1. Primers of hormone-related genes in tobacco plants used for RT-PCR 38
Table 3-1. Gene specific primers pairs for RT-PCR analysis 61
Table 3-2. Gene expression profile by volatiles of Bacillus subtilis JS 64
Table 4-1. Sequences of pathogenesis-related (PR) genes in tobacco plants used for RT-PCR 81
Figure 2-1. Image of Bacillus subtilis JS. 39
Figure 2-2. Comparasion of nucleotide sequence and phylogenetic tree of 16S rRNA 40
Figure 2-3. Growth promotion on tobacco with exposure to volatiles of Bacillus subtilis JS. The asterisk indicate a significantly difference according to Independent Samples t-Test at P=0.01. Data represent the average of five replicates. 41
Figure 2-4. Growth promotion on tobacco in vertical plate with Bacillus subtilis JS. Con: Water, JS: Bacillus subtilis JS; DH5α, Escherichia coli DH5α; PS, Pseudomonas syringae pv. Tomato; XC, Xanthomonas campestris pv. vesicatoria;... 42
Figure 2-5. Analysis of hormone related gene expression by RT-PCR. Plants were harvested after 24, 48, 72, 96 hours from the treatment of Bacillus subtilis JS volatiles. Actin was included as a constitutive control. 43
Figure 2-6. Growth promotion on Arabidopsis mutant by volatiles of Bacillus subtilis JS. Col-0: Arabidopsis thaliana ecotypes Columbia; Ler: Landsberg erecta; aba3-1 and abi1-1: ABA deficient line; abi4-1:ABA insensitive line; eir1-1: auxin... 44
Figure 2-7. Comparison on growth of Arabidopsis wild type Col-0. 45
Figure 2-8. Effect of the growth promoting Bacillus subtilis JS on shoot length and weight of sweet potato. Each data point represents the mean ±standard error. Different alphabetical letters are significantly different according to Tukey HSD at... 46
Figure 2-9. Effect of the Bacillus subtilis JS on the growth of sweet potato. 47
Figure 3-1. Overview of the suppression subtractive hybridization procedure by PCR-Select cDNA Subtraction Kit: cDNA synthesis, The tester and driver cDNA are synthesized from 2 μg of each sample mRNA; Restriction digestion, The tester... 66
Figure 3-2. Pie chart of second level gene ontology (GO) terms for biological process in ESTs of tobacco treated with volatiles of Bacillus subtilis JS. 68
Figure 3-3. RT-PCR analysis of gene expression profile affected by volatiles of Bacillus subtilis JS (up-regulated). 69
Figure 4-1. Analysis of PR genes expression levels by RT-PCR. Plants were harvested after 24, 48, 72, 96 hours from volatiles of Bacillus subtilis JS treatment. Actin was included as a constitutive control. 82
Figure 4-2. Disease severity of tobacco inoculated with R. solani AG-1 (IB) (A, circle) and P. nicotianae (B, square). control (left), and treatment with Bacillus subtilis JS (right). 83
Figure 4-3. Enhanced disease resistance in tobacco treated with volatiles of Bacillus subtilis JS. A. R. solani AG-1 (IB) B. P. nicotianae. The disease evaluation was presented with the lesion size at 3 days after inoculation.... 84
Figure 5-1. Putative mechanism of growth promotion and enhanced disease resistance on plant by volatiles of Bacillus subtilis JS. Volatiles of Bacillus subtilis JS play a role as elicitors.... 88