Title: Analysis of the Induced Systemic Resistance Triggered by Trichoderma spp. in Solanum lycopersicum against Botrytis cinerea Infection
Other Titles: Analyse van de systemische resistentie geïnduceerd door Trichoderma spp. in Solanum lycopersicon tegen infectie door Botrytis cinerea
Authors: Yang, Yuxia
Issue Date: 18-Dec-2013
Abstract: Tomato is one of the most important vegetables in the world, with the annual production reaching up to 160 million tons in 2011 (FAOSTAT 2013) and a 3 % annual increase in consumption. Like many other crops, tomato yield is hampered by various plant pathogens and pests. One of the most notorious fungal pathogens of tomato is Botrytis cinerea, the causative agent of grey mould that is able to infect more than 200 plant species. Nowadays B. cinerea management is still largely relying on chemical treatments, which do not always lead to a successful outcome, mainly due to the multiple modes of infection of B. cinerea, its ability to survive as sclerotia in plants debris for extended periods, as well as the high frequency of fungicide resistance development. Hence, seeking alternative or complementary approaches to control B. cinerea is an urgent matter. Trichoderma spp. could contribute to such an alternative management approach. They are cosmopolitan soil fungi that can be isolated from all latitudes of the earth. Being beneficial to plants, some Trichoderma strains are already being used in agriculture as biocontrol organisms (BCOs). Previously it was found that Trichoderma spp. have direct antagonistic effects on many plant pathogens, including B. cinerea, which has been a focal point of the biocontrol community for a long time. To date, evidence is accumulating that Trichoderma spp. can also have indirect effects on pathogens, through triggering induced systemic resistance (ISR) in plants. In the first part of this doctoral thesis, we provide an overview of fungal biocontrol organisms in disease control of tomato, including Trichoderma-mediated ISR in tomato, as well as a more specific description of the biocontrol research in our group, which is centered around the Trichoderma hamatum T382-B.cinerea interaction. In order to unravel the ISR molecular mechanisms in tomato triggered by our model BCO Trichoderma hamatum T382 against B. cinerea, we first developed a robust lab-scale hydroponics system for ISR disease assays with the tripartite interaction T. hamatum T382 - tomato - B. cinerea, with ISR being characterized by significantly smaller B. cinerea lesion diameters. After confirming the robustness and reliability of the hydroponics system, we extrapolated our findings by replacing the components in the tripartite model system, using different Trichoderma strains and tomato cultivars. Several ISR-positive and ISR-negative strains were identified. In a next step, the further B. cinerea disease development in tomato plants pre-treated with two ISR-positive Trichoderma strains was monitored, and a reduction in leaf chlorosis and sporulating lesions was demonstrated in plants pre-treated by ISR-positive Trichoderma strains. Additionally, we found that the efficiency of Trichoderma-mediated ISR is dependent on the tomato genotype. The molecular basis of ISR in tomato mediated by T. hamatum T382 was unraveled by a transcriptome analysis. To elucidate the mechanisms behind this complex tripartite interaction, a transcriptome study of leaves from tomato plants pre-treated or not with T. hamatum T382 was carried out, both before (ISR-prime) and after (ISR-boost) B. cinerea infection. The large number of differentially expressed genes obtained allowed us to classify them according to the biological pathways in which they are involved. By focusing on pathways instead of genes, a holistic picture of the mechanisms underlying ISR emerged. More specifically, metabolism of jasmonic acid, ethylene, abscisic acid and phenylpropanoids, as well as several MAMPs (microbe associated molecular pattern) related signaling events were found to be highly induced. Since our research group performed a similar transcriptome analysis on the tripartite interaction T. hamatum T382 - Arabidopsis thaliana - B. cinerea, a comparison of the ISR mechanisms between these two plant species was made. In order to validate the putative role of hormones and secondary metabolism in T. hamatum T382-mediated ISR in tomato, several mutants and a transgenic line were employed for B. cinerea disease assays. The mutants were impaired in a hormone-related pathway (systemin signaling, jasmonic acid biosynthesis, ethylene signaling, abscisic acid biosynthesis) or in the phenylpropanoid biosynthesis pathway. The results of the disease assays suggest that systemin signaling, biosynthesis of jasmonic acid and abscisic acid, as well as phenylpropanoid biosynthesis indeed are essential components in T. hamatum T382-mediated ISR in tomato. Given that the genes categorized in most of these biological processes were up-regulated in our transcriptome data in ISR-prime, the results obtained from the mutant disease assays confirm the results from the transcriptome analysis. The ISR inducing ability of Trichoderma spp. has been reported to be closely associated with their rhizosphere-competence, which refers to their ability to colonize plant roots. Therefore, root colonization patterns by selected Trichoderma strains (both ISR-positive and ISR-negative ones) were first characterized by bright-field microscopy of tomato transverse root sections. However, we could not relate the colonization pattern of the tested strains to their ISR-inducing capacity in tomato, as this pattern was the same for both ISR-positive and ISR-negative strains. In a next step, in order to better visualize the growth of T. hamatum T382 in the vicinity of tomato roots, an autotrophic in vitro system was optimized. The ISR phenotype in this system was confirmed before proceeding to an in-depth microscopic study. Using scanning electron microscopy, we were able to observe the propagules of T. hamatum T382 (both conidia and hyphae) with high magnification and great clarity. Moreover, the green fluorescent protein (GFP) tagged T. hamatum T382 was constructed, which provided us with an additional tool to investigate whether T. hamatum T382 could enter tomato roots. Observation with confocal laser scanning microscopy revealed that T. hamatum T382 mainly closely adhered to the root surface, although appressoria-like structures commonly used by fungi to penetrate plant tissues, could be observed, in addition to occasional root penetrating hyphae. Finally, a general discussion on the results obtained in this thesis is presented, as well as some outlooks for future continuation of this research. By switching the plant partner from the originally studied model system T. hamatum T382 - Arabidopsis thaliana - B. cinerea to a major crop plant tomato, this study not only provides extensive insight on molecular mechanism of T. hamatum T382-mediated ISR in tomato against B. cinerea, but also contributed to the translation of knowledge on basic biological processes (on model plants) to efficient applications in the field.
Table of Contents: Table of Contents
Table of contents i
Summary v
Samenvatting viii
List of abbreviations xi
Chapter I Fungal biocontrol organisms in disease control of tomato
1.1 Introduction 2
1.2 Microbial antagonism 5
1.2.1 Mycoparasitism 5
1.2.2 Antibiosis 6
1.2.3 Competition 7
1.3 Plant-mediated effects 8
1.3.1 AMF 10 AMF - tomato colonization pattern 10 AMF - tomato recognition and interaction 11 AMF - tomato-pathogen ISR 13
1.3.2 Piriformospora indica 14 P. indica colonization pattern 14 P. indica - host recognition and interaction 15 P. indica - host - pathogen ISR 16
1.3.3 Trichoderma 17 Trichoderma colonization pattern 17 Trichoderma - tomato recognition and interaction 18 Trichoderma - tomato-pathogen ISR 20
1.3.4 Fusarium oxysporum 21 F. oxysporum colonization pattern 21 F. oxysporum - tomato recognition and interaction 21 F. oxysporum - tomato-pathogen ISR 23
1.3.5 Pythium oligandrum 23 P. oligandrum colonization pattern 23 P. oligandrum - tomato recognition and interaction 24 P. oligandrum - tomato-pathogen ISR 25
1.4 Other plant-mediated effects 25
1.5 Conclusions 28
1.6 Rationale and objectives of the doctoral project 32
1.6.1 Rationale 32
1.6.2 Objectives 36
1.7 References 38
Chapter II Development of an experimental system for Trichoderma-mediated ISR in Solanum lycopersicum against Botrytis cinerea
2.1 Introduction 50
2.2 Results 52
2.2.1 ISR in tomato triggered by T. hamatum T382 in soil 52
2.2.2 ISR in tomato triggered by Trichoderma spp. in hydroponics 53 Optimization of the lab-scale hydroponics system for ISR testing 53 Characterization of ISR-triggering ability of various Trichoderma spp. against B. cinerea
in tomato 57
2.2.3 Analysis of B. cinerea infection development in tomato after Trichoderma spp.
treatment 58
2.2.4 Trichoderma-mediated ISR in tomato is modulated by the plant genotype 61
2.3 Discussion 63
2.4 Materials and Methods 67
2.4.1 Plant and fungal materials 67
2.4.2 Disease assay in potting soil 67
2.4.3 Disease assay in the hydroponics system 68
2.5 References 69
Chapter III Genome-wide transcription analysis of ISR mediated by Trichoderma hamatum T382 in Solanum lycopersicum against Botrytis cinerea infection
3.1 Introduction 74
3.2 Results 77
3.2.1 Gene specific determination of relative time points for further transcriptome analysis 77
3.2.2 Transcriptome analysis of ISR in tomato triggered by T. hamatum T382 against
B. cinerea 78 Biological processes enriched in tomato leaves during ISR-prime and ISR-boost 79 Visualization of ISR-prime differential expressed genes in metabolic pathways 87
3.2.3 Validation of microarray data by qRT-PCR 91
3.3 Discussion 94
3.3.1 The role of plant hormones in T. hamatum T382-mediated ISR 94
iii JA and ET 94 SA 96 Abscisic acid 98 Gibberellin 99 Brassinosteroids 99 Auxin 100 Cytokinin 101
3.3.2 The role of secondary metabolism in T. hamatum T382-mediated ISR 102 Terpenoids 102 Phenolic compounds 103
3.3.3 The possible role of MTI in T. hamatum T382-mediated ISR 104 Perception the MAMPs of T. hamatum T382 104 Transducing the MTI signaling--Ca2+ and ROS 105
3.3.4 Regulation of photosynthesis and carbohydrate metabolism in T. hamatum T382-mediated
ISR 107
3.3.5 Comparison of ISR mechanisms in tomato and A. thaliana triggered by T. hamatum
T382 during ISR-prime 108
3.4 Conclusions 110
3.5 Materials and methods 111
3.5.1 Plant materials 111
3.5.2 Microarray 111
3.5.3 MapMan based transcriptome analysis 111 Biological processes enrichment analysis 111 visualization of transcriptome changes 112
3.5.4 qRT-PCR 112
3.6 References 113
Chapter IV Signaling pathways of Trichoderma hamatum T382-mediated ISR in Solanum lycopersicum against Botrytis cinerea
4.1 Introduction 122
4.2 Results 125
4.2.1 Requirement of systemin signaling and JA biosynthesis pathway for T. hamatum
T382- mediated ISR in tomato against B. cinerea 125
4.2.2 T. hamatum T382-mediated ISR in tomato against B. cinerea appears independent from SA
and ET signaling pathways 127
4.2.3 Involvement of ABA biosynthesis in T. hamatum T382-mediated ISR in tomato against
B. cinerea 128
4.2.4 Role of the phenylpropanoid biosynthesis in T. hamatum T382-mediated ISR in tomato
against B. cinerea 128
4.3 Discussion 133
4.4 Materials and methods 138
4.4.1 Biological Materials 138
4.4.2 Disease assays using a hydroponic system 138
4.5 References 139
Chapter V Microscopic study of Trichoderma spp. colonization of Solanum lycopersicum roots
5.1 Introduction 144
5.2 Results 146
5.2.1 Bright field microscope analysis of Trichoderma spp. colonization of tomato roots in
the hydroponics system 146
5.2.2 Histological study in optimized in vitro system 150 Observation of T. hamatum T382-mediated ISR in autotrophic in vitro system 150 Scanning electron microscopy study of T. hamatum T382 - tomato root association 151 Confocal laser scanning microscopy study of T. hamatum T382 - tomato root
association 154
5.3 Discussion 159
5.4 Materials and Methods 164
5.4.1 Tomato growth in the hydroponic system 164
5.4.2 Disease assays in autotrophic in vitro system 164
5.4.3 Bright field microscope observation 164
5.4.4 Scanning electron microscope observation 165
5.4.5 Agrobacterium tumefaciens mediated transformation 165
5.4.6 Confocal laser scanning microscope observation 166
5.5 References 167
Chapter VI General discussion and perspectives
6.1 Lab-scale analysis of Trichoderma-mediated ISR 172
6.2 The molecular mechanisms of T. hamatum T382-mediated ISR against B. cinerea
infection of tomato 173
6.3 Hormonal crosstalk in the plant immune system 175
6.4 Concluding remarks and general perspectives 177
6.5 References 180
Appendix xiii
List of publications xvii
ISBN: 978-90-8826-340-8
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Centre of Microbial and Plant Genetics

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