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Plants as primary producers in terrestrial ecosystems are under constant threat from a multitude of attackers, which include insect herbivores. In addition to interactions with detrimental organisms, plants host a diversity of beneficial organisms, which include microbes in the rhizosphere. Furthermore, the interactions between plants and several groups of root-associated microbes such as mycorrhizae, plant growth promoting rhizobacteria (PGPR) and plant growth promoting fungi (PGPF) can affect plant interactions with foliar insect herbivores. The beneficial root-associated microbes are able to modify plant physiology by promoting plant growth and induced systemic resistance (ISR), in which the balance between both effects will determine the final impact on the insect herbivores. Using Arabidopsis thaliana Col-0, this thesis explores the molecular mechanisms on how plants integrate responses when simultaneously interacting with the rhizobacterium Pseudomonas fluorescens and the generalist and the specialist leaf-chewing insects Mamestra brassicae and Pieris brassicae respectively.
A literature review on the state-of-the-art in the field of microbe-plant-insect interactions (Chapter 2) explores how root-associated microbes and insect folivores can influence each other via a shared host plant. For more than a decade, both ecological and mechanistic studies mostly focused on exploring these belowground and aboveground interactions using single microbe and single herbivore species. The importance of increasing the complexity of the study system in order to understand the interactions in more natural situations is being emphasized. Furthermore, this review discusses the role of plant hormones in regulating plant growth and defense against folivores, while simultaneously being involved in associations with root-associated microbes.
Experimental evidence has shown patterns on the effects of mycorrhizal colonization on plant interactions with insect herbivores, and raises the question whether plant colonization by different groups of root-associated microbes has similar effects on particular categories of insect herbivores. In Chapter 3, plant-mediated effects of a non-pathogenic rhizobacterium on the performance of leaf-chewing insects, and the underlying mechanisms modulating the interactions, have been examined. Colonization of A. thaliana Col-0 roots by the bacterium P. fluorescens strain WCS417r resulted in decreased larval weight of the generalist leaf-chewing M. brassicae, and had no effect on larval weight of the specialist leaf-chewing P. brassicae. The crucial role of jasmonic acid (JA) in regulating rhizobacteria-mediated induced systemic resistance (ISR) against M. brassicae is confirmed by including plant mutants in the study. Interestingly, I also observed that rhizobacteria can induce systemic susceptibility to M. brassicae caterpillars. Comparison of M. brassicae performance and gene transcription in A. thaliana plants, grown in potting soil or a mixture of potting soil and sand in a 1:1 ratio, shows that in a mixture of potting soil and sand, rhizobacterial treatment had a consistently negative effect on M. brassicae, whereas the effect is more variable in potting soil. Rhizobacterial treatment primed plants grown in potting soil and sand for stronger expression of JA- and ethylene-regulated genes PDF1.2 and HEL, supporting stronger resistance to M. brassicae. Taken together, the results show that soil composition can be one of the factors modulating the outcome of microbe-plant-insect interactions.
Chapter 4 further addresses the mechanisms underlying rhizobacteria-mediated ISR against the generalist leaf-chewing M. brassicae by integrating plant gene transcription, chemistry and performance of M. brassicae in wild type A. thaliana Col-0 plants and mutants defective in the JA-pathway, i.e. dde2-2 and myc2, in the ET pathway, i.e. ein2-1, and in the JA-/ET-pathway, i.e. ora59. Results of this study show that rhizobacterial colonization alone or in combination with herbivore infestation induced the expression of the defense-associated genes ORA59 and PDF1.2 at higher levels than activation by herbivore feeding alone, and the expression of both genes is suppressed in the knock-out mutant ora59. Interestingly, the colonization of plant roots by rhizobacteria alters the levels of plant defense compounds, i.e. camalexin and glucosinolates (GLS), by enhancing the synthesis of constitutive and induced levels of camalexin and aliphatic GLS while suppressing the induced levels of indole GLS. The changes are associated with modulation of the JA-/ET-signaling pathways as shown by investigating mutants. Furthermore, the herbivore performance results show that functional JA- and ET-signaling pathways are required for rhizobacteria-mediated ISR against leaf-chewing insects as observed in the knock-out mutants dde2-2 and ein2-1. The results indicate that colonization of plant roots by rhizobacteria modulates plant-insect interactions by prioritizing the ORA59-branch over the MYC2-branch, although the transcription factor ORA59 is not the only one responsible for the observed effects of rhizobacteria-mediated ISR against leaf-chewing insects.
Taking a step further in increasing the complexity of the study system, Chapter 5 investigates how co-cultivation of P. fluorescens strains WCS417r and SS101 affects direct plant defense to the caterpillar M. brassicae. Inoculation of either P. fluorescens WCS417r or SS101 singly at root tips or simultaneously at two different positions along the roots resulted in a similar level of rhizobacterial colonization by each strain, whereas co-cultivation of both strains at either the root tips or at two different positions along the roots resulted in a higher colonization level of strain WCS417r compared to colonization by SS101. The results suggest that the site of inoculation influences the direct interactions between the two strains in the rhizosphere, as also confirmed by in vitro antagonism assays in the absence of plants. Both upon single inoculation and co-cultivation of both strains at the same or different sites along the roots, the two rhizobacterial strains induced the same strength of ISR against the caterpillar M. brassicae and the same degree of plant growth promotion. In the roots, colonization by the two strains as single or mixed culture resulted in a similar gene expression pattern of up-regulation of MYC2, down-regulation of WRKY70 and no effect on NPR1 expression, genes representing JA-signaling, SA-signaling and the node of crosstalk between the two pathways, respectively. We hypothesize that both rhizobacterial strains use negative crosstalk between JA- and SA-pathways as mechanism to suppress plant immunity and establish colonization. This study shows that competitive interactions between rhizobacterial strains known to induce plant defense in systemic tissue via different signaling pathways, may interfere with synergistic effects on ISR and plant growth promotion.
While the effect of root-associated microbes on direct plant defense against insect herbivores has been studied previously, the effect of these microbes on indirect plant defense to herbivores is much less known. Chapter 6 explores how colonization by the rhizobacterium P. fluorescens strain WCS417r affects indirect plant defense against the generalist herbivore M. brassicae by combining behavioral, chemical and gene transcriptional approaches. The results show that rhizobacterial colonization of A. thaliana roots results in an increased attraction of the parasitoid Microplitis mediator to caterpillar-infested plants. Volatile analysis revealed that rhizobacterial colonization suppressed emission of the terpene (E)-α-bergamotene, and the aromatics methyl salicylate and lilial in response to caterpillar feeding. Rhizobacterial colonization decreased the caterpillar-induced transcription of the terpene synthase genes TPS03 and TPS04. Rhizobacteria enhanced both growth and indirect defense of plants under caterpillar attack. This study shows that rhizobacteria have a high potential to enhance the biocontrol of leaf-chewing herbivores based on enhanced attraction of parasitoids.
Taken together, the research presented in this thesis has shown how single or combined applications of rhizobacteria affect interactions of plants with leaf-chewing insects in terms of direct and indirect resistance. Furthermore, results presented in this thesis have revealed some of the molecular mechanisms underlying plant-mediated interactions between rhizobacteria and leaf-chewing insects that can be used in developing practical approaches by applying beneficial root-associated microbes for improving plant resistance.
|Doctor of Philosophy
|20 May 2015
|Place of Publication
|Published - 20 May 2015
- rhizosphere bacteria
- multitrophic interactions
- defence mechanisms
- pseudomonas fluorescens
- mamestra brassicae
- pieris brassicae
- plant-microbe interactions
- insect plant relations
- plant-herbivore interactions
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- 1 Finished
Multidisciplinary approach to study effects of beneficial rhizobacteria on induced plant defences to aboveground herbivorous insects
1/06/10 → 20/05/15