Microbiome dynamics of disease suppresive soils
Gómez Expósito, Ruth - \ 2017
Wageningen University. Promotor(en): F.P.M. Govers; J.M. Raaijmakers, co-promotor(en): J. Postma; I. de Bruijn. - Wageningen : Wageningen University - ISBN 9789463431774 - 267
suppressive soils - soil suppressiveness - plant diseases - thanatephorus cucumeris - microbial ecology - soil microbiology - rhizosphere bacteria - soil bacteria - community ecology - soil fungi - transcriptomics - taxonomy - ziektewerende gronden - bodemweerbaarheid - plantenziekten - thanatephorus cucumeris - microbiële ecologie - bodemmicrobiologie - rizosfeerbacteriën - bodembacteriën - gemeenschapsecologie - bodemschimmels - transcriptomica - taxonomie
Disease suppressive soils are soils in which plants do not get diseased from plant pathogens due to the presence (and activities) of the microbes present in the soil. Understanding which microbes contribute to confer suppression and through which mechanisms they can protect plants is crucial for a sustainable control of plant diseases. In the research conducted in this thesis, I first examined the role of Lysobacter species, previously associated with disease suppressive soils, in suppressing damping-off disease caused by the soil-borne fungal pathogen Rhizoctonia solani on sugar beet. The majority of the Lysobacter strains tested revealed a broad metabolic potential in producing a variety of enzymes and secondary metabolites able to suppress R. solani in vitro. However, any of these strains could consistently suppress damping-off disease when applied in soil bioassays. Their ability to promote plant growth was also tested for sugar beet, cauliflower, onion or Arabidopsis thaliana. Results indicated that any of the Lysobacter strains could consistently promote plant growth, neither via direct contact nor via volatile production. Second, I investigated whether the antagonistic activity of Lysobacter species could be triggered when applied as bacterial consortia, together with Pseudomonas and Streptomyces species. Although several bacterial combinations showed an increased antagonistic effect towards R. solani in vitro, no consistent effects were observed when these bacterial consortia were applied in vivo. Third, I investigated the dynamical changes in the bacterial community composition and functions occurring during the process of disease suppressiveness induction by performing whole community analyses using next-generation sequencing techniques. Results indicated that suppressiveness induction was most associated with changes in certain bacterial traits rather than changes in the bacteria community composition itself. Among the functions found as more active in suppressive soils were several ‘classic’ mechanisms of disease suppression, including competition for nutrients, iron and space and production of extracellular enzymes, indol-acetic-acid and hydrogen cyanide. Among the enzymes found in higher abundance in suppressive soil were these ones involved in the degradation of oxalic acid, a pathogenicity factor produced by pathogenic fungi to help infecting the host plant. Hence, I finally studied the role of bacteria able to produce enzymes able to degrade oxalic acid in suppressing R. solani disease. Enrichment of native oxalotrophic bacteria existing in soil, their isolation and further application into soil revealed that they could effectively suppress Rhizoctonia disease. Characterization of these oxalotrophic bacteria revealed that members within the Caulobacter and Nocardioides species could suppress R. solani disease by their own. Furthermore, the research done in this thesis highlights the importance of combining different techniques to unravel the mechanisms underlying disease suppression and the importance of studying function-over-phylogeny. Additionally, it also highlights the importance of organic amendments (such as oxalic acid) directly into soils in order to “engineer” the bacterial functions towards the control of diseases caused by R. solani.
Volatile-mediated interactions in the rhizosphere
Cordovez da Cunha, Viviane - \ 2016
Wageningen University. Promotor(en): Francine Govers; Jos Raaijmakers, co-promotor(en): V.J. Carrion. - Wageningen : Wageningen University - ISBN 9789462579019 - 219
rhizosphere bacteria - rhizosphere fungi - microbial interactions - volatile compounds - suppressive soils - actinobacteria - streptomyces - microbacterium - thanatephorus cucumeris - growth stimulators - biological control - defence mechanisms - genomics - transcriptomics - rizosfeerbacteriën - rizosfeerschimmels - microbiële interacties - vluchtige verbindingen - ziektewerende gronden - actinobacteria - streptomyces - microbacterium - thanatephorus cucumeris - groeistimulatoren - biologische bestrijding - verdedigingsmechanismen - genomica - transcriptomica
Plants and microorganisms are constantly engaged in highly dynamic interactions both above- and belowground. Several of these interactions are mediated by volatile organic compounds (VOCs), small carbon-based compounds with high vapor pressure at ambient temperature. In the rhizosphere, VOCs have an advantage in intra- and interorganismal signaling since they can diffuse through soil pores over longer distances than other metabolites and are not dependent on water availability. The research described in this PhD thesis explored how beneficial and pathogenic microorganisms that live in the rhizosphere and endosphere modulate plant growth, development and resistance via the production of VOCs. In vitro and in vivo bioassays as well as different ‘omic’ approaches, such as volatomics, transcriptomics and genomics, were employed to investigate underlying mechanisms of VOC-mediated microbe-microbe and microbe-plant interactions.
To investigate the diversity and functions of microbial VOCs, a disease-suppressive soil was used as the source of the VOC-producing microorganisms. Previous metagenomics studies reported Actinobacteria, in particular Streptomyces and Microbacterium species, as the most abundant bacterial genera found in a soil naturally suppressive to the fungal root pathogen Rhizoctonia solani. VOCs of several Streptomyces isolates inhibited hyphal growth of R. solani and in addition, promoted plant growth. Coupling the Streptomyces VOC profiles with their effects on fungal growth pinpointed methyl 2-methylpentanoate and 1,3,5-trichloro-2-methoxy benzene as antifungal VOCs. Also Microbacterium isolates showed VOC-mediated antifungal activity and plant growth promotion. VOC profiling of Microbacterium sp. EC8 revealed several sulfur-containing compounds and ketones such as dimethyl disulfide, trimethyl trisulfide and 3,3,6-trimethylhepta-1,5-dien-4-one (also known as Artemisia ketone). Genome analysis of strain EC8 revealed genes involved in sulfur metabolism. Resolving the role of the identified compounds and genes in VOC-mediated plant growth promotion and induced resistance will be subject of future studies. VOC-mediated chemical warfare underground has been proposed as a key mechanism of natural disease-suppressive soils. The results presented in this thesis indeed point in that direction. However, to more conclusively determine the role of the identified Actinobacterial VOCs in soil suppressiveness to R. solani, it will be important to demonstrate that the fungicidal VOCs are actually produced in situ at the right place and at sufficient concentrations to suppress plant infection by the pathogenic fungus.
In agriculture, VOCs and VOC-producing microorganisms provide a potential alternative to the use of pesticides to protect plants and to improve crop production. In the past decades, several in vitro studies have described the effects of microbial VOCs on other (micro)organisms. However, little is still known on the potential of VOCs in large-scale agriculture and horticulture. The results described in this thesis show that VOCs from Microbacterium sp. EC8 stimulate the growth of Arabidopsis, lettuce and tomato, but do not control damping-off disease of lettuce caused by R. solani. Significant biomass increases were also observed for plants exposed only shortly to the bacterial VOCs prior to transplantation of the seedlings to soil. These results indicate that VOCs from strain EC8 can prime plants for growth promotion without direct contact and prolonged colonization. Furthermore, the induction of the plant growth-promoting effects appeared to be plant tissue specific. Root exposure to the bacterial VOCs led to a significant increase in plant biomass whereas shoot exposure did not result in significant biomass increase of lettuce and tomato seedlings. Genome-wide transcriptome analysis of Arabidopsis seedlings exposed to VOCs from this bacterium showed an up-regulation of genes involved in sulfur and nitrogen metabolism and in ethylene and jasmonic acid signaling. These results suggest that the blend of VOCs of strain EC8 favors, in part, the plant’s assimilation of sulfate and nitrogen, essential nutrients for plant growth, development and also resistance.
Similar to beneficial microorganisms, plant pathogenic microorganisms have also evolved strategies to modulate growth and defense of their hosts. For instance, compounds secreted by pathogens may suppress or interfere with plant defense. In this thesis I show that R. solani produces an array of VOCs that promote growth, accelerate development, change VOC emission and reduce insect resistance of plants. Plant growth-promoting effects induced by the fungal VOCs were not transgenerational. Genome-wide transcriptome analysis of Arabidopsis seedlings revealed that exposure to fungal VOCs caused up-regulation of genes involved in auxin signaling, but down-regulation of genes involved in ethylene and jasmonic acid signaling. These findings suggest that this soil-borne pathogen uses VOCs to predispose plants for infection by stimulating lateral root formation and enhancing root biomass while suppressing defense mechanisms. Alternatively, upon perception of VOCs from soil-borne pathogens, plants may invest in root biomass while minimizing investments in defense, a trade-off that helps them to speed up growth and reproduction and to survive pathogen attack.
In conclusion, the research presented in this thesis shows that both plants and microorganisms engage via VOCs in long-distance interactions and that beneficial and pathogenic soil microorganisms can alter plant physiology in different ways. Here, I provided a first step in identifying microbial genes involved in the regulation of biologically active VOCs as well as candidate plant genes involved in VOC perception and signal transduction. How plants sense and differentiate among VOCs from beneficial and pathogenic soil microorganisms will be an intriguing subject for future studies.
Rhizobacterial modification of plant defenses against insect herbivores: from molecular mechanisms to tritrophic interactions
Pangesti, N.P.D. - \ 2015
Wageningen University. Promotor(en): Marcel Dicke; Joop van Loon. - Wageningen : Wageningen University - ISBN 9789462572836 - 224
planten - rizosfeerbacteriën - insecten - multitrofe interacties - verdedigingsmechanismen - pseudomonas fluorescens - mamestra brassicae - pieris brassicae - plant-microbe interacties - insect-plant relaties - plant-herbivoor relaties - plants - rhizosphere bacteria - insects - multitrophic interactions - defence mechanisms - pseudomonas fluorescens - mamestra brassicae - pieris brassicae - plant-microbe interactions - insect plant relations - plant-herbivore interactions
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.
Bodembacterie helpt plant tegen rupsenvraat
Sikkema, A. ; Pangesti, N.P.D. - \ 2015
Wageningen : St. voor Duurzame Ontwikkeling
arabidopsis - bodembacteriën - rizosfeerbacteriën - pseudomonas - gewasbescherming - rupsen - plaagresistentie - biologische bestrijding - landbouwkundig onderzoek - arabidopsis - soil bacteria - rhizosphere bacteria - pseudomonas - plant protection - caterpillars - pest resistance - biological control - agricultural research
Bodembacteriën die in het wortelmilieu van planten leven, verminderen de vatbaarheid van planten voor rupsenvraat. Dat blijkt uit onderzoek van Wageningse entomologen. In de modelplant Arabidopsis konden ze aantonen dat rhizobacteriën de plant in verhoogde staat van paraatheid brengen.
Biet gebaat bij juiste bacteriemix (interview met J. Raaijmakers)
Scharroo, J. ; Raaijmakers, J.M. - \ 2011
Bionieuws 21 (2011)10. - ISSN 0924-7734 - p. 5 - 5.
rizosfeerbacteriën - bodemweerbaarheid - bodembiologie - rizosfeer - suikerbieten - plantenziekten - akkerbouw - rhizosphere bacteria - soil suppressiveness - soil biology - rhizosphere - sugarbeet - plant diseases - arable farming
Aantallen bacteriën bepalen het ziektewerende vermogen van de bodem
Genotypic diversity and rhizosphere competence of antibiotic-producing Pseudomonas species
Bergsma-Vlami, M. - \ 2008
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jos Raaijmakers. - [S.l. : S.n. - ISBN 9789085049524 - 192
pseudomonas - antibiotica - rizosfeerbacteriën - rizosfeer - populatiedynamica - genetische diversiteit - biologische bestrijding - suikerbieten - pseudomonas - antibiotics - rhizosphere bacteria - rhizosphere - population dynamics - genetic diversity - biological control - sugarbeet
The phenolic antibiotic 2,4-diacetylphloroglucinol (DAPG) has been implicated in biological control of multiple plant pathogens by fluorescent Pseudomonas species. DAPG-producing Pseudomonas strains are effective biocontrol agents, however, their ecological performance is often highly variable resulting in inconsistent disease suppression. The ecological performance is complex and determined by many bacterial traits and environmental factors, including the host plant. In this thesis, several genotypic and phenotypic characteristics underlying the ecological performance of DAPG-producing Pseudomonas were investigated.
To discriminate between genotypically different DAPG-producing Pseudomonas strains directly in rhizosphere samples without their prior isolation or enrichment on nutrient media, a simple and rapid method was developed based on polymorphisms in the polyketide synthase gene phlD. Denaturing Gradient Gel Electrophoresis (DGGE) analysis, sequencing and phylogenetic analyses of indigenous phlD+ isolates obtained from the rhizosphere of wheat, sugar beet and potato plants, resulted in the identification of seven phlD+ genotypes, designated A, B, C, D, E, F, and Z, five of which were not described previously (C, D, E, F and Z). The phlD-DGGE analysis allowed simultaneous detection of multiple phlD+ isolates in the rhizosphere and, compared to cultivation-based approaches, this technique does not have the bias toward detecting either the most dominant genotype or the genotype with higher growth rates or competitive abilities during cultivation.
Subsequent studies with representative strains of each of the Pseudomonas genotypes showed that three genotypes (A, Z and G) were superior in long-term colonization of roots of wheat, sugar beet and potato plants. These results suggest that their rhizosphere competence is not linked to a specific plant species, but is due to yet unknown characteristics that enable these strains to be competitive in different rhizosphere environments. In contrast, the rhizosphere competence of Pseudomonas genotypes E, C and F was dependent on the plant species and, therefore, these strains are considered to be specialists instead of generalists.
Results of this thesis further showed that the host plant species also have a significant effect on DAPG-production by indigenous phlD+ Pseudomonas: the wheat and potato rhizospheres supported significantly higher amounts of DAPG produced per cell basis than the rhizospheres of sugar beet and lily. In the same context, the eight Pseudomonas genotypes differed significantly in their ability to produce DAPG in the rhizosphere of sugar beet plants with in situ DAPG concentrations ranging from 1 to 144 ng per 105 cells. Based on these data, significant correlations were established between the rhizosphere competence of a genotype and in situ DAPG production levels. In general, these correlations suggest that Pseudomonas genotypes that produce high amounts of DAPG per cell basis in situ establish lower population densities in the sugar beet rhizosphere than genotypes that produce small amounts of DAPG. To our knowledge, this is the first study that shows an inverse correlation between rhizosphere competence of Pseudomonas strains and in situ antibiotic production.
Biocontrol assays showed that P. ultimum was effectively controlled by all eight Pseudomonas strains and differential effects were observed in biocontrol activity against A. cochlioides. Pseudomonas genotype G was the most effective in biocontrol of Pythium and Aphanomyces damping-off, and its biocontrol activity was due, at least in part, to DAPG production as its DAPG-deficient mutant was significantly less effective. Comparative analysis of the eight DAPG-producing Pseudomonas genotypes revealed a highly significant correlation between their rhizosphere competence and efficacy to control Aphanomyces damping-off of sugar beet. These results indicate that the more rhizosphere competent DAPG-producing Pseudomonas strains are, the higher their efficacy is to control A. cochlioides in sugar beet. The promising results obtained with genotypes A, Z and G in the sugar beet bioassays provide a strong basis for their implementation in the current integrated disease management strategies in sugar beet.
The results acquired in this thesis have shown that the identification of the genotypic diversity and rhizosphere competence of antibiotic-producing Pseudomonas species is of great value, because it may allow maximizing root colonization and disease suppression. Knowledge of genetic traits involved in host preference of these antagonistic bacteria will help to identify strains that are adequately adapted to specific host-pathogen systems. Similarly, looking into plant traits that promote the growth and activity of introduced biocontrol strains can be highly complementary and further contribute to sustainability in agriculture.