R.F. Doornbos's research while affiliated with Utrecht University and other places

Microbial communities that are associated with plant roots are highly diverse and harbor tens of thousands of species. This so-called microbiome controls plant health through several mechanisms including the suppression of infectious diseases, which is especially prominent in disease suppressive soils. The mechanisms implicated in disease suppression include competition for nutrients, antibiosis, and induced systemic resistance (ISR). For many biological control agents ISR has been recognized as the mechanism that at least partly explains disease suppression. Implications of ISR on recruitment and functioning of the rhizosphere microbiome are discussed.

The rhizosphere was defined over 100 years ago as the zone around the root where microorganisms and processes important for plant growth and health are located. Recent studies show that the diversity of microorganisms associated with the root system is enormous. This rhizosphere microbiome extends the functional repertoire of the plant beyond imagination. The rhizosphere microbiome of Arabidopsis thaliana is currently being studied for the obvious reason that it allows the use of the extensive toolbox that comes with this model plant. Deciphering plant traits that drive selection and activities of the microbiome is now a major challenge in which Arabidopsis will undoubtedly be a major research object. Here we review recent microbiome studies and discuss future research directions and applicability of the generated knowledge.

Despite significant advances in crop protection, plant diseases cause a 20% yield loss in food and cash crops worldwide. Therefore, interactions between plants and pathogens have been studied in great detail. In contrast, the interplay between plants and non-pathogenic microorganisms has received scant attention, and differential responses of plants to pathogenic and non-pathogenic microorganisms are as yet not well understood. Plants affect their rhizosphere microbial communities that can contain beneficial, neutral and pathogenic elements. Interactions between the different elements of these communities have been studied in relation to biological control of plant pathogens. One of the mechanisms of disease control is induced systemic resistance (ISR). Studies on biological control of plant diseases have focused on ISR the last decade, because ISR is effective against a wide range of pathogens and thus offers serious potential for practical applications in crop protection. Such applications may however affect microbial communities associated with plant roots and interfere with the functioning of the root microbiota. Here, we review the possible impact of plant defense signaling on bacterial communities in the rhizosphere. To better assess implications of shifts in the rhizosphere microflora we first review effects of root exudates on soil microbial communities. Current knowledge on inducible defense signaling in plants is discussed in the context of recognition and systemic responses to pathogenic and beneficial microorganisms. Finally, the as yet limited knowledge on effects of plant defense on rhizosphere microbial communities is reviewed and we discuss future directions of research that will contribute to unravel the molecular interplay of plants and their beneficial microflora.

Systemically induced resistance is a promising strategy to control plant diseases, as it affects numerous pathogens. However, since induced resistance reduces one or both growth and activity of plant pathogens, the indigenous microflora may also be affected by an enhanced defensive state of the plant. The aim of this study was to elucidate how much the bacterial rhizosphere microflora of Arabidopsis is affected by induced systemic resistance (ISR) or systemic acquired resistance (SAR). Therefore, the bacterial microflora of wild-type plants and plants affected in their defense signaling was compared. Additionally, ISR was induced by application of methyl jasmonate and SAR by treatment with salicylic acid or benzothiadiazole. As a comparative model, we also used wild type and ethylene-insensitive tobacco. Some of the Arabidopsis genotypes affected in defense signaling showed altered numbers of culturable bacteria in their rhizospheres; however, effects were dependent on soil type. Effects of plant genotype on rhizosphere bacterial community structure could not be related to plant defense because chemical activation of ISR or SAR had no significant effects on density and structure of the rhizosphere bacterial community. These findings support the notion that control of plant diseases by elicitation of systemic resistance will not significantly affect the resident soil bacterial microflora.

Pseudomonas putida KT2440 is an efficient colonizer of the rhizosphere of plants of agronomical and basic interest. We have demonstrated that KT2440 can protect the model plant Arabidopsis thaliana against infection by the phytopathogen Pseudomonas syringae pv. tomato DC3000. P. putida extracellular haem-peroxidase (PP2561) was found to be important for competitive colonization and essential for the induction of plant systemic resistance. Root exudates of plants elicited by KT2440 exhibited distinct patterns of metabolites compared with those of non-elicited plants. The levels of some of these compounds were dramatically reduced in axenic plants or plants colonized by a mutant defective in PP2561, which has increased sensitiveness to oxidative stress with respect to the wild type. Thus high-level oxidative stress resistance is a bacterial driving force in the rhizosphere for efficient colonization and to induce systemic resistance. These results provide important new insight into the complex events that occur in order for plants to attain resistance against foliar pathogens.

In the rhizosphere, numerous microbial and plant-microbe interactions occur. Of special interest is the ability of specific rhizosphere bacteria to elicit induced systemic resistance (ISR), a state of enhanced defensive capacity of the plant that is effective against a wide range of pathogens. The goal to minimize the use of agrochemicals in crop protection stimulates the development of practical applications of ISR-eliciting bacteria as an environmentally friendly alternative. However, application of these bacteria on a large scale and at high densities may perturb the indigenous microflora. This thesis is focused on effects of plant defense signaling on the indigenous bacterial rhizosphere microflora. Population densities of the bacterial and Pseudomonas spp. microflora were determined by selective dilution plating, whereas bacterial community structures were studied by DGGE analysis of amplified 16S rDNA, obtained from DNA directly extracted from rhizosphere and bulk soil. As model plant we used Arabidopsis thaliana accession Col-0, since numerous defense signaling mutants and transgenic lines are available and rhizobacteria-mediated ISR has been characterized in detail in this species. To determine if the indigenous bacterial microflora in the rhizosphere is affected by plant defenses, Arabidopsis genotypes with altered defense signaling were used. Whereas no differences were observed on microbial community structures, in some defense signal-transduction mutants rhizosphere population densities of culturable bacteria or Pseudomonas spp. were different from those of the parent Col-0. These differences were observed only in one type of soil. Apparently, soil is a predominant factor shaping microbial communities. In a complementary approach, jasmonic acid (JA)- or salicylic acid (SA)-dependent defenses were chemically activated by application of these hormones. Neither the abundance, nor the community structure of the bacterial rhizosphere microflora was affected by activation of the JA- or SA-dependent responses. Whereas Pseudomonas putida WCS358r and Pseudomonas fluorescens WCS417r elicit ISR against the bacterial leaf pathogen Pseudomonas syringae pv. tomato (Pst) in Arabidopsis, P. fluorescens WCS374r does not. The root-colonizing capacity of these three bacterial strains was studied on wild-type Arabidopsis and on a non ISR-expressing mutant, myb72. Whereas WCS358r and WCS417r proliferated on the roots of the wild type, this was not the case for WCS374r. However, none of the strains proliferated on the roots of the myb72 mutant. Apparently, MYB72 is not only essential for the expression of ISR, but also influences root colonization by rhizobacteria. Metabolic profiling revealed that treatment of wild-type plants and the myb72 mutant with the Pseudomonas spp. strains significantly altered the amounts of sugars, organic acids and amino acids. Most annotated metabolite fragments could be linked to known plant-microbe or plant-pathogen interactions, but not to the expression of ISR. Finally, population densities of total culturable bacteria and Pseudomonas spp. in the phyllosphere were determined upon infection with Pst. Arabidopsis mutants differed in their sensitivity to Pst and the most sensitive mutants also had the highest bacterial and Pseudomonas spp. populations on their leaves. Collectively, these results suggest that control of plant diseases by elicitation of induced systemic resistance will not significantly affect the indigenous rhizosphere bacterial microflora.

A long-term field experiment (1999-2002) was conducted to monitor effects on the indigenous microflora of Pseudomonas putida WCS358r and two transgenic derivatives constitutively producing phenazine-1-carboxylic acid (PCA) or 2,4-diacetylphloroglucinol (DAPG). The strains were introduced as seed coating on wheat into the same field plots each year. Rhizosphere populations of ascomycetes were analysed using denaturing gradient gel electrophoresis (DGGE). To evaluate the significance of changes caused by the genetically modified microorganisms (GMMs), they were compared with effects caused by a crop rotation from wheat to potato. In the first year, only the combination of both GMMs caused a significant shift in the ascomycete community. After the repeated introductions this effect was no longer evident. However, cropping potato significantly affected the ascomycete community. This effect persisted into the next year when wheat was grown. Clone libraries were constructed from samples taken in 1999 and 2000, and sequence analysis indicated ascomycetes of common genera to be present. Most species occurred in low frequencies, distributed almost evenly in all treatments. However, in 1999 Microdochium occurred in relatively high frequencies, whereas in the following year no Microdochium species were detected. On the other hand, Fusarium-like organisms were low in 1999, and increased in 2000. Both the DGGE and the sequence analysis revealed that repeated introduction of P. putida WCS358r had no major effects on the ascomycete community in the wheat rhizosphere, but demonstrated a persistent difference between the rhizospheres of potato and wheat.