Volatile-mediated interactions in the rhizosphere

<p>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 <em>via</em> the production of VOCs. <em>In vitro</em> and <em>in vivo</em> 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.</p> <p>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 <em>Streptomyces</em> and <em>Microbacterium</em> species, as the most abundant bacterial genera found in a soil naturally suppressive to the fungal root pathogen <em>Rhizoctonia solani</em>. VOCs of several <em>Streptomyces</em> isolates inhibited hyphal growth of <em>R. solani</em> and in addition, promoted plant growth. Coupling the <em>Streptomyces</em> VOC profiles with their effects on fungal growth pinpointed methyl 2-methylpentanoate and 1,3,5-trichloro-2-methoxy benzene as antifungal VOCs. Also <em>Microbacterium</em> isolates showed VOC-mediated antifungal activity and plant growth promotion. VOC profiling of <em>Microbacterium</em> 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 <em>R. solani</em>, it will be important to demonstrate that the fungicidal VOCs are actually produced <em>in situ</em> at the right place and at sufficient concentrations to suppress plant infection by the pathogenic fungus.</p> <p>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 <em>in vitro</em> 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 <em>Microbacterium</em> sp. EC8 stimulate the growth of <em>Arabidopsis</em>, lettuce and tomato, but do not control damping-off disease of lettuce caused by <em>R. solani</em>. 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 <em>Arabidopsis</em> 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.</p> <p>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 <em>R. solani</em> 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 <em>Arabidopsis</em> 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.</p> <p>In conclusion, the research presented in this thesis shows that both plants and microorganisms engage <em>via </em>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.</p> <p>