The root rot complex of pea - Screening for resistance and quantification of microbial key players in the rhizosphere

Pea (Pisum sativum L.) is a valuable and healthy protein source for food and feed. In addition to the nutritional benefits, pea is an invaluable agro-ecological asset for sustainable cropping systems through positive effects on soil fertility and soil microbial diversity. The symbiosis with nitrogen-fixing bacteria allows pea and other legume crops to supply the soil with nitrogen and, therefore, to significantly reduce the application of external nitrogen fertilisers. Therefore, pea plays an important role especially in low-input farming systems. The growing market for plant- based protein supply is likely to promote pea cultivation in the near future. However, pea production is severely challenged by various soil-borne pathogens that form a Pea Root Rot Complex (PRRC) causing root-rot diseases. Despite considerable progress in resistance breeding against individual pathogens, current pea varieties lack resistance against multiple interacting pathogens. The overall goal of this thesis was to contribute to the understanding of resistance against root rot pathogen complexes in pea. Chapter 1 gives an overview of the importance of pea as a future key player in agricultural systems and the food sector before introducing the pea root rot complex concept and its relevance for research on resistance. Furthermore, the most recent developments in molecular biology relevant for molecular plant breeding of pea are briefly summarised and an overview of quantitative real-time PCR relevant for research on microbial interactions in the pea root rot complex is given. Chapter 2 reviews the current knowledge of resistance against root- rot pathogens in major grain legumes, highlights the importance of the host genotype in determining the composition of plant-associated microbial communities and how the root associated microbiome relates to plant health. In addition, major findings on the role of root exudation in disease susceptibility and resistance of grain legumes are summarised. Finally, it delineates how this knowledge could be integrated in resistance breeding of grain legumes. In Chapter 3, a resistance screening assay was established based on infested soil from an agricultural field that showed severe pea root rot pressure. This approach was chosen in order to account for the whole rhizosphere microbiome - including the naturally occuring pathogen complex - in the assessment of root rot resistance in pea. The initial ITS- amplicon sequencing of the fungal rhizosphere community of diseased pea roots grown in the infested soil showed a root community of evenly abundant fungal taxonomic units not dominated by a few taxa. This finding points at complex interactions within the PRRC. Two hundred and sixty-one pea cultivars, landraces and breeding lines were screened for resistance on the naturally infested field soil in a controlled conditions experiment. The screening system allowed for a reproducible assessment of disease parameters among the tested genotypes. Broad sense heritabilities on the infested soil were H2 = 0.89 for plant emergence, H2 = 0.43 for root rot index and H2 = 0.51 for relative shoot dry weight. The resistance ranking was verified in an on-farm experiment with nine pea genotypes in two field sites: The controlled conditions root rot index showed a significant correlation with the resistance ranking in the field site with high PRRC infestation (Spearman's ρ = 0.73, p = .03). The screening system offers a tool for selection at early stages of the plant development, and for the study of plant resistance in the light of complex plant-microbe interactions. For Chapter 4, a subset of five resistant and three susceptible pea genotypes was selected based on the initial screening. In analogy to the previous experiment, a controlled conditions experiment was setup up in order to assess and validate resistance of the eight pea genotypes on four soils. Plant growth was significantly reduced on the three sick soils compared to the healthy soil. Despite the significantly different levels of disease pressure in the three infested soils (ANOVA: p < .001) and the strong genotype effect (p < .001), no significant soil × genotype interaction (p < .342) was found for plant growth reduction. In addition to disease assessments, ten key microbial taxa (eight putative pea pathogens and two putative beneficials) were quantified in the roots by quantitative real-time PCR (qPCR). Fusarium solani, F. oxysporum and Aphanomyces euteiches were the most abundant pathogens in diseased roots from the three sick soils. Further, various levels of the pathogens F. avenaceum, F. redolens, Rhizoctonia solani, D. pinodella and Pythium sp. as well as the potential antagonist Clonostachys rosea were quantified by qPCR. The contribution of individual pathogens to root rot and growth reduction differed among the three sick soils: F. solani and F. oxysporum showed significant correlations (Spearman correlations; p < 0.05) with root rot index and relative shoot dry weight in the two soils with the highest infestation level; A. euteiches showed significant relations with disease in two sick soils from Germany. The quantities of arbuscular mycorrhizal fungi were negatively correlated with root rot index and positively correlated with relative shoot dry weight in all sick soils. Furthermore, the root microbial composition differed significantly among the pea genotypes (PERMANOVA; p < .0001) and the soils (p < .0001) and a significant pea genotype × soil interaction was evidenced (p < .0001). In addition, resistant pea genotypes showed significantly lower F. solani and A. euteiches, and higher arbuscular mycorrhizal fungi abundance in the roots (Wilcoxon rank-sum test; p < .05). These results give insights into the complex interaction between key microorganisms of the PRRC and the plant, by pointing out potential key microorganisms in the root rot pathobiome. Further disentanglement of this complex and the validation of key microbial players can be harnessed by resistance breeding. Chapter 5 reviews the experimental approaches and results from the previous chapters before discussing the major findings and implications for future research and resistance breeding. I also raise the question if and how knowledge about complex soil microorganisms-plant feedbacks can be incorporated in resistance screenings and breeding efforts to conclude that today we are at a point where information on microbial complexes could indeed assist resistance breeding. However, our current state of knowledge does not yet allow to design specific microbiome-enabled selection-tools. This last chapter will also give short outlooks and indicate possible future lines of research in the field of microbe-mediated plant resistance