Pim Goossens

Plants can respond to pathogen attack by assembling disease-suppressive microbiomes. In Arabidopsis thaliana, infection by the obligate foliar downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) consistently led to the formation of a soil microbial community, referred to as “soilborne legacy” (SBL), that enhanced resistance in subsequent plant populations grown in the same soil. Previous work identified an enrichment of specific “Hpa-associated microbiota” (HAM) in the phyllospheres of infected plants, which suppressed pathogen proliferation. Here, we demonstrate how the assembly of protective HAM in the phyllosphere contributes to a disease-suppressive SBL. We identified a community of 25 core-HAM that consistently dominated the phyllospheres of 14 sets of distinct Hpa-infected plant populations across six independent experiments. Using HAM-free, gnotobiotic Hpa spores, the infection-driven assembly of a core-HAM representative was recapitulated, showing de novo and progressive enrichment under sustained disease pressure. Despite being transmitted via soil as SBL, HAM are phyllosphere specialists with infected leaves as their primary niche. Disease-induced HAM assembly is initiated in the phyllosphere rather than the rhizosphere, and once transmitted, they particularly accumulate on aboveground tissues. Leaf wash-offs from plant populations that inherited SBL were shown to effectively suppress downy mildew disease when applied to leaves of plants grown in unconditioned soil. These findings reveal that downy mildew disease–suppressive soils transmit a protective core microbiome to the phyllosphere, highlighting a crucial link between belowground and aboveground plant-driven microbiome assembly processes. Paradoxically, the phyllosphere thus emerges as a key assembly hub for disease-suppressive soil microbiomes.

Plants host diverse microbial communities that can be influenced by their hosts to mitigate biotic stress. Previous research demonstrated that distinct laboratory cultures of Hyaloperonospora arabidopsidis (Hpa) on Arabidopsis thaliana , consistently harbor nearly identical bacteria. In this study, we analyzed the bacterial phyllosphere communities of laboratory-grown spinach plants infected by the downy mildew pathogen Peronospora effusa (Pe). Using 16S amplicon sequencing, we identified 14 Amplicon Sequence Variants (ASVs), with diverse taxonomies, that were enriched in at least 3 out of 5 investigated Pe cultures. This small set of 14 ASVs occupied on average 6.9% of the total bacterial communities in healthy spinach plants, and 43.1% in Pe-inoculated plants. A specific Rhodococcus and a Paenarthrobacter ASV were particularly prevalent and abundant. To validate these findings outside of the laboratory, we planted a susceptible variety of spinach in 4 agricultural fields and sampled leaves from Pe-infected plants in 2 fields where this pathogen naturally occurred. Comparative microbiome analysis of diseased and healthy plants revealed significant enrichment of 16 and 31 ASVs in these 2 fields, respectively. Among these, the Paenarthrobacter ASV was enriched in one field and the Rhodococcus ASV in the other field, suggesting that disease-associated microbiota that are abundantly detected in Pe laboratory cultures are also associated with Pe-infected field plants. Additionally, we observed an overlap of ASVs that were associated with both Pe and Hpa, indicating that similar bacteria are linked to downy mildew disease across different hosts.

Hyaloperonospora arabidopsidis (Hpa) is an obligately biotrophic downy mildew that is routinely cultured on Arabidopsis thaliana hosts that harbour complex microbiomes. We hypothesized that the culturing procedure proliferates Hpa-associated microbiota (HAM) in addition to the pathogen and exploited this model system to investigate which microorganisms consistently associate with Hpa. Using amplicon sequencing, we found nine bacterial sequence variants that are shared between at least three out of four Hpa cultures in the Netherlands and Germany and comprise 34% of the phyllosphere community of the infected plants. Whole-genome sequencing showed that representative HAM bacterial isolates from these distinct Hpa cultures are isogenic and that an additional seven published Hpa metagenomes contain numerous sequences of the HAM. Although we showed that HAM benefit from Hpa infection, HAM negatively affect Hpa spore formation. Moreover, we show that pathogen-infected plants can selectively recruit HAM to both their roots and shoots and form a soil-borne infection-associated microbiome that helps resist the pathogen. Understanding the mechanisms by which infection-associated microbiomes are formed might enable breeding of crop varieties that select for protective microbiomes.