Characterization of Plant-Microbial Interactions in the Thousand Cankers Disease Complex using Amplicon Sequencing, Transcriptomics, and Genomics

Walnut (Juglans spp.) trees grown for timber and nut production are threatened by thousand cankers disease in parts of the United States and Europe. The disease is caused by the fungus Geosmithia morbida and bark beetle vector, Pityophthorus juglandis. The most susceptible host, eastern black walnut (J. nigra), typically dies 2-to-3 years following infection and is more severely affected outside of its native range. Despite initial description of the disease in 2009, our understanding of the ecology of the disease and methods for disease management are limited. Thus, our overarching objective was to characterize pathogen interactions with J. nigra and its associated microorganisms and assess the efficacy of potential methods for disease management.

We determined that J. nigra microbial communities differed between parts of the host’s native and introduced ranges and that these differences may modulate disease severity. In support of this, we recovered endophytic Trichoderma isolates that produced metabolites capable of inhibiting G. morbida growth in vitro from trees in the native range. We also found that the commercial biological control strain, T. afroharzianum KRL-AG2 (RootShield®) suppressed G. morbida in dual-plate assays, making it a possible biological management tool. When assessing the efficacy of RootShield® and the phosphorous acid fungicide, PHOSPHO-jet™, we found that neither treatment reduced canker sizes and found no detectable differences in host defense gene expression following treatment. However, PHOSPHO-jet™ treatments had non-target effects on J. nigra microbial communities, potentially affecting host health. From this study, we also determined that J. nigra defense responses to G. morbida were mediated by salicylic acid and dampen after a single inoculation event. We also identified downregulated putative G. morbida effectors that were absent from the genomes of other Geosmithia species. Exploration of G. morbida genomes resulted in the detection of genetic polymorphisms in these unique effectors that were predicted to result in missense mutations, potentially influencing G. morbida virulence. This research has deepened our understanding of molecular host-pathogen interactions, the roles of host-associated microorganisms in modulating thousand cankers disease severity and allowed us to identify how potential disease management strategies affect host health holistically.