Nicotiana attenuata microbiome characterization and plant-bacterial interactions from single isolates to consortia

Santhanam, Rakesh GND

Plants rely on soil microbes for beneficial interactions i.e., growth promotion, nutrient availability and disease suppression. Biotic and abiotic factors shaping the root microbiome are still largely unknown. Furthermore, why and how plants acquire soil microbes during germination and their ecological and functional roles remain elusive. Most of the high-throughput sequencing studies are limited to model or crop plants and lack the complementation of a culture-dependent approach to reveal functional traits of dominant and rare microbial communities. Until now, poly-microbial disease complex and poly-microbial biocontrol application received little attention in the scientific community. In this dissertation, my work aims to address these intriguing questions. During my research work, I investigated the influence of the phytohormone jasmonic acid (JA) and plant developmental stages on leaf and root bacterial communities in wild tobacco N. attenuata. Roots and leaves of field grown plants of a previously characterized, isogenic line impaired in JA biosynthesis (irAOC) and an empty vector (EV) control line were harvested over five different developmental stages from rosette to flowering stages. A 454- pyrosequencing and culture dependent method were employed to characterize the bacterial communities. Neither JA nor plant developmental stages shaped the bacterial communities; however, tissue type had a major effect on community composition. Roots harbored higher bacterial diversity compared to leaves, irrespective of genotypes. The culture-independent results were further validated by a culture-dependent approach and demonstrated that JA does not influence the bacterial community composition of N. attenuata. Furthermore, bacterial plant growth promoting (PGP) effects are independent of the plant’s ability to produce endogenous JA. Moreover, I hypothesized abiotic factor such as soil types and the plant’s response to UVB exposure shape the N. attenuata root microbiome. To test this hypothesis, we harvested native grown N. attenuata from different locations at the Great Basin Desert, Utah. Deep-sequencing analysis revealed that root bacterial communities were independent of soil types. Moreover, root bacterial communities from five different locations clustered separately compared to soil types in a non-metric multi-dimensional scaling plot (NMDS), although chemical properties of soils are different. Diversity of soil samples is higher compared to roots and plants selectively recruit the microbial communities from soil, irrespective of location and genotypes. However, fungal recruitment is less specific by plants. Interestingly, in N. attenuata, the phylum Deinococcus-Thermus is unique compared to other plant species and more abundant in roots than in soils, which indicates selective enrichment of Deinococcus members in N. attenuata roots. Based on the UV resistant trait of Deinococcus-Thermus and the native habitat of N. attenuata characterized by high UVB fluence rates.We hypothesized that, plants response to UVB exposure and its UVB perception receptor UV RESISTANCE LOCUS 8 (UVR8) and response -flavonoid biosynthesis enzyme chalcone synthase (CHAL) – influences the colonization of Deinococcus- Thermus. We generated an isogenic line impaired in UVR8 expression (irUVR8) and used it along with a previously characterized UVB response line (irCHAL). In a microcosm experiment with a synthetic bacterial community, Deinococcus highly colonized wild type roots under UVB exposure, but not the isogenic lines impaired in UVB perception (irUVR8) and response (irCHAL). In a consecutive study, I addressed the current agricultural dilemma and long festering problem of monoculture practice and fungal outbreaks. The continuous usage of a field plot in N. attenuata’s native habitat using an agricultural set-up for the past 15 years led to the emergence of sudden wilt disease caused by fungal pathogen Fusarium and Alternaria disease complex. Sudden wilt disease is characterized by sudden collapse of the vascular system associated with roots turning black. Three different strategies such as microbial biocontrol, chemical fungicide and soil amendment were tested under field conditions. Among 7 different treatments, only bacterial consortia protected the plant from sudden wilt disease in 2013 field trial. In the consecutive year field trial 2014, bacterial consortia attenuated the sudden wilt disease, demonstrating the robustness of the protection effect. Bacterial protection effect is independent of genotypes and does not influence ecological traits of N. attenuata. Furthermore, bacterial consortia can be re-isolated from previously bacterial inoculated healthy plants indicating that these native consortia are excellent root colonizers. In conclusion, this work demonstrates that N. attenuata’s microbiome is not significantly sculpted by JA, plant developmental stages and soil types, whereas, tissue types and UVB supplementation influence it. In addition, this thesis addresses the importance of poly-microbial solutions to enhance the plants’ tolerance against poly-microbial disease complexes in an agricultural context; and it delineates the functional and ecological role of procurement of microbial communities by plants during germination.

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Santhanam, Rakesh: Nicotiana attenuata microbiome characterization and plant-bacterial interactions from single isolates to consortia. Jena 2016.

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