Inter-species interactions between two bacterial flower commensals and a floral pathogen reduce disease incidence and alter pathogen activity.

Flowers are colonized by a diverse community of microorganisms that can alter plant health and interact with floral pathogens. Erwinia amylovora is a flower-inhabiting bacterium and a pathogen that infects different plant species, including Malus × domestica (apple). Previously, we showed that the co-inoculation of two bacterial strains, members of the genera Pseudomonas and Pantoea, isolated from apple flowers, reduced disease incidence caused by this floral pathogen. Here, we decipher the ecological interactions between the two flower-associated bacteria and E. amylovora in field experimentation and in vitro co-cultures. The two flower commensal strains did not competitively exclude E. amylovora from the stigma habitat, as both bacteria and the pathogen co-existed on the stigma of apple flowers and in vitro. This suggests that plant protection might be mediated by other mechanisms than competitive niche exclusion. Using a synthetic stigma exudation medium, ternary co-culture of the bacterial strains led to a substantial alteration of gene expression in both the pathogen and the two microbiota members. Importantly, the gene expression profiles for the ternary co-culture were not just additive from binary co-cultures, suggesting that some functions only emerged in multipartite co-culture. Additionally, the ternary co-culture of the strains resulted in a stronger acidification of the growth milieu than mono- or binary co-cultures, pointing to another emergent property of co-inoculation. Our study emphasizes the critical role of emergent properties mediated by inter-species interactions within the plant holobiont and their potential impact on plant health and pathogen behavior. IMPORTANCE: Fire blight, caused by Erwinia amylovora, is one of the most important plant diseases of pome fruits. Previous work largely suggested plant microbiota commensals suppressed disease by antagonizing pathogen growth. However, inter-species interactions of multiple flower commensals and their influence on pathogen activity and behavior have not been well studied. Here, we show that co-inoculating two bacterial strains that naturally colonize the apple flowers reduces disease incidence. We further demonstrate that the interactions between these two microbiota commensals and the floral pathogen led to the emergence of new gene expression patterns and a strong alteration of the external pH, factors that may modify the pathogen's behavior. Our findings emphasize the critical role of emergent properties mediated by inter-species interactions between plant microbiota and plant pathogens and their impact on plant health.

Nanoscale sulfur alters the bacterial and eukaryotic communities of the tomato rhizosphere and their interactions with a fungal pathogen.

Nanoformulations of sulfur have demonstrated the potential to enhance plant growth and reduce disease incidence when plants are confronted with pathogens. However, the impact of nanoscale sulfur on microbial communities in close contact with the plant root, known as the rhizosphere, remain poorly characterized. In this study, we investigate the impact of three formulations of sulfur; bulk sulfur, uncoated (pristine) sulfur nanoparticles, and stearic acid coated sulfur nanoparticles, on the rhizosphere of tomato plants. Tomato plants were additionally challenged by the pathogenic fungus Fusarium oxysporum f. sp. Lycopersici. Employing bacterial 16S rRNA gene sequencing, along with recently in-house designed peptide nucleic acid clamps to facilitate the recovery of microeukaryote sequences, we performed a comprehensive survey of rhizosphere microbial populations. We found the largest influence on the composition of the rhizosphere microbiome was the presence of the fungal pathogen. However, sulfur amendments also drove state changes in the rhizosphere populations; for example, enriching the relative abundance of the plant-beneficial sulfur-oxidizing bacterium Thiobacillus. Notably, when investigating the response of the rhizosphere community to the different sulfur amendments, there was a strong interaction between the fungal pathogen and sulfur treatments. This resulted in different bacterial and eukaryotic taxa being enriched in association with the different forms of sulfur, which was dependent on the presence of the pathogen. These data point to nano formulations of sulfur exerting unique shifts in the rhizosphere community compared to bulk sulfur, particularly in association with a plant pathogen, and have implications for the sustainable use of nanoscale strategies in sustainable agriculture.

Introducing an environmental microbiome to axenic Aedes aegypti mosquitoes documents bacterial responses to a blood meal.

IMPORTANCE: The blood meal of the female mosquito serves as a nutrition source to support egg development, so is an important aspect of its biology. Yet, the roles the microbiome may play in blood digestion are poorly characterized. We employed axenic mosquitoes to investigate how the microbiome differs between mosquitoes reared in the insectary versus mosquitoes that acquire their microbiome from the environment. Environmental microbiomes were more diverse and showed larger temporal shifts over the course of blood digestion. Importantly, only bacteria from the environmental microbiome performed hemolysis in culture, pointing to functional differences between bacterial populations. These data highlight that taxonomic differences between the microbiomes of insectary-reared and wild mosquitoes are potentially also related to their functional ecology. Thus, axenic mosquitoes colonized with environmental bacteria offer a way to investigate the role of bacteria from the wild in mosquito processes such as blood digestion, under controlled laboratory conditions.

Therapeutic Delivery of Nanoscale Sulfur to Suppress Disease in Tomatoes: In Vitro Imaging and Orthogonal Mechanistic Investigation.

Nanoscale sulfur can be a multifunctional agricultural amendment to enhance crop nutrition and suppress disease. Pristine (nS) and stearic acid coated (cS) sulfur nanoparticles were added to soil planted with tomatoes (Solanum lycopersicum) at 200 mg/L soil and infested with Fusarium oxysporum. Bulk sulfur, ionic sulfate, and healthy controls were included. Orthogonal end points were measured in two greenhouse experiments, including agronomic and photosynthetic parameters, disease severity/suppression, mechanistic biochemical and molecular end points including the time-dependent expression of 13 genes related to two S bioassimilation and pathogenesis-response, and metabolomic profiles. Disease reduced the plant biomass by up to 87%, but nS and cS amendment significantly reduced disease as determined by area-under-the-disease-progress curve by 54 and 56%, respectively. An increase in planta S accumulation was evident, with size-specific translocation ratios suggesting different uptake mechanisms. In vivo two-photon microscopy and time-dependent gene expression revealed a nanoscale-specific elemental S bioassimilation pathway within the plant that is separate from traditional sulfate accumulation. These findings correlate well with time-dependent metabolomic profiling, which exhibited increased disease resistance and plant immunity related metabolites only with nanoscale treatment. The linked gene expression and metabolomics data demonstrate a time-sensitive physiological window where nanoscale stimulation of plant immunity will be effective. These findings provide mechanistic understandings of nonmetal nanomaterial-based suppression of plant disease and significantly advance sustainable nanoenabled agricultural strategies to increase food production.

The Axenic and Gnotobiotic Mosquito: Emerging Models for Microbiome Host Interactions.

The increasing availability of modern research tools has enabled a revolution in studies of non-model organisms. Yet, one aspect that remains difficult or impossible to control in many model and most non-model organisms is the presence and composition of the host-associated microbiota or the microbiome. In this review, we explore the development of axenic (microbe-free) mosquito models and what these systems reveal about the role of the microbiome in mosquito biology. Additionally, the axenic host is a blank template on which a microbiome of known composition can be introduced, also known as a gnotobiotic organism. Finally, we identify a "most wanted" list of common mosquito microbiome members that show the greatest potential to influence host phenotypes. We propose that these are high-value targets to be employed in future gnotobiotic studies. The use of axenic and gnotobiotic organisms will transition the microbiome into another experimental variable that can be manipulated and controlled. Through these efforts, the mosquito will be a true model for examining host microbiome interactions.

What's under the Christmas Tree? A Soil Sulfur Amendment Lowers Soil pH and Alters Fir Tree Rhizosphere Bacterial and Eukaryotic Communities, Their Interactions, and Functional Traits.

In this study, we describe the legacy effects of a soil sulfur amendment experiment performed 6 years prior and the resulting alterations to the rhizosphere communities of fir trees on a Christmas tree plantation. The pH of bulk soil was ∼1.4 pH units lower than that of untreated soils and was associated with reduced Ca, Mg, and organic matter contents. Similarly, root chemistry differed due to the treatment, with roots in sulfur-amended soils showing significantly higher Al, Mn, and Zn contents and reduced levels of B and Ca. 16S rRNA and 18S rRNA gene sequencing was pursued to characterize the bacterial/archaeal and eukaryotic communities in the rhizosphere soils. The treatment induced dramatic and significant changes in the microbial populations, with thousands of 16S rRNA gene sequence variants and hundreds of 18S rRNA gene variants being significantly different in relative abundances between the treatments. Additionally, co-occurrence networks showed that bacterial and eukaryotic interactions, network topology, and hub taxa were significantly different when constructed from the control and treated soil 16S and 18S rRNA gene amplicon libraries. Metagenome sequencing identified several genes related to transport proteins that differentiated the functional potentials of the communities between treatments, pointing to physiological adaptations in the microbial communities for living at altered pH. These data show that a legacy of soil acidification increased the heterogeneity of the soil communities as well as decreasing taxon connections, pointing to a state of ecosystem instability that has potentially persisted for 6 years. IMPORTANCE We used sulfur incorporation to investigate the legacy effects of lowered soil pH on the bacterial and eukaryotic populations in the rhizosphere of Christmas trees. Acidification of the soils drove alterations of fir tree root chemistry and large shifts in the taxonomic and functional compositions of the communities. These data demonstrate that soil pH influences are manifest across all organisms inhabiting the soil, from the host plant to the microorganisms inhabiting the rhizosphere soils. Thus, this study highlights the long-lasting influence of altering soil pH on soil and plant health as well as the status of the microbiome.