Defining Composition and Function of the Rhizosphere Microbiota of Barley Genotypes Exposed to Growth-Limiting Nitrogen Supplies.

The microbiota populating the rhizosphere, the interface between roots and soil, can modulate plant growth, development, and health. These microbial communities are not stochastically assembled from the surrounding soil, but their composition and putative function are controlled, at least partially, by the host plant. Here, we use the staple cereal barley as a model to gain novel insights into the impact of differential applications of nitrogen, a rate-limiting step for global crop production, on the host genetic control of the rhizosphere microbiota. Using a high-throughput amplicon sequencing survey, we determined that nitrogen availability for plant uptake is a factor promoting the selective enrichment of individual taxa in the rhizosphere of wild and domesticated barley genotypes. Shotgun sequencing and metagenome-assembled genomes revealed that this taxonomic diversification is mirrored by a functional specialization, manifested by the differential enrichment of multiple Gene Ontology terms, of the microbiota of plants exposed to nitrogen conditions limiting barley growth. Finally, a plant soil feedback experiment revealed that host control of the barley microbiota underpins the assembly of a phylogenetically diverse group of bacteria putatively required to sustain plant performance under nitrogen-limiting supplies. Taken together, our observations indicate that under nitrogen conditions limiting plant growth, host-microbe and microbe-microbe interactions fine-tune the host genetic selection of the barley microbiota at both taxonomic and functional levels. The disruption of these recruitment cues negatively impacts plant growth. IMPORTANCE The microbiota inhabiting the rhizosphere, the thin layer of soil surrounding plant roots, can promote the growth, development, and health of their host plants. Previous research indicated that differences in the genetic composition of the host plant coincide with variations in the composition of the rhizosphere microbiota. This is particularly evident when looking at the microbiota associated with input-demanding modern cultivated varieties and their wild relatives, which have evolved under marginal conditions. However, the functional significance of these differences remains to be fully elucidated. We investigated the rhizosphere microbiota of wild and cultivated genotypes of the global crop barley and determined that nutrient conditions limiting plant growth amplify the host control on microbes at the root-soil interface. This is reflected in a plant- and genotype-dependent functional specialization of the rhizosphere microbiota, which appears to be required for optimal plant growth. These findings provide novel insights into the significance of the rhizosphere microbiota for plant growth and sustainable agriculture.

First come, first served: superinfection exclusion in Deformed wing virus is dependent upon sequence identity and not the order of virus acquisition.

Deformed wing virus (DWV) is the most important globally distributed pathogen of honey bees and, when vectored by the ectoparasite Varroa destructor, is associated with high levels of colony losses. Divergent DWV types may differ in their pathogenicity and are reported to exhibit superinfection exclusion upon sequential infections, an inevitability in a Varroa-infested colony. We used a reverse genetic approach to investigate competition and interactions between genetically distinct or related virus strains, analysing viral load over time, tissue distribution with reporter gene-expressing viruses and recombination between virus variants. Transient competition occurred irrespective of the order of virus acquisition, indicating no directionality or dominance. Over longer periods, the ability to compete with a pre-existing infection correlated with the genetic divergence of the inoculae. Genetic recombination was observed throughout the DWV genome with recombinants accounting for ~2% of the population as determined by deep sequencing. We propose that superinfection exclusion, if it occurs at all, is a consequence of a cross-reactive RNAi response to the viruses involved, explaining the lack of dominance of one virus type over another. A better understanding of the consequences of dual- and superinfection will inform development of cross-protective honey bee vaccines and landscape-scale DWV transmission and evolution.

Evidence for and against deformed wing virus spillover from honey bees to bumble bees: a reverse genetic analysis.

Deformed wing virus (DWV) is a persistent pathogen of European honey bees and the major contributor to overwintering colony losses. The prevalence of DWV in honey bees has led to significant concerns about spillover of the virus to other pollinating species. Bumble bees are both a major group of wild and commercially-reared pollinators. Several studies have reported pathogen spillover of DWV from honey bees to bumble bees, but evidence of a sustained viral infection characterized by virus replication and accumulation has yet to be demonstrated. Here we investigate the infectivity and transmission of DWV in bumble bees using the buff-tailed bumble bee Bombus terrestris as a model. We apply a reverse genetics approach combined with controlled laboratory conditions to detect and monitor DWV infection. A novel reverse genetics system for three representative DWV variants, including the two master variants of DWV-type A and B-was used. Our results directly confirm DWV replication in bumble bees but also demonstrate striking resistance to infection by certain transmission routes. Bumble bees may support DWV replication but it is not clear how infection could occur under natural environmental conditions.

A footprint of plant eco-geographic adaptation on the composition of the barley rhizosphere bacterial microbiota.

The microbiota thriving in the rhizosphere, the thin layer of soil surrounding plant roots, plays a critical role in plant's adaptation to the environment. Domestication and breeding selection have progressively differentiated the microbiota of modern crops from the ones of their wild ancestors. However, the impact of eco-geographical constraints faced by domesticated plants and crop wild relatives on recruitment and maintenance of the rhizosphere microbiota remains to be fully elucidated. Here we performed a comparative 16S rRNA gene survey of the rhizosphere of 4 domesticated and 20 wild barley (Hordeum vulgare) genotypes grown in an agricultural soil under controlled environmental conditions. We demonstrated the enrichment of individual bacteria mirrored the distinct eco-geographical constraints faced by their host plants. Unexpectedly, Elite varieties exerted a stronger genotype effect on the rhizosphere microbiota when compared with wild barley genotypes adapted to desert environments with a preferential enrichment for members of Actinobacteria. Finally, in wild barley genotypes, we discovered a limited, but significant, correlation between microbiota diversity and host genomic diversity. Our results revealed a footprint of the host's adaptation to the environment on the assembly of the bacteria thriving at the root-soil interface. In the tested conditions, this recruitment cue layered atop of the distinct evolutionary trajectories of wild and domesticated plants and, at least in part, is encoded by the barley genome. This knowledge will be critical to design experimental approaches aimed at elucidating the recruitment cues of the barley microbiota across a range of soil types.

Green Bees: Reverse Genetic Analysis of Deformed Wing Virus Transmission, Replication, and Tropism.

Environmental and agricultural pollination services by honey bees, Apis mellifera, and honey production are compromised by high levels of annual colony losses globally. The majority are associated with disease caused by deformed wing virus (DWV), a positive-strand RNA virus, exacerbated by the ectoparasitic mite Varroa destructor. To improve honey bee health, a better understanding of virus transmission and pathogenesis is needed which requires the development of tools to study virus replication, transmission, and localisation. We report the use of reverse genetic (RG) systems for the predominant genetically distinct variants of DWV to address these questions. All RG-recovered viruses replicate within 24 h post-inoculation of pupae and could recapitulate the characteristic symptoms of DWV disease upon eclosion. Larvae were significantly less susceptible but could be infected orally and subsequently developed disease. Using genetically tagged RG DWV and an in vitro Varroa feeding system, we demonstrate virus replication in the mite by accumulation of tagged negative-strand viral replication intermediates. We additionally apply a modified DWV genome expressing a fluorescent reporter protein for direct in vivo observation of virus distribution in injected pupae or fed larvae. Using this, we demonstrate extensive sites of virus replication in a range of pupal tissues and organs and in the nascent wing buds in larvae fed high levels of virus, indicative of a direct association between virus replication and pathogenesis. These studies provide insights into virus replication kinetics, tropism, transmission, and pathogenesis, and produce new tools to help develop the understanding needed to control DWV-mediated colony losses.