Crop management shapes the diversity and activity of DNA and RNA viruses in the rhizosphere.

BACKGROUND: The rhizosphere is a hotspot for microbial activity and contributes to ecosystem services including plant health and biogeochemical cycling. The activity of microbial viruses, and their influence on plant-microbe interactions in the rhizosphere, remains undetermined. Given the impact of viruses on the ecology and evolution of their host communities, determining how soil viruses influence microbiome dynamics is crucial to build a holistic understanding of rhizosphere functions. RESULTS: Here, we aimed to investigate the influence of crop management on the composition and activity of bulk soil, rhizosphere soil, and root viral communities. We combined viromics, metagenomics, and metatranscriptomics on soil samples collected from a 3-year crop rotation field trial of oilseed rape (Brassica napus L.). By recovering 1059 dsDNA viral populations and 16,541 ssRNA bacteriophage populations, we expanded the number of underexplored Leviviricetes genomes by > 5 times. Through detection of viral activity in metatranscriptomes, we uncovered evidence of "Kill-the-Winner" dynamics, implicating soil bacteriophages in driving bacterial community succession. Moreover, we found the activity of viruses increased with proximity to crop roots, and identified that soil viruses may influence plant-microbe interactions through the reprogramming of bacterial host metabolism. We have provided the first evidence of crop rotation-driven impacts on soil microbial communities extending to viruses. To this aim, we present the novel principal of "viral priming," which describes how the consecutive growth of the same crop species primes viral activity in the rhizosphere through local adaptation. CONCLUSIONS: Overall, we reveal unprecedented spatial and temporal diversity in viral community composition and activity across root, rhizosphere soil, and bulk soil compartments. Our work demonstrates that the roles of soil viruses need greater consideration to exploit the rhizosphere microbiome for food security, food safety, and environmental sustainability. Video Abstract.

2-Aminoethylphosphonate utilization in Pseudomonas putida BIRD-1 is controlled by multiple master regulators.

Bacteria possess various regulatory mechanisms to detect and coordinate a response to elemental nutrient limitation. In pseudomonads, the two-component system regulators CbrAB, NtrBC and PhoBR, are responsible for regulating cellular response to carbon (C), nitrogen (N) and phosphorus (P) respectively. Phosphonates are reduced organophosphorus compounds produced by a broad range of biota and typified by a direct C-P bond. Numerous pseudomonads can use the environmentally abundant phosphonate species 2-aminoethylphosphonate (2AEP) as a source of C, N, or P, but only PhoBR has been shown to play a role in 2AEP utilization. On the other hand, utilization of 2AEP as a C and N source is considered substrate inducible. Here, using the plant-growth-promoting rhizobacterium Pseudomonas putida BIRD-1 we present evidence that 2AEP utilization is under dual regulation and only occurs upon depletion of C, N, or P, controlled by CbrAB, NtrBC, or PhoBR respectively. However, the presence of 2AEP was necessary for full gene expression, i.e. expression was substrate inducible. Mutation of a LysR-type regulator, termed AepR, upstream of the 2AEP transaminase-phosphonatase system (PhnWX), confirmed this dual regulatory mechanism. To our knowledge, this is the first study identifying coordination between global stress response and substrate-specific regulators in phosphonate metabolism.

Natural attenuation of legacy hydrocarbon spills in pristine soils is feasible despite difficult environmental conditions in the monsoon tropics.

The Kimberley region of Western Australia is a National Heritage listed region that is internationally recognised for its environmental and cultural significance. However, petroleum spills have been reported at a number of sites across the region, representing an environmental concern. The region is also characterised as having low soil nutrients, high temperatures and monsoonal rain - all of which may limit the potential for natural biodegradation of petroleum. Therefore, this work evaluated the effect of legacy petroleum hydrocarbons on the indigenous soil microbial community (across the domains Archaea, Bacteria and Fungi) at three sites in the Kimberley region. At each site, soil cores were removed from contaminated and control areas and analysed for total petroleum hydrocarbons, soil nutrients, pH and microbial community profiling (using16S rRNA and ITS sequencing on the Illumina MiSeq Platform). The presence of petroleum hydrocarbons decreased microbial diversity across all kingdoms, altered the structure of microbial communities and increased the abundance of putative hydrocarbon degraders (e.g. Mycobacterium, Acremonium, Penicillium, Bjerkandera and Candida). Microbial community shifts from contaminated soils were also associated with an increase in soil nutrients (notably Colwell P and S). Our study highlights the long-term effect of legacy hydrocarbon spills on soil microbial communities and their diversity in remote, infertile monsoonal soils, but also highlights the potential for natural attenuation to occur in these environments.

Transporter characterisation reveals aminoethylphosphonate mineralisation as a key step in the marine phosphorus redox cycle.

The planktonic synthesis of reduced organophosphorus molecules, such as alkylphosphonates and aminophosphonates, represents one half of a vast global oceanic phosphorus redox cycle. Whilst alkylphosphonates tend to accumulate in recalcitrant dissolved organic matter, aminophosphonates do not. Here, we identify three bacterial 2-aminoethylphosphonate (2AEP) transporters, named AepXVW, AepP and AepSTU, whose synthesis is independent of phosphate concentrations (phosphate-insensitive). AepXVW is found in diverse marine heterotrophs and is ubiquitously distributed in mesopelagic and epipelagic waters. Unlike the archetypal phosphonate binding protein, PhnD, AepX has high affinity and high specificity for 2AEP (Stappia stellulata AepX Kd 23 ± 4 nM; methylphosphonate Kd 3.4 ± 0.3 mM). In the global ocean, aepX is heavily transcribed (~100-fold>phnD) independently of phosphate and nitrogen concentrations. Collectively, our data identifies a mechanism responsible for a major oxidation process in the marine phosphorus redox cycle and suggests 2AEP may be an important source of regenerated phosphate and ammonium, which are required for oceanic primary production.

Identification of microbial signatures linked to oilseed rape yield decline at the landscape scale.

BACKGROUND: The plant microbiome plays a vital role in determining host health and productivity. However, we lack real-world comparative understanding of the factors which shape assembly of its diverse biota, and crucially relationships between microbiota composition and plant health. Here we investigated landscape scale rhizosphere microbial assembly processes in oilseed rape (OSR), the UK's third most cultivated crop by area and the world's third largest source of vegetable oil, which suffers from yield decline associated with the frequency it is grown in rotations. By including 37 conventional farmers' fields with varying OSR rotation frequencies, we present an innovative approach to identify microbial signatures characteristic of microbiomes which are beneficial and harmful to the host. RESULTS: We show that OSR yield decline is linked to rotation frequency in real-world agricultural systems. We demonstrate fundamental differences in the environmental and agronomic drivers of protist, bacterial and fungal communities between root, rhizosphere soil and bulk soil compartments. We further discovered that the assembly of fungi, but neither bacteria nor protists, was influenced by OSR rotation frequency. However, there were individual abundant bacterial OTUs that correlated with either yield or rotation frequency. A variety of fungal and protist pathogens were detected in roots and rhizosphere soil of OSR, and several increased relative abundance in root or rhizosphere compartments as OSR rotation frequency increased. Importantly, the relative abundance of the fungal pathogen Olpidium brassicae both increased with short rotations and was significantly associated with low yield. In contrast, the root endophyte Tetracladium spp. showed the reverse associations with both rotation frequency and yield to O. brassicae, suggesting that they are signatures of a microbiome which benefits the host. We also identified a variety of novel protist and fungal clades which are highly connected within the microbiome and could play a role in determining microbiome composition. CONCLUSIONS: We show that at the landscape scale, OSR crop yield is governed by interplay between complex communities of both pathogens and beneficial biota which is modulated by rotation frequency. Our comprehensive study has identified signatures of dysbiosis within the OSR microbiome, grown in real-world agricultural systems, which could be used in strategies to promote crop yield. Video abstract.

Niche-adaptation in plant-associated Bacteroidetes favours specialisation in organic phosphorus mineralisation.

Bacteroidetes are abundant pathogen-suppressing members of the plant microbiome that contribute prominently to rhizosphere phosphorus mobilisation, a frequent growth-limiting nutrient in this niche. However, the genetic traits underpinning their success in this niche remain largely unknown, particularly regarding their phosphorus acquisition strategies. By combining cultivation, multi-layered omics and biochemical analyses we first discovered that all plant-associated Bacteroidetes express constitutive phosphatase activity, linked to the ubiquitous possession of a unique phosphatase, PafA. For the first time, we also reveal a subset of Bacteroidetes outer membrane SusCD-like complexes, typically associated with carbon acquisition, and several TonB-dependent transporters, are induced during Pi-depletion. Furthermore, in response to phosphate depletion, the plant-associated Flavobacterium used in this study expressed many previously characterised and novel proteins targeting organic phosphorus. Collectively, these enzymes exhibited superior phosphatase activity compared to plant-associated Pseudomonas spp. Importantly, several of the novel low-Pi-inducible phosphatases and transporters, belong to the Bacteroidetes auxiliary genome and are an adaptive genomic signature of plant-associated strains. In conclusion, niche adaptation to the plant microbiome thus appears to have resulted in the acquisition of unique phosphorus scavenging loci in Bacteroidetes, enhancing their phosphorus acquisition capabilities. These traits may enable their success in the rhizosphere and also present exciting avenues to develop sustainable agriculture.

Identification of extracellular glycerophosphodiesterases in Pseudomonas and their role in soil organic phosphorus remineralisation.

In soils, phosphorus (P) exists in numerous organic and inorganic forms. However, plants can only acquire inorganic orthophosphate (Pi), meaning global crop production is frequently limited by P availability. To overcome this problem, rock phosphate fertilisers are heavily applied, often with negative environmental and socio-economic consequences. The organic P fraction of soil contains phospholipids that are rapidly degraded resulting in the release of bioavailable Pi. However, the mechanisms behind this process remain unknown. We identified and experimentally confirmed the function of two secreted glycerolphosphodiesterases, GlpQI and GlpQII, found in Pseudomonas stutzeri DSM4166 and Pseudomonas fluorescens SBW25, respectively. A series of co-cultivation experiments revealed that in these Pseudomonas strains, cleavage of glycerolphosphorylcholine and its breakdown product G3P occurs extracellularly allowing other bacteria to benefit from this metabolism. Analyses of metagenomic and metatranscriptomic datasets revealed that this trait is widespread among soil bacteria with Actinobacteria and Proteobacteria, specifically Betaproteobacteria and Gammaproteobacteria, the likely major players.

The 'known' genetic potential for microbial communities to degrade organic phosphorus is reduced in low-pH soils.

In soil, bioavailable inorganic orthophosphate is found at low concentrations and thus limits biological growth. To overcome this phosphorus scarcity, plants and bacteria secrete numerous enzymes, namely acid and alkaline phosphatases, which cleave orthophosphate from various organic phosphorus substrates. Using profile hidden Markov modeling approaches, we investigated the abundance of various non specific phosphatases, both acid and alkaline, in metagenomes retrieved from soils with contrasting pH regimes. This analysis uncovered a marked reduction in the abundance and diversity of various alkaline phosphatases in low-pH soils that was not counterbalanced by an increase in acid phosphatases. Furthermore, it was also discovered that only half of the bacterial strains from different phyla deposited in the Integrated Microbial Genomes database harbor alkaline phosphatases. Taken together, our data suggests that these 'phosphatase lacking' isolates likely increase in low-pH soils and future research should ascertain how these bacteria overcome phosphorus scarcity.

Comparative genomic, proteomic and exoproteomic analyses of three Pseudomonas strains reveals novel insights into the phosphorus scavenging capabilities of soil bacteria.

Bacteria that inhabit the rhizosphere of agricultural crops can have a beneficial effect on crop growth. One such mechanism is the microbial-driven solubilization and remineralization of complex forms of phosphorus (P). It is known that bacteria secrete various phosphatases in response to low P conditions. However, our understanding of their global proteomic response to P stress is limited. Here, exoproteomic analysis of Pseudomonas putida BIRD-1 (BIRD-1), Pseudomonas fluorescens SBW25 and Pseudomonas stutzeri DSM4166 was performed in unison with whole-cell proteomic analysis of BIRD-1 grown under phosphate (Pi) replete and Pi deplete conditions. Comparative exoproteomics revealed marked heterogeneity in the exoproteomes of each Pseudomonas strain in response to Pi depletion. In addition to well-characterized members of the PHO regulon such as alkaline phosphatases, several proteins, previously not associated with the response to Pi depletion, were also identified. These included putative nucleases, phosphotriesterases, putative phosphonate transporters and outer membrane proteins. Moreover, in BIRD-1, mutagenesis of the master regulator, phoBR, led us to confirm the addition of several novel PHO-dependent proteins. Our data expands knowledge of the Pseudomonas PHO regulon, including species that are frequently used as bioinoculants, opening up the potential for more efficient and complete use of soil complexed P.

Evidence for functional redundancy in arbuscular mycorrhizal fungi and implications for agroecosystem management.

Arbuscular mycorrhizal (AM) fungi provide benefits to host plants and show functional diversity, with evidence of functional trait conservation at the family level. Diverse communities of AM fungi ought therefore to provide increased benefits to the host, with implications for the management of sustainable agroecosystems. However, this is often not evident in the literature, with diversity saturation at low species number. Growth and nutrient uptake were measured in onions in the glasshouse on AM-free phosphorus (P)-poor soil, inoculated with between one and seven species of AM fungi in all possible combinations. Inoculation with AM fungi increased shoot dry weight as well as P and copper concentrations in shoots but reduced the concentration of potassium and sulphur. There was little evidence of increased benefit from high AM fungal diversity, and increasing diversity beyond three species did not result in significantly higher shoot weight or P or Cu concentrations. Species of Glomeraceae had the greatest impact on growth and nutrient uptake, while species of Acaulospora and Racocetra did not have a significant impact. Failure to show a benefit from high AM fungal diversity in this and other studies may be the result of experimental conditions, with the benefits of AM fungal diversity only becoming apparent when the host plant is faced with multiple stress factors. Replicating the complex interactions between AM fungi, the host plant and their environment in the laboratory in order to fully understand these interactions is a major challenge to AM research.

Contrasting arbuscular mycorrhizal communities colonizing different host plants show a similar response to a soil phosphorus concentration gradient.

High soil phosphorus (P) concentration is frequently shown to reduce root colonization by arbuscular mycorrhizal (AM) fungi, but the influence of P on the diversity of colonizing AM fungi is uncertain. We used terminal restriction fragment length polymorphism (T-RFLP) of 18S rDNA and cloning to assess diversity of AM fungi colonizing maize (Zea mays), soybean (Glycene max) and field violet (Viola arvensis) at three time points in one season along a P gradient of 10-280 mg l(-1) in the field. Percentage AM colonization changed between sampling time points but was not reduced by high soil P except in maize. There was no significant difference in AM diversity between sampling time points. Diversity was reduced at concentrations of P > 25 mg l(-1), particularly in maize and soybean. Both cloning and T-RFLP indicated differences between AM communities in the different host species. Host species was more important than soil P in determining the AM community, except at the highest P concentration. Our results show that the impact of soil P on the diversity of AM fungi colonizing plants was broadly similar, despite the fact that different plants contained different communities. However, subtle differences in the response of the AM community in each host were evident.

Mycorrhizas and biomass crops: opportunities for future sustainable development.

Central to soil health and plant productivity in natural ecosystems are in situ soil microbial communities, of which mycorrhizal fungi are an integral component, regulating nutrient transfer between plants and the surrounding soil via extensive mycelial networks. Such networks are supported by plant-derived carbon and are likely to be enhanced under coppiced biomass plantations, a forestry practice that has been highlighted recently as a viable means of providing an alternative source of energy to fossil fuels, with potentially favourable consequences for carbon mitigation. Here, we explore ways in which biomass forestry, in conjunction with mycorrhizal fungi, can offer a more holistic approach to addressing several topical environmental issues, including 'carbon-neutral' energy, ecologically sustainable land management and CO(2) sequestration.