Circadian rhythms in the plant host influence rhythmicity of rhizosphere microbiota.

BACKGROUND: Recent studies demonstrated that microbiota inhabiting the plant rhizosphere exhibit diel changes in abundance. To investigate the impact of plant circadian rhythms on bacterial and fungal rhythms in the rhizosphere, we analysed temporal changes in fungal and bacterial communities in the rhizosphere of Arabidopsis plants overexpressing or lacking function of the circadian clock gene LATE ELONGATED HYPOCOTYL (LHY). RESULTS: Under diel light-dark cycles, the knock-out mutant lhy-11 and the gain-of-function mutant lhy-ox both exhibited gene expression rhythms with altered timing and amplitude compared to wild-type plants. Distinct sets of bacteria and fungi were found to display rhythmic changes in abundance in the rhizosphere of both of these mutants, suggesting that abnormal patterns of rhythmicity in the plant host caused temporal reprogramming of the rhizosphere microbiome. This was associated with changes in microbial community structure, including changes in the abundance of fungal guilds known to impact on plant health. Under constant environmental conditions, microbial rhythmicity persisted in the rhizosphere of wild-type plants, indicating control by a circadian oscillator. In contrast, loss of rhythmicity in lhy-ox plants was associated with disrupted rhythms for the majority of rhizosphere microbiota. CONCLUSIONS: These results show that aberrant function of the plant circadian clock is associated with altered rhythmicity of rhizosphere bacteria and fungi. In the long term, this leads to changes in composition of the rhizosphere microbiome, with potential consequences for plant health. Further research will be required to understand the functional implications of these changes and how they impact on plant health and productivity.

Agricultural land-use favours Mucoromycotinian, but not Glomeromycotinian, arbuscular mycorrhizal fungi across ten biomes.

Globally, agricultural land-use negatively affects soil biota that contribute to ecosystem functions such as nutrient cycling, yet arbuscular mycorrhizal fungi (AMF) are promoted as essential components of agroecosystems. Arbuscular mycorrhizal fungi include Glomeromycotinian AMF (G-AMF) and the arbuscule-producing fine root endophytes, recently re-classified into the Endogonales order within Mucoromycotina. The correct classification of Mucoromycotinian AMF (M-AMF) and the availability of new molecular tools can guide research to better the understanding of their diversity and ecology. To investigate the impact on G-AMF and M-AMF of agricultural land-use at a continental scale, we sampled DNA from paired farm and native sites across 10 Australian biomes. Glomeromycotinian AMF were present in both native and farm sites in all biomes. Putative M-AMF were favoured by farm sites, rare or absent in native sites, and almost entirely absent in tropical biomes. Temperature, rainfall, and soil pH were strong drivers of richness and community composition of both groups, and plant richness was an important mediator. Both fungal groups occupy different, but overlapping, ecological niches, with M-AMF thriving in temperate agricultural landscapes. Our findings invite exploration of the origin and spread of M-AMF and continued efforts to resolve the phylogeny of this newly reclassified group of AMF.

A Novel Signaling Pathway Required for Arabidopsis Endodermal Root Organization Shapes the Rhizosphere Microbiome.

The Casparian strip (CS) constitutes a physical diffusion barrier to water and nutrients in plant roots, which is formed by the polar deposition of lignin polymer in the endodermis tissue. The precise pattern of lignin deposition is determined by the scaffolding activity of membrane-bound Casparian Strip domain proteins (CASPs), but little is known of the mechanism(s) directing this process. Here, we demonstrate that Endodermis-specific Receptor-like Kinase 1 (ERK1) and, to a lesser extent, ROP Binding Kinase1 (RBK1) are also involved in regulating CS formation, with the former playing an essential role in lignin deposition as well as in the localization of CASP1. We show that ERK1 is localized to the cytoplasm and nucleus of the endodermis and that together with the circadian clock regulator, Time for Coffee (TIC), forms part of a novel signaling pathway necessary for correct CS organization and suberization of the endodermis, with their single or combined loss of function resulting in altered root microbiome composition. In addition, we found that other mutants displaying defects in suberin deposition at the CS also display altered root exudates and microbiome composition. Thus, our work reveals a complex network of signaling factors operating within the root endodermis that establish both the CS diffusion barrier and influence the microbial composition of the rhizosphere.

Tree phyllospheres are a habitat for diverse populations of CO-oxidizing bacteria.

Carbon monoxide (CO) is both a ubiquitous atmospheric trace gas and an air pollutant. While aerobic CO-degrading microorganisms in soils and oceans are estimated to remove ~370 Tg of CO per year, the presence of CO-degrading microorganisms in above-ground habitats, such as the phyllosphere, and their potential role in CO cycling remains unknown. CO-degradation by leaf washes of two common British trees, Ilex aquifolium and Crataegus monogyna, demonstrated CO uptake in all samples investigated. Based on the analyses of taxonomic and functional genes, diverse communities of candidate CO-oxidizing taxa were identified, including members of Rhizobiales and Burkholderiales which were abundant in the phyllosphere at the time of sampling. Based on predicted genomes of phyllosphere community members, an estimated 21% of phyllosphere bacteria contained CoxL, the large subunit of CO-dehydrogenase. In support of this, data mining of publicly available phyllosphere metagenomes for genes encoding CO-dehydrogenase subunits demonstrated that, on average, 25% of phyllosphere bacteria contained CO-dehydrogenase gene homologues. A CO-oxidizing Phyllobacteriaceae strain was also isolated from phyllosphere samples which contains genes encoding both CO-dehydrogenase as well as a ribulose-1,5-bisphosphate carboxylase-oxygenase. These results suggest that the phyllosphere supports diverse and potentially abundant CO-oxidizing bacteria, which are a potential sink for atmospheric CO.

Evidence for Niche Differentiation in the Environmental Responses of Co-occurring Mucoromycotinian Fine Root Endophytes and Glomeromycotinian Arbuscular Mycorrhizal Fungi.

Fine root endophytes (FRE) were traditionally considered a morphotype of arbuscular mycorrhizal fungi (AMF), but recent genetic studies demonstrate that FRE belong within the subphylum Mucoromycotina, rather than in the subphylum Glomeromycotina with the AMF. These findings prompt enquiry into the fundamental ecology of FRE and AMF. We sampled FRE and AMF in roots of Trifolium subterraneum from 58 sites across temperate southern Australia. We investigated the environmental drivers of composition, richness, and root colonization of FRE and AMF by using structural equation modelling and canonical correspondence analyses. Root colonization by FRE increased with increasing temperature and rainfall but decreased with increasing phosphorus (P). Root colonization by AMF increased with increasing soil organic carbon but decreased with increasing P. Richness of FRE decreased with increasing temperature and soil pH. Richness of AMF increased with increasing temperature and rainfall but decreased with increasing soil aluminium (Al) and pH. Aluminium, soil pH, and rainfall were, in decreasing order, the strongest drivers of community composition of FRE; they were also important drivers of community composition of AMF, along with temperature, in decreasing order: rainfall, Al, temperature, and soil pH. Thus, FRE and AMF showed the same responses to some (e.g. soil P, soil pH) and different responses to other (e.g. temperature) key environmental factors. Overall, our data are evidence for niche differentiation among these co-occurring mycorrhizal associates.

Partial maintenance of organ-specific epigenetic marks during plant asexual reproduction leads to heritable phenotypic variation.

Plants differ from animals in their capability to easily regenerate fertile adult individuals from terminally differentiated cells. This unique developmental plasticity is commonly observed in nature, where many species can reproduce asexually through the ectopic initiation of organogenic or embryogenic developmental programs. While organ-specific epigenetic marks are not passed on during sexual reproduction, the fate of epigenetic marks during asexual reproduction and the implications for clonal progeny remain unclear. Here we report that organ-specific epigenetic imprints in Arabidopsis thaliana can be partially maintained during asexual propagation from somatic cells in which a zygotic program is artificially induced. The altered marks are inherited even over multiple rounds of sexual reproduction, becoming fixed in hybrids and resulting in heritable molecular and physiological phenotypes that depend on the identity of the founder tissue. Consequently, clonal plants display distinct interactions with beneficial and pathogenic microorganisms. Our results demonstrate how novel phenotypic variation in plants can be unlocked through altered inheritance of epigenetic marks upon asexual propagation.

Urban meadows as an alternative to short mown grassland: effects of composition and height on biodiversity.

There are increasing calls to provide greenspace in urban areas, yet the ecological quality, as well as quantity, of greenspace is important. Short mown grassland designed for recreational use is the dominant form of urban greenspace in temperate regions but requires considerable maintenance and typically provides limited habitat value for most taxa. Alternatives are increasingly proposed, but the biodiversity potential of these is not well understood. In a replicated experiment across six public urban greenspaces, we used nine different perennial meadow plantings to quantify the relative roles of floristic diversity and height of sown meadows on the richness and composition of three taxonomic groups: plants, invertebrates, and soil microbes. We found that all meadow treatments were colonized by plant species not sown in the plots, suggesting that establishing sown meadows does not preclude further locally determined grassland development if management is appropriate. Colonizing species were rarer in taller and more diverse plots, indicating competition may limit invasion rates. Urban meadow treatments contained invertebrate and microbial communities that differed from mown grassland. Invertebrate taxa responded to changes in both height and richness of meadow vegetation, but most orders were more abundant where vegetation height was longer than mown grassland. Order richness also increased in longer vegetation and Coleoptera family richness increased with plant diversity in summer. Microbial community composition seems sensitive to plant species composition at the soil surface (0-10 cm), but in deeper soils (11-20 cm) community variation was most responsive to plant height, with bacteria and fungi responding differently. In addition to improving local residents' site satisfaction, native perennial meadow plantings can produce biologically diverse grasslands that support richer and more abundant invertebrate communities, and restructured plant, invertebrate, and soil microbial communities compared with short mown grassland. Our results suggest that diversification of urban greenspace by planting urban meadows in place of some mown amenity grassland is likely to generate substantial biodiversity benefits, with a mosaic of meadow types likely to maximize such benefits.

New fungal primers reveal the diversity of Mucoromycotinian arbuscular mycorrhizal fungi and their response to nitrogen application.

BACKGROUND: Arbuscular mycorrhizas (AM) are the most widespread terrestrial symbiosis and are both a key determinant of plant health and a major contributor to ecosystem processes through their role in biogeochemical cycling. Until recently, it was assumed that the fungi which form AM comprise the subphylum Glomeromycotina (G-AMF), and our understanding of the diversity and ecosystem roles of AM is based almost exclusively on this group. However recent evidence shows that fungi which form the distinctive 'fine root endophyte' (FRE) AM morphotype are members of the subphylum Mucoromycotina (M-AMF), so that AM symbioses are actually formed by two distinct groups of fungi. RESULTS: We investigated the influence of nitrogen (N) addition and wheat variety on the assembly of AM communities under field conditions. Visual assessment of roots showed co-occurrence of G-AMF and M-AMF, providing an opportunity to compare the responses of these two groups. Existing 'AM' 18S rRNA primers which co-amplify G-AMF and M-AMF were modified to reduce bias against Mucoromycotina, and compared against a new 'FRE' primer set which selectively amplifies Mucoromycotina. Using the AM-primers, no significant effect of either N-addition or wheat variety on G-AMF or M-AMF diversity or community composition was detected. In contrast, using the FRE-primers, N-addition was shown to reduce M-AMF diversity and altered community composition. The ASV which responded to N-addition were closely related, demonstrating a clear phylogenetic signal which was identified only by the new FRE-primers. The most abundant Mucoromycotina sequences we detected belonged to the same Endogonales clades as dominant sequences associated with FRE morphology in Australia, indicating that closely related M-AMF may be globally distributed. CONCLUSIONS: The results demonstrate the need to consider both G-AMF and M-AMF when investigating AM communities, and highlight the importance of primer choice when investigating AMF community dynamics.

Water flow plays a key role in determining chemical biodegradation in water-sediment systems.

Before agrochemicals can be registered and sold, the chemical industry is required to perform regulatory tests to assess their environmental persistence, using defined guidelines. Aquatic fate tests (e.g. OECD 308) lack environmental realism as they are conducted under dark conditions and in small-scale static systems, which can affect microbial diversity and functionality. In this study, water-sediment microflumes were used to investigate the impact of these deficiencies in environmental realism on the fate of the fungicide, isopyrazam. Although on a large-scale, these systems aimed to retain the key aspects of OECD 308 tests. Tests were carried out under both a non-UV light-dark cycle and continuous darkness and under both static and flowing water conditions, to investigate how light and water flow affect isopyrazam biodegradation pathways. In static systems, light treatment played a significant role, with faster dissipation in illuminated compared to dark microflumes (DT50s = 20.6 vs. 47.7 days). In flowing systems (DT50s = 16.8 and 15.3 days), light did not play a significant role in dissipation, which was comparable between the two light treatments, and faster than in dark static microflumes. Microbial phototroph biomass was significantly reduced by water flow in the illuminated systems, thereby reducing their contribution to dissipation. Comprehensive analysis of bacterial and eukaryotic community composition identified treatment specific changes following incubation, with light promoting relative abundance of Cyanobacteria and eukaryotic algae, and flow increasing relative abundance of fungi. We conclude that both water velocity and non-UV light increased isopyrazam dissipation, but the contribution of light depended on the flow conditions. These differences may have resulted from impacts on microbial communities and via mixing processes, particularly hyporheic exchange. Inclusion of both light and flow in studies could improve the extent they mimic natural environments and predict chemical environmental persistence, thus bridging the gap between laboratory and field studies.

Diversity and Ecological Guild Analysis of the Oil Palm Fungal Microbiome Across Root, Rhizosphere, and Soil Compartments.

The rhizosphere microbiome is a major determinant of plant health, which can interact with the host directly and indirectly to promote or suppress productivity. Oil palm is one of the world's most important crops, constituting over a third of global vegetable oil production. Currently there is little understanding of the oil palm microbiome and its contribution to plant health and productivity, with existing knowledge based almost entirely on culture dependent studies. We investigated the diversity and composition of the oil palm fungal microbiome in the bulk soil, rhizosphere soil, and roots of 2-, 18-, and 35-year old plantations in Selangor, Malaysia. The fungal community showed substantial variation between the plantations, accounting for 19.7% of community composition, with compartment (root, rhizosphere soil, and bulk soil), and soil properties (pH, C, N, and P) contributing 6.5 and 7.2% of community variation, respectively. Rhizosphere soil and roots supported distinct communities compared to the bulk soil, with significant enrichment of Agaricomycetes, Glomeromycetes, and Lecanoromycetes in roots. Several putative plant pathogens were abundant in roots in all the plantations, including taxa related to Prospodicola mexicana and Pleurostoma sp. The mycorrhizal status and dependency of oil palm has yet to be established, and using 18S rRNA primers we found considerable between-site variation in Glomeromycotinian community composition, accounting for 31.2% of variation. There was evidence for the selection of Glomeromycotinian communities in oil palm roots in the older plantations but compartment had a weak effect on community composition, accounting for 3.9% of variation, while soil variables accounted for 9% of community variation. While diverse Mucoromycotinian fungi were detected, they showed very low abundance and diversity within roots compared to bulk soil, and were not closely related to taxa which have been linked to fine root endophyte mycorrhizal morphology. Many of the fungal sequences showed low similarity to established genera, indicating the presence of substantial novel diversity with significance for plant health within the oil palm microbiome.

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.

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.

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.

Long-read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity.

High-throughput DNA metabarcoding of amplicon sizes below 500 bp has revolutionized the analysis of environmental microbial diversity. However, these short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. This lesser phylogenetic resolution of short amplicons may be overcome by new long-read sequencing technologies. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain an ~4500-bp region spanning most of the eukaryotic small subunit (18S) and large subunit (28S) ribosomal DNA genes. We first treated the CCS reads with a novel curation workflow, generating 650 high-quality operational taxonomic units (OTUs) containing the physically linked 18S and 28S regions. To assign taxonomy to these OTUs, we developed a phylogeny-aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity-based methods. The taxonomically annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well-resolved phylogeny spanning all major groups of eukaryotes, allowing us to accurately derive the evolutionary origin of environmental diversity. A total of 1,019 sequences were included, of which a majority (58%) corresponded to the new long environmental OTUs. The long reads also allowed us to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Together, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.

Inclusion of seasonal variation in river system microbial communities and phototroph activity increases environmental relevance of laboratory chemical persistence tests.

Regulatory tests assess crop protection product environmental fate and toxicity before approval for commercial use. Although globally applied laboratory tests can assess biodegradation, they lack environmental complexity. Microbial communities are subject to temporal and spatial variation, but there is little consideration of these microbial dynamics in the laboratory. Here, we investigated seasonal variation in the microbial composition of water and sediment from a UK river across a two-year time course and determined its effect on the outcome of water-sediment (OECD 308) and water-only (OECD 309) biodegradation tests, using the fungicide isopyrazam. These OECD tests are performed under dark conditions, so test systems incubated under non-UV light:dark cycles were also included to determine the impact on both inoculum characteristics and biodegradation. Isopyrazam degradation was faster when incubated under non-UV light at all collection times in water-sediment microcosms, suggesting that phototrophic communities can metabolise isopyrazam throughout the year. Degradation rate varied seasonally between inoculum collection times only in microcosms incubated in the light, but isopyrazam mineralisation to 14CO2 varied seasonally under both light and dark conditions, suggesting that heterotrophic communities may also play a role in degradation. Bacterial and phototroph communities varied across time, but there was no clear link between water or sediment microbial composition and variation in degradation rate. During the test period, inoculum microbial community composition changed, particularly in non-UV light incubated microcosms. Overall, we show that regulatory test outcome is not influenced by temporal variation in microbial community structure; however, biodegradation rates from higher tier studies with improved environmental realism, e.g. through addition of non-UV light, may be more variable. These data suggest that standardised OECD tests can provide a conservative estimate of pesticide persistence end points and that additional tests including non-UV light could help bridge the gap between standard tests and field studies.

First Cryo-Scanning Electron Microscopy Images and X-Ray Microanalyses of Mucoromycotinian Fine Root Endophytes in Vascular Plants.

AIMS: Arbuscule-producing fine root endophytes (FRE) (previously incorrectly Glomus tenue) were recently placed within subphylum Mucoromycotina; the first report of arbuscules outside subphylum Glomeromycotina. Here, we aimed to estimate nutrient concentrations in plant and fungal structures of FRE and to test the utility of cryo-scanning electron microscopy (cryoSEM) for studying these fungi. METHODS: We used replicated cryoSEM and X-ray microanalysis of heavily colonized roots of Trifolium subterraneum. RESULTS: Intercellular hyphae and hyphae in developed arbuscules were consistently very thin; 1.35 ± 0.03 µm and 0.99 ± 0.03 µm in diameter, respectively (mean ± SE). Several intercellular hyphae were often adjacent to each other forming "hyphal ropes." Developed arbuscules showed higher phosphorus concentrations than senesced arbuscules and non-colonized structures. Senesced arbuscules showed greatly elevated concentrations of calcium and magnesium. CONCLUSION: While uniformly thin hyphae and hyphal ropes are distinct features of FRE, the morphology of fully developed arbuscules, elevated phosphorus in fungal structures, and accumulation of calcium with loss of structural integrity in senesced arbuscules are similar to glomeromycotinian fungi. Thus, we provide evidence that FRE may respond to similar host-plant signals or that the host plant may employ a similar mechanism of association with FRE and AMF.

A bacmid approach to the genetic manipulation of granuloviruses.

A Cydia pomonella granulovirus (CpGV) bacmid has been constructed, which allows rapid and efficient production of recombinant baculoviruses in Escherichia coli. An 8.6kbp bacterial DNA cassette derived from the AcMNPV Bac-to-Bac system was ligated into a unique PacI restriction site within an intergenic region flanking the DNA ligase gene of the CpGV genome. The CpGV bacmids produced in E. coli were transfected into a CpGV-permissive C. pomonella cell line and the transfected cells fed to larvae to amplify the virus. The enhanced green fluorescent protein (EGFP) gene under the constitutive Drosophila heat-shock promoter was transposed into the mini-attTn7 transposition site, using a modified pFASTBAC donor plasmid, to generate a recombinant CpGV bacmid which caused infected larvae to glow under UV light. Targeted homologous recombination was also achieved in a recombinant proficient E. coli strain (BJ5183). A chloramphenicol acetyl transferase (CAT) gene replaced the cathepsin (v-cath) gene in the bacmid to produce a v-cath-deletion mutant. This is the first published report of a granulovirus bacmid, which will allow easy manipulation of the CpGV genome, enabling future studies on granulovirus genes and biology.

Plant Rhizosphere Selection of Plasmodiophorid Lineages from Bulk Soil: The Importance of "Hidden" Diversity.

Microbial communities closely associated with the rhizosphere can have strong positive and negative impacts on plant health and growth. We used a group-specific amplicon approach to investigate local scale drivers in the diversity and distribution of plasmodiophorids in rhizosphere/root and bulk soil samples from oilseed rape (OSR) and wheat agri-systems. Plasmodiophorids are plant- and stramenopile-associated protists including well known plant pathogens as well as symptomless endobiotic species. We detected 28 plasmodiophorid lineages (OTUs), many of them novel, and showed that plasmodiophorid communities were highly dissimilar and significantly divergent between wheat and OSR rhizospheres and between rhizosphere and bulk soil samples. Bulk soil communities were not significantly different between OSR and wheat systems. Wheat and OSR rhizospheres selected for different plasmodiophorid lineages. An OTU corresponding to Spongospora nasturtii was positively selected in the OSR rhizosphere, as were two genetically distinct OTUs. Two novel lineages related to Sorosphaerula veronicae were significantly associated with wheat rhizosphere samples, indicating unknown plant-protist relationships. We show that group-targeted eDNA approaches to microbial symbiont-host ecology reveal significant novel diversity and enable inference of differential activity and potential interactions between sequence types, as well as their presence.

Resistance and resilience responses of a range of soil eukaryote and bacterial taxa to fungicide application.

The application of plant protection products has the potential to significantly affect soil microbial community structure and function. However, the extent to which soil microbial communities from different trophic levels exhibit resistance and resilience to such compounds remains poorly understood. The resistance and resilience responses of a range of microbial communities (bacteria, fungi, archaea, pseudomonads, and nematodes) to different concentrations of the strobilurin fungicide, azoxystrobin were studied. A significant concentration-dependent decrease, and subsequent recovery in soil dehydrogenase activity was recorded, but no significant impact on total microbial biomass was observed. Impacts on specific microbial communities were studied using small subunit (SSU) rRNA terminal restriction fragment length polymorphism (T-RFLP) profiling using soil DNA and RNA. The application of azoxystrobin significantly affected fungal and nematode community structure and diversity but had no impact on other communities. Community impacts were more pronounced in the RNA-derived T-RFLP profiles than in the DNA-derived profiles. qPCR confirmed that azoxystrobin application significantly reduced fungal, but not bacterial, SSU rRNA gene copy number. Azoxystrobin application reduced the prevalence of ascomycete fungi, but increased the relative abundance of zygomycetes. Azoxystrobin amendment also reduced the relative abundance of nematodes in the order Enoplia, but stimulated a large increase in the relative abundance of nematodes from the order Araeolaimida.