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Behaviours such as chemotaxis can facilitate metabolic exchanges between phytoplankton and heterotrophic bacteria, which ultimately regulate oceanic productivity and biogeochemistry. However, numerically dominant picophytoplankton have been considered too small to be detected by chemotactic bacteria, implying that cell–cell interactions might not be possible between some of the most abundant organisms in the ocean. Here we examined how bacterial behaviour influences metabolic exchanges at the single-cell level between the ubiquitous picophytoplankton Synechococcus and the heterotrophic bacterium Marinobacter adhaerens, using bacterial mutants deficient in motility and chemotaxis. Stable-isotope tracking revealed that chemotaxis increased nitrogen and carbon uptake of both partners by up to 4.4-fold. A mathematical model following thousands of cells confirmed that short periods of exposure to small but nutrient-rich microenvironments surrounding Synechococcus cells provide a considerable competitive advantage to chemotactic bacteria. These findings reveal that transient interactions mediated by chemotaxis can underpin metabolic relationships among the ocean’s most abundant microorganisms. ISSN:2058-5276

The skeleton of reef-building coral harbors diverse microbial communities that could compensate for metabolic deficiencies caused by the loss of algal endosymbionts, i.e., coral bleaching. However, it is unknown to what extent endolith taxonomic diversity and functional potential might contribute to thermal resilience. Here we exposed Goniastrea edwardsi and Porites lutea, two common reef‐building corals from the central Red Sea to a 17-day long heat stress. Using hyperspectral imaging, marker gene/metagenomic sequencing, and NanoSIMS, we characterized their endolithic microbiomes together with 15N and 13C assimilation of two skeletal compartments: the endolithic band directly below the coral tissue and the deep skeleton. The bleaching-resistant G. edwardsi was associated with endolithic microbiomes of greater functional diversity and redundancy that exhibited lower N and C assimilation than endoliths in the bleaching-sensitive P. lutea. We propose that the lower endolithic primary productivity in G. edwardsi can be attributed to the dominance of chemolithotrophs. Lower primary production within the skeleton may prevent unbalanced nutrient fluxes to coral tissues under heat stress, thereby preserving nutrient-limiting conditions characteristic of a stable coral-algal symbiosis. Our findings link coral endolithic microbiome structure and function to bleaching susceptibility, providing new avenues for understanding and eventually mitigating reef loss.

Symbiotic cnidarians such as corals and anemones form highly productive and biodiverse coral-reef ecosystems in nutrient-poor ocean environments, a phenomenon known as Darwin’s Paradox. Resolving this paradox requires elucidating the molecular bases of efficient nutrient distribution and recycling in the cnidarian-dinoflagellate symbiosis. Using the sea anemone Aiptasia, we show that during symbiosis, the increased availability of glucose and the presence of the algae jointly induce the coordinated upregulation and re-localization of glucose and ammonium transporters. These molecular responses are critical to support symbiont functioning and organism-wide nitrogen assimilation through GS/GOGAT-mediated amino-acid biosynthesis. Our results reveal crucial aspects of the molecular mechanisms underlying nitrogen conservation and recycling in these organisms that allow them to thrive in the nitrogen-poor ocean environments.One-sentence summaryWhole-organism nitrogen assimilation fueled by glucose from symbiotic algae enables corals to flourish in oligotrophic waters.

The ability of organisms to combine autotrophy and heterotrophy gives rise to one of the most successful nutritional strategies on Earth: mixotrophy. Sponges are integral members of shallow-water ecosystems and many host photosynthetic symbionts, but studies on mixotrophic sponges have focused primarily on species residing in high-light environments. Here, we quantify the contribution of photoautotrophy to the respiratory demand and total carbon diet of the sponge Chondrilla caribensis, which hosts symbiotic cyanobacteria and lives in low-light environments. Although the sponge is net heterotrophic at 20 m water depth, photosynthetically fixed carbon potentially provides up to 52% of the holobiont’s respiratory demand. When considering the total mixotrophic diet, photoautotrophy contributed an estimated 7% to total daily carbon uptake. Visualization of inorganic 13C- and 15N-incorporation using nanoscale secondary ion mass spectrometry (NanoSIMS) at the single-cell level confirmed that a portion of nutrients assimilated by the prokaryotic community was translocated to host cells. Photoautotrophy can thus provide an important supplemental source of carbon for sponges, even in low-light habitats. This trophic plasticity may represent a widespread strategy for net heterotrophic sponges hosting photosymbionts, enabling the host to buffer against periods of nutritional stress.

Parasites are widespread and diverse in oceanic plankton and many of them infect single-celled algae for survival. How these parasites develop and scavenge energy within the host and how the cellular organization and metabolism of the host is altered remain open questions. Combining quantitative structural and chemical imaging with time-resolved transcriptomics, we unveil dramatic morphological and metabolic changes of the marine parasite Amoebophrya (Syndiniales) during intracellular infection, particularly following engulfment and digestion of nutrient-rich host chromosomes. Changes include a sequential acristate and cristate mitochondrion with a 200-fold increase in volume, a 13-fold increase in nucleus volume, development of Golgi apparatus and a metabolic switch from glycolysis (within the host) to TCA (free-living dinospore). Similar changes are seen in apicomplexan parasites, thus underlining convergent traits driven by metabolic constraints and the infection cycle. In the algal host, energy-producing organelles (plastid, mitochondria) remain relatively intact during most of the infection. We also observed that sugar reserves diminish while lipid droplets increase. Rapid infection of the host nucleus could be a “zombifying” strategy, allowing the parasite to digest nutrient-rich chromosomes and escape cytoplasmic defense, whilst benefiting from maintained carbon-energy production of the host cell.

Symbiosis between reef-building corals and unicellular algae (Symbiodiniaceae) fuels the growth and productivity of corals reefs. Capacity for Symbiodiniaceae to fix inorganic carbon (Ci) and translocate carbon compounds to the host is central to coral health, but how these processes change for corals thriving in environmental extremes remains largely unresolved. We investigate how a model coral—Pocillopora acuta—persists from a reef habitat into an adjacent extreme mangrove lagoon on the Great Barrier Reef. We combine respirometry and photophysiology measurements, Symbiodiniaceae genotyping, and 13C labelling to compare P. acuta metabolic performance across habitats, in relation to the Ci uptake and translocation capacity by symbionts’ autotrophy. We show that differences in P. acuta metabolic strategies across habitats align with a shift in dominant host-associated Symbiodiniaceae taxon, from Cladocopium in the reef to Durusdinium in the mangroves. This shift corresponded with a change in “photosynthetic strategy”, with P. acuta in the mangroves utilising absorbed light for photochemistry over non-photochemical quenching. Mangrove corals translocated similar proportions of carbon compared to the reefs, despite a lower Ci uptake. These trends indicate that coral survival in mangrove environments occurs through sustained translocation rate of organic compounds from coral symbionts to host.

Efficient nutrient cycling in the coral-algal symbiosis requires constant but limited nitrogen availability. Coral-associated diazotrophs, i.e., prokaryotes capable of fixing dinitrogen, may thus support productivity in a stable coral-algal symbiosis but could contribute to its breakdown when overstimulated. However, the effects of environmental conditions on diazotroph communities and their interaction with other members of the coral holobiont remain poorly understood. Here we assessed the effects of heat stress on diazotroph diversity and their contribution to holobiont nutrient cycling in the reef-building coral Stylophora pistillata from the central Red Sea. In a stable symbiotic state, we found that nitrogen fixation by coral-associated diazotrophs constitutes a source of nitrogen to the algal symbionts. Heat stress caused an increase in nitrogen fixation concomitant with a change in diazotroph communities. Yet, this additional fixed nitrogen was not assimilated by the coral tissue or the algal symbionts. We conclude that although diazotrophs may support coral holobiont functioning under low nitrogen availability, altered nutrient cycling during heat stress abates the dependence of the coral host and its algal symbionts on diazotroph-derived nitrogen. Consequently, the role of nitrogen fixation in the coral holobiont is strongly dependent on its nutritional status and varies dynamically with environmental conditions.

Strategies for remediation of per- and polyfluoroalkyl substances (PFAS) generally prioritise highly contaminated source areas. However, the mobility of PFAS in the environment often results in extensive low-level contamination of surface waters across broad areas. Constructed Floating Wetlands (CFWs) promote the growth of plants in buoyant structures where pollutants are assimilated into plant biomass. This study examined the hydroponic growth of Juncus krausii, Baumea articulata and Phragmites australis over a 28-day period for remediation of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) contaminated (0.2 µg/L to 30 µg/L) urban stormwater. With increasing PFOA and PFOS concentrations, accumulation in plant species increased although root and shoot distribution varied depending on PFAS functional group. Less PFOA than PFOS accumulated in plant roots (0.006–0.16 versus 0.008–0.68 µg/g), while more PFOA accumulated in the plant shoots (0.02–0.55 versus 0.01–0.16 µg/g) indicating translocation to upper plant portions. Phragmites australis accumulated the highest overall plant tissue concentrations of PFOA and PFOS. The NanoSIMS data demonstrated that PFAS associated with roots and shoots was absorbed and not just surface bound. These results illustrate that CFWs have the potential to be used to reduce PFAS contaminants in surface waters. Refereed/Peer-reviewed

Roots are a primary source of organic carbon input in most soils. The consumption of living and detrital root inputs involves multi-trophic processes and multiple kingdoms of microbial life, but typical microbial ecology studies focus on only one or two major lineages. We used Illumina shotgun RNA sequencing to conduct PCR-independent SSU rRNA community analysis (“community RNA-Seq”) and simultaneously assess the bacteria, archaea, fungi, and microfauna surrounding both living and decomposing roots of the annual grass, Avena fatua. Plants were grown in 13CO2-labeled microcosms amended with 15N-root litter to identify the preferences of rhizosphere organisms for root exudates (13C) versus decaying root biomass (15N) using NanoSIMS microarray imaging (Chip-SIP). When litter was available, rhizosphere and bulk soil had significantly more Amoebozoa, which are potentially important yet often overlooked top-down drivers of detritusphere community dynamics and nutrient cycling. Bulk soil containing litter was depleted in Actinobacteria but had significantly more Bacteroidetes and Proteobacteria. While Actinobacteria were abundant in the rhizosphere, Chip-SIP showed Actinobacteria preferentially incorporated litter relative to root exudates, indicating this group’s more prominent role in detritus elemental cycling in the rhizosphere. Our results emphasize that decomposition is a multi-trophic process involving complex interactions, and our methodology can be used to track the trajectory of carbon through multi-kingdom soil food webs.

SummaryParasites are widespread and diverse in the oceanic plankton, and many of them infect single-celled algae for survival. How these parasites develop and scavenge energy within the host and whether the cellular organization and metabolism of the host is altered remain open questions. Combining quantitative structural and chemical imaging with time-resolved transcriptomics, we unveil dramatic morphological and metabolic changes of the parasite Amoebophrya (Syndiniales) during intracellular infection (e.g. 200-fold increase of mitochondrion volume), particularly following digestion of nutrient-rich host chromosomes. Some of these changes are also found in the apicomplexan parasites (e.g. sequential acristate and cristate mitochondrion, switch from glycolysis to TCA), thus underlining key evolutionary-conserved mechanisms. In the algal host, energy-producing organelles (chloroplast) remain intact during most of the infection, but sugar reserves diminish while lipid droplets increase. Thus, rapid infection of the host nucleus could be a zombifying strategy to digest nutrient-rich chromosomes and escape cytoplasmic defense while benefiting from the maintained C-energy production of the host cell.

Orogenic Au deposits have contributed the majority of Au recovered globally throughout history. However, the mechanism that concentrates Au to extremely high bonanza grades in small domains within these deposits remains enigmatic. The volume of fluid required to provide extreme Au endowments in localized occurrences is not reflected in field observations (e.g., in the extent of quartz veining or hydrothermal alteration). Detailed optical, scanning and transmission electron microscopy, nanoscale secondary ion mass spectrometry, and 3-D neutron tomography have been used to investigate the processes responsible for development of anomalously high grade ore (upward of 3% Au) found in quartz veins at Fosterville gold mine (Victoria, Australia). Distinct textural settings of visible Au include (1) Au concentrated along pressure solution seams associated with wall-rock selvages, (2) as nano- to microscale dusty Au seams parallel to pressure solution seams, and (3) in microscale tension fractures perpendicular to stylolitic seams. The distribution of Au in arsenopyrite and pyrite hosted within pressure solution seams changes as a function of the extent of deformation. Sulfides in highly deformed pressure solution seams exclusively host Au as nano- to micrometer-sized clusters within features associated with corrosion and brittle failure, whereas sulfides in mildly deformed pressure solution seams have Au bound in the crystal structure. It is proposed that Au supersaturation in fluids introduced during seismic periods led to the deposition of abundant Au nanoparticles in quartz-carbonate veins. Subsequent pressure dissolution of vein quartz and carbonate during interseismic intervals allowed for episodic increase in the Au/quartz ratio and permitted liberation and migration of Au nanoparticles, promoting Au grain growth in favorable textural settings. Galvanic corrosion and brittle fracturing of auriferous sulfides during the interseismic period allowed additional remobilization and/or enrichment of sulfide-hosted Au. Repetition of this mechanism over the time scale of deposit formation acted to concentrate Au within the lodes. This Au ore upgrading model, referred to as “aseismic refinement,” provides a new insight for the genesis of ultrarich Au mineralization and, based on textures reported from many Au deposits, may be a globally significant component in the formation of orogenic Au deposits.

Roots are the primary source of organic carbon inputs to most soils. Decomposition is a multi-trophic process involving multiple kingdoms of microbial life, but typically microbial ecology studies focus on one or two major lineages in isolation. We used Illumina shotgun RNA sequencing to conduct PCR-independent SSU rRNA community analysis (“community RNA-Seq”) to simultaneously study the bacteria, archaea, fungi, and microfauna surrounding both living and decomposing roots of the annual grass, Avena fatua. Plants were grown in 13CO2-labeled microcosms amended with 15N-root litter. We identified rhizosphere substrate preferences for 13C-exudates versus 15N-litter using NanoSIMS microarray imaging (Chip-SIP). When litter was available, rhizosphere and bulk soil had significantly more Amoebozoa, which are potentially important yet often overlooked top-down drivers of detritusphere community dynamics and nutrient cycling. Bulk soil containing litter was depleted in Actinobacteria but had significantly more Bacteroidetes and Proteobacteria. While Actinobacteria were abundant in the rhizosphere, Chip-SIP showed Actinobacteria preferentially incorporated litter relative to root exudates, indicating this group’s more prominent role in detritus elemental cycling in the rhizosphere. Our results emphasize that decomposition is a multi-trophic process involving cross-kingdom interactions, and the trajectory of carbon through this soil food web likely impacts the fate of carbon in soil.

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.

Background Sponges are increasingly recognised as key ecosystem engineers in many aquatic habitats. They play an important role in nutrient cycling due to their unrivalled capacity for processing both dissolved and particulate organic matter (DOM and POM) and the exceptional metabolic repertoire of their diverse and abundant microbial communities. Functional studies determining the role of host and microbiome in organic nutrient uptake and exchange, however, are limited. Therefore, we coupled pulse-chase isotopic tracer techniques with nanoscale secondary ion mass spectrometry (NanoSIMS) to visualise the uptake and translocation of 13C- and 15N-labelled dissolved and particulate organic food at subcellular level in the high microbial abundance sponge Plakortis angulospiculatus and the low microbial abundance sponge Halisarca caerulea. Results The two sponge species showed significant enrichment of DOM- and POM-derived 13C and 15N into their tissue over time. Microbial symbionts were actively involved in the assimilation of DOM, but host filtering cells (choanocytes) appeared to be the primary site of DOM and POM uptake in both sponge species overall, via pinocytosis and phagocytosis, respectively. Translocation of carbon and nitrogen from choanocytes to microbial symbionts occurred over time, irrespective of microbial abundance, reflecting recycling of host waste products by the microbiome. Conclusions Here, we provide empirical evidence indicating that the prokaryotic communities of a high and a low microbial abundance sponge obtain nutritional benefits from their host-associated lifestyle. The metabolic interaction between the highly efficient filter-feeding host and its microbial symbionts likely provides a competitive advantage to the sponge holobiont in the oligotrophic environments in which they thrive, by retaining and recycling limiting nutrients. Sponges present a unique model to link nutritional symbiotic interactions to holobiont function, and, via cascading effects, ecosystem functioning, in one of the earliest metazoan–microbe symbioses.

Bromine and peroxidasin (an extracellular peroxidase) are essential for generating sulfilimine cross-links between a methionine and a hydroxylysine within collagen IV, a basement membrane protein. The sulfilimine cross-links increase the structural integrity of basement membranes. The formation of sulfilimine cross-links depends on the ability of peroxidasin to use bromide and hydrogen peroxide substrates to produce hypobromous acid (HOBr). Once a sulfilimine cross-link is created, bromide is released into the extracellular space and becomes available for reutilization. Whether the HOBr generated by peroxidasin is used very selectively for creating sulfilimine cross-links or whether it also causes oxidative damage to bystander molecules (e.g., generating bromotyrosine residues in basement membrane proteins) is unclear. To examine this issue, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to define the distribution of bromine in mammalian tissues. We observed striking enrichment of bromine ((79)Br, (81)Br) in basement membranes of normal human and mouse kidneys. In peroxidasin knockout mice, bromine enrichment of basement membranes of kidneys was reduced by ∼85%. Proteomic studies revealed bromination of tyrosine-1485 in the NC1 domain of α2 collagen IV from kidneys of wild-type mice; the same tyrosine was brominated in collagen IV from human kidney. Bromination of tyrosine-1485 was reduced by >90% in kidneys of peroxidasin knockout mice. Thus, in addition to promoting sulfilimine cross-links in collagen IV, peroxidasin can also brominate a bystander tyrosine. Also, the fact that bromine enrichment is largely confined to basement membranes implies that peroxidasin activity is largely restricted to basement membranes in mammalian tissues.

In contrast to the situation in plants inhabiting most of the world’s ecosystems, mycorrhizal fungi are usually absent from roots of the only two native vascular plant species of maritime Antarctica, Deschampsia antarctica and Colobanthus quitensis. Instead, a range of ascomycete fungi, termed dark septate endophytes (DSEs), frequently colonise the roots of these plant species. We demonstrate that colonisation of Antarctic vascular plants by DSEs facilitates not only the acquisition of organic nitrogen as early protein breakdown products, but also as non‐proteinaceous d‐amino acids and their short peptides, accumulated in slowly‐decomposing organic matter, such as moss peat. Our findings suggest that, in a warming maritime Antarctic, this symbiosis has a key role in accelerating the replacement of formerly dominant moss communities by vascular plants, and in increasing the rate at which ancient carbon stores laid down as moss peat over centuries or millennia are returned to the atmosphere as CO2., In contrast to the situation in plants inhabiting most of the world's ecosystems, mycorrhizal fungi are usually absent from roots of the only two native vascular plant species of maritime Antarctica, Deschampsia antarctica and Colobanthus quitensis. Instead, a range of ascomycete fungi, termed dark septate endophytes (DSEs), frequently colonise the roots of these plant species. We demonstrate that colonisation of Antarctic vascular plants by DSEs facilitates not only the acquisition of organic nitrogen as early protein breakdown products, but also as non‐proteinaceous D‐amino acids and their short peptides, accumulated in slowly‐decomposing organic matter, such as moss peat.

Ongoing impacts of climatic change especially moisture stress remain a global challenge to agricultural production and food security. Such abiotic effects directly influence soil microbial communities. Previously we demonstrated that polymeric hydrogels, able to provide specific interactions with soil microbial communities, enhance the dynamics and selectivity of ingress and colonisation of plant beneficial bacteria at soil microcosm scales. We now show that small quantities of osmotic hydrogels containing mannose nanofibrils specifically situated in close proximity to developing root zones provide enhanced microbial ingress, colonisation and continuous wetting pathways when contacting developing wheat root systems. These effectively create extensions to the natural rhizosphere sustaining significant beneficial rhizobacterial communities when under moisture stress. Sequencing studies, on wheat production soils undergoing a season of low average rainfall clearly showed microbial abundance increases and taxa selectivity in the rhizosphere-hydrogel regions compared to controls. Here wheat yields increased by about 20% with hydrogel addition compared to controls, which represented a 15% increase in the overall average yield in the region. This represents the first reported demonstration that developing rhizospheres may be readily engineered to consistently select their own distinct microbiome as nodes of functional microbial abundance significantly benefiting grain yield during abiotic stress. Thereby suggesting new opportunities to maintain grain yields during periods of drying climate via these fibrillar hydrogels.

Seagrasses thrive in anoxic sediments where sulphide can accumulate to phytotoxic levels. So how do seagrasses persist in this environment? Here, we propose that radial oxygen loss (ROL) from actively growing root tips protects seagrasses from sulphide intrusion not only by abiotically oxidising sulphides in the rhizosphere of young roots, but also by influencing the abundance and spatial distribution of sulphate-reducing and sulphide-oxidising bacteria. We used a novel multifaceted approach combining imaging techniques (confocal fluorescence in situ hybridisation, oxygen planar optodes, and sulphide diffusive gradients in thin films) with microbial community profiling to build a complete picture of the microenvironment of growing roots of the seagrasses Halophila ovalis and Zostera muelleri. ROL was restricted to young root tips, indicating that seagrasses will have limited ability to influence sulphide oxidation in bulk sediments. On the microscale, however, ROL corresponded with decreased abundance of potential sulphate-reducing bacteria and decreased sulphide concentrations in the rhizosphere surrounding young roots. Furthermore, roots leaking oxygen had a higher abundance of sulphide-oxidising cable bacteria; which is the first direct observation of these bacteria on seagrass roots. Thus, ROL may enhance both abiotic and bacterial sulphide oxidation and restrict bacterial sulphide production around vulnerable roots, thereby helping seagrasses to colonise sulphide-rich anoxic sediments.

Climate change enhances root exudation of organic compounds into soils and can lead to loss of soil carbon. Research now shows that oxalic acid (a common exudate) releases organic compounds from protective mineral associations.

Phytoplankton-bacteria interactions drive the surface ocean sulfur cycle and local climatic processes through the production and exchange of a key compound: dimethylsulfoniopropionate (DMSP). Despite their large-scale implications, these interactions remain unquantified at the cellular-scale. Here we use secondary-ion mass spectrometry to provide the first visualization of DMSP at sub-cellular levels, tracking the fate of a stable sulfur isotope (34S) from its incorporation by microalgae as inorganic sulfate to its biosynthesis and exudation as DMSP, and finally its uptake and degradation by bacteria. Our results identify for the first time the storage locations of DMSP in microalgae, with high enrichments present in vacuoles, cytoplasm and chloroplasts. In addition, we quantify DMSP incorporation at the single-cell level, with DMSP-degrading bacteria containing seven times more 34S than the control strain. This study provides an unprecedented methodology to label, retain, and image small diffusible molecules, which can be transposable to other symbiotic systems. DOI: http://dx.doi.org/10.7554/eLife.23008.001, eLife digest Sulfur is an essential element for many organisms and environmental processes. Every year, organisms including microalgae produce more than one billion tons of a sulfur-containing compound called DMSP. Some of this DMSP is released into seawater, where it acts as a key nutrient for microscopic organisms and as a foraging cue to attract fish. DMSP is also the precursor of a gas that helps to form clouds. Despite DMSP’s potential large-scale effects, it is still not clear what role it plays in the organisms that produce it, or how it is transferred from the microalgae that produce it to the bacteria that use it. It is thought that DMSP could potentially protect the cells from sudden changes in the amount of salt in the seawater (salinity) or from other damage, such as oxidative stress – a build-up of harmful chemicals inside cells. In a controlled setting using artificial seawater, Raina et al. used high-resolution imaging and chemical analysis to track the journey of DMSP from microalgae to recipient bacteria. The results show that similar to land plants, algae store DMSP in the compartments that regulate cell pressure and photosynthesis. The presence of DMSP in these locations also supports its proposed role in protecting cells from changes in salinity or oxidative damage. A future step will be to identify the genes involved in producing DMSP in microalgae. This knowledge could be used to create mutants that are either incapable of producing this molecule or that overproduce it. In combination with the high-resolution imaging techniques described here, this will allow researchers to fully understand the role that DMSP plays in these organisms. DOI: http://dx.doi.org/10.7554/eLife.23008.002

Mycorrhiza formation represents a significant carbon (C) acquisition alternative for orchid species, particularly those that remain achlorophyllous through all life stages. As it is known that orchid mycorrhizas facilitate nutrient transfer (most notably of C), it has not been resolved if C transfer occurs only after lysis of mycorrhizal structures (fungal pelotons) or also across the mycorrhizal interface of pre-lysed pelotons. We used high-resolution secondary ion mass spectrometry (nanoSIMS) and labelling with enriched (13) CO2 to trace C transfers, at subcellular scale, across mycorrhizal interfaces formed by Rhizanthella gardneri, an achlorphyllous orchid. Carbon was successfully traced in to the fungal portion of orchid mycorrhizas. However, we did not detect C movement across intact mycorrhizal interfaces up to 216 h post (13) CO2 labelling. Our findings provide support for the hypothesis that C transfer from the mycorrhizal fungus to orchid, at least for R. gardneri, likely occurs after lysis of the fungal peloton.

Amino sugars in fungal cell walls (such as chitin) represent an important source of nitrogen (N) in many forest soil ecosystems. Despite the importance of this material in soil nitrogen cycling, comparatively little is known about abiotic and biotic controls on and the timescale of its turnover. Part of the reason for this lack of information is the inaccessibility of these materials to classic bulk extraction methods. To address this issue, we used advanced visualization tools to examine transformation pathways of chitin-rich fungal cell wall residues as they interact with microorganisms, soil organic matter and mineral surfaces. Our goal was to document initial micro-scale dynamics of the incorporation of 13 C- and 15 N-labeled chitin into fungi-dominated microenvironments in O-horizons of old-growth forest soils. At the end of a 3-week incubation experiment, high-resolution secondary ion mass spectrometry imaging of hyphae-associated soil microstructures revealed a preferential association of 15 N with Fe-rich particles. Synchrotron-based scanning transmission X-ray spectromicroscopy (STXM/NEXAFS) of the same samples showed that thin organic coatings on these soil microstructures are enriched in aliphatic C and amide N on Fe (hydr)oxides, suggesting a concentration of microbial lipids and proteins on these surfaces. A possible explanation for the results of our micro-scale investigation of chemical and spatial patterns is that amide N from chitinous fungal cell walls was assimilated by hyphae-associated bacteria, resynthesized into proteinaceous amide N, and subsequently concentrated onto Fe (hydr)oxide surfaces. If confirmed in other soil ecosystems, such rapid association of microbial N with hydroxylated Fe oxide surfaces may have important implications for mechanistic models of microbial cycling of C and N.

Gastrodia sesamoides, a common obligate mycoheterotrophic orchid species found in eastern Australia relies on a soil fungus to provide a source of carbon nutrition. The identity of this fungus is not known although in other studies of Gastrodia species a number of mycobionts have been suggested including Fomes and Mycena. In this study the fungal community of rhizomes of G. sesamoides has been identified via fungal ITS-DNA PCR amplification, cloning and sequencing. Although a number of fungi were identified by this approach the most common fungal ITS DNA within the orchid were saprotrophic members of the Marasmiaceae (Campanella and Marasmius spp.). Analysis of the natural carbon and nitrogen stable isotope abundances of stems of G. sesamoides showed an enrichment in 13C and low levels of 15N. These data suggest that G. sesamoides obtains its carbon parasitically from free-living saprotrophic fungi and not from an ectomycorrhizal fungal partner of a photosynthetic plant, as is common for other obligate mycoheterotrophic orchid species.

In this study, we have identified the root-associated fungi of a common species of terrestrial orchid across its range in eastern Australia. We have amplified and cloned fungal ITS DNA extracted from roots of 15 Pterostylis nutans R. Br. plants from six separate geographic localities. Sequencing and GenBank comparison demonstrated two species of Ceratobasidium fungi as the main fungal partners of the orchid. Uncommon fungal associates included homobasidiomycete species such as a Gymnomyces sp. and a Tricholoma sp., Leptodontidium orchidicola, and an unidentified soil fungus. These results demonstrate that specificity for fungal partners occurs in P. nutans and reinforces the idea that conservation measures for endangered Australian orchids must include ex situ perpetuation of fungal symbionts as well as plant material.

We used ITS-RFLP and sequence analysis to determine the identities of the fungal endophytes of six terrestrial orchid species from southeastern Queensland, a region previously unexplored in this context. Pure cultures of orchid - colonising fungi were obtained and fungal identities were assessed by means of ITS-PCR, RFLP analysis, sequence comparison, and protocorm colonisation tests. ITS-PCR and RFLP analysis resulted in five main groupings. Sequencing and GenBank comparison of these five groups showed that the fungal endophytes isolated from the three Pterostylis species were probably Thanatephorus species. There was close sequence identity (90%) of the fungus isolated from Acianthus spp. to Epulorhiza repens, suggesting these may be the same fungal species. However, that only E. repens succeeded in colonising protocorms of Thelymitra pauciflora suggests these may be different species of Epulorhiza. Analysis of the ITS and LSU sequences of the fungus isolated from Caladenia carnea showed high identities with a sequence from a Sebacina vermifera originally isolated from Caladenia dilatata. These results show that there is specificity for fungal partners within the orchid genera Acianthus, Caladenia and Pterostylis.

Mycorrhiza formation represents a significant carbon (C) acquisition alternative for orchid species, particularly those that remain achlorophyllous through all life stages. As it is known that orchid mycorrhizas facilitate nutrient transfer (most notably of C), it has not been resolved if C transfer occurs only after lysis of mycorrhizal structures (fungal pelotons) or also across the mycorrhizal interface of pre-lysed pelotons. We used high-resolution secondary ion mass spectrometry (nanoSIMS) and labelling with enriched (13) CO2 to trace C transfers, at subcellular scale, across mycorrhizal interfaces formed by Rhizanthella gardneri, an achlorphyllous orchid. Carbon was successfully traced in to the fungal portion of orchid mycorrhizas. However, we did not detect C movement across intact mycorrhizal interfaces up to 216 h post (13) CO2 labelling. Our findings provide support for the hypothesis that C transfer from the mycorrhizal fungus to orchid, at least for R. gardneri, likely occurs after lysis of the fungal peloton.

Summary • Rhizanthella gardneri is a rare and fully subterranean orchid that is presumably obligately mycoheterotrophic. R. gardneri is thought to be linked via a common mycorrhizal fungus to co-occurring autotrophic shrubs, but there is no experimental evidence to support this supposition. • We used compartmentalized microcosms to investigate the R. gardneri tripartite relationship. 13CO2 was applied to foliage of Melaleuca scalena plants and [13C-15N]glycine was fed to the common mycorrhizal fungus, and both sources traced to R. gardneri plants. • In our microcosm trial, up to 5% of carbon (C) fed as 13CO2 to the autotrophic shrub was transferred to R. gardneri. R. gardneri also readily acquired soil C and nitrogen (N), where up to 6.2% of C and 22.5% of N fed as labelled glycine to soil was transferred via the fungus to R. gardneri after 240 h. • Our study confirms that R. gardneri is mycoheterotrophic and acquires nutrients via mycorrhizal fungus connections from an ectomycorrhizal autotrophic shrub and directly from the soil via the same fungus. This connection with a specific fungus is key to explaining why R. gardneri occurs exclusively under certain Melaleuca species at a very limited number of sites in Western Australia.

Fully subterranean Rhizanthella gardneri (Orchidaceae) is obligately mycoheterotrophic meaning it is nutritionally dependent on the fungus it forms mycorrhizas with. Furthermore, R. gardneri purportedly participates in a nutrient sharing tripartite relationship where its mycorrhizal fungus simultaneously forms ectomycorrhizas with species of Melaleuca uncinata s.l. Although the mycorrhizal fungus of R. gardneri has been morphologically identified as Thanatephorus gardneri (from a single isolate), this identification has been recently questioned. We sought to clarify the identification of the mycorrhizal fungus of R. gardneri, using molecular methods, and to identify how specific its mycorrhizal relationship is. Fungal isolates taken from all sites where R. gardneri is known to occur shared almost identical ribosomal DNA (rDNA) sequences. The fungal isolate rDNA most closely matched that of other Ceratobasidiales species, particularly within the Ceratobasidium genus. However, interpretation of results was difficult as we found two distinct ITS sequences within all mycorrhizal fungal isolates of R. gardneri that we assessed. All mycorrhizal fungal isolates of R. gardneri readily formed ectomycorrhizas with a range of M. uncinata s.l. species. Consequently, it is likely that R. gardneri can form a nutrient sharing tripartite relationship where R. gardneri is connected to autotrophic M. uncinata s.l. by a common mycorrhizal fungus. These findings have implications for better understanding R. gardneri distribution, evolution and the ecological significance of its mycorrhizal fungus, particularly in relation to nutrient acquisition.

Rhizanthella gardneri R.S.Rogers is an entirely subterranean mycoheterotrophic orchid known only from two isolated populations within south-western Western Australia (WA). This rare species appears restricted to habitats dominated by species of the Melaleuca uncinata complex. R. gardneri purportedly forms a tripartite relationship with Melaleuca1, via a connecting mycorrhizal fungus, for the purpose of carbohydrate and nutrient acquisition. Here, we quantify key climate, soil and vegetation characteristics of known R. gardneri habitats to provide baseline data for monitoring of known R. gardneri populations, to better understand how R. gardneri interacts with its habitat and to identify possible new sites for R. gardneri introduction. We found that the habitats of the two known R. gardneri populations show considerable differences in soil chemistry, Melaleuca structure and Melaleuca productivity. Multivariate analyses showed that both multidimensional scaling (MDS) and principal components analysis (PCA) ordinations of soil chemical characteristics were very similar. Individual sites within populations were relatively similar in all attributes measured, whereas overall northern and southern habitats were distinct from each other. These results suggest that R. gardneri can tolerate a range of conditions and may be more widespread than previously thought, given that there are extensive areas of Melaleuca thickets with similar habitat characteristics across south-western WA. Variability within the habitats of known R. gardneri populations suggests translocation of this species into sites with similar vegetation may be a viable option for the survival of this species.