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Sugarcane is a globally important food, biofuel and biomaterials crop. High nitrogen (N) fertilizer rates aimed at increasing yield often result in environmental damage because of excess and inefficient application. Inoculation with diazotrophic bacteria is an attractive option for reducing N fertilizer needs. However, the efficacy of bacterial inoculants is variable, and their effective formulation remains a knowledge frontier. Here, we take a new approach to investigating diazotrophic bacteria associated with roots using culture-independent microbial community profiling of a commercial sugarcane variety (Q208(A) ) in a field setting. We first identified bacteria that were markedly enriched in the rhizosphere to guide isolation and then tested putative diazotrophs for the ability to colonize axenic sugarcane plantlets (Q208(A) ) and promote growth in suboptimal N supply. One isolate readily colonized roots, fixed N2 and stimulated growth of plantlets, and was classified as a new species, Burkholderia australis sp. nov. Draft genome sequencing of the isolate confirmed the presence of nitrogen fixation. We propose that culture-independent identification and isolation of bacteria that are enriched in rhizosphere and roots, followed by systematic testing and confirming their growth-promoting capacity, is a necessary step towards designing effective microbial inoculants.

Maintaining a high rate of water uptake is crucial for maximum longevity of cut stems. Physiological gel/tylosis formation decreases water transport efficiency in the xylem. The primary mechanism of action for post-harvest Cu2+ treatments in improving cut flower and foliage longevity has been elusive. The effect of Cu2+ on wound-induced xylem vessel occlusion was investigated for Acacia holosericea A. Cunn. ex G. Don. Experiments were conducted using a Cu2+ pulse (5 h, 2.2 mM) and a Cu2+ vase solution (0.5 mM) vs a deionized water (DIW) control. Development of xylem blockage in the stem-end region 10 mm proximal to the wounded stem surface was examined over 21 days by light and transmission electron microscopy. Xylem vessels of stems stood into DIW were occluded with gels secreted into vessel lumens via pits from surrounding axial parenchyma cells. Gel secretion was initiated within 1-2 days post-wounding and gels were detected in the xylem from day 3. In contrast, Cu2+ treatments disrupted the surrounding parenchyma cells, thereby inhibiting gel secretion and maintaining the vessel lumens devoid of occlusions. The Cu2+ treatments significantly improved water uptake by the cut stems as compared to the control. © 2013 Scandinavian Plant Physiology Society.

Transmission electron microscopy (TEM) can provide ultrastructural information for cells in microbial community samples and phylogenetic information can be recovered via molecular surveys. Here we report an approach to link these data sets by coupling fluorescence in situ hybridization (FISH) with either conventional biological or cryogenic TEM. The method could fundamentally improve our understanding of the organization and functioning of microbial communities in natural systems.

Freeze substitution is a process for low temperature dehydration and fixation of rapidly frozen cells that usually takes days to complete. New methods for freeze substitution have been developed that require only basic laboratory tools: a platform shaker, liquid nitrogen, a metal block with holes for cryotubes and an insulated container such as an ice bucket. With this equipment, excellent freeze substitution results can be obtained in as little as 90 min for cells of small volume such as bacteria and tissue culture cells. For cells of greater volume or that have significant diffusion barriers such as cuticles or thick cell walls, one can extend the time to 3 h or more with dry ice. The 3-h method works well for all manner of specimens, including plants and Caenorhabditis elegans as well as smaller samples. Here, we present the basics of the techniques and some results from Nicotiana leaves, C. elegans adult worms, Escherichia coli and baby hamster kidney tissue culture cells. © 2011 The Authors. Journal of Microscopy

Anaerobic ammonium-oxidizing (anammox) bacteria are divided into three compartments by bilayer membranes (from out- to inside): paryphoplasm, riboplasm and anammoxosome. It is proposed that the anammox reaction is performed by proteins located in the anammoxosome and on its membrane giving rise to a proton-motive-force and subsequent ATP synthesis by membrane-bound ATPases. To test this hypothesis, we investigated the location of membrane-bound ATPases in the anammox bacterium 'Candidatus Kuenenia stuttgartiensis'. Four ATPase gene clusters were identified in the K. stuttgartiensis genome: one typical F-ATPase, two atypical F-ATPases and a prokaryotic V-ATPase. K. stuttgartiensis transcriptomic and proteomic analysis and immunoblotting using antisera directed at catalytic subunits of the ATPase gene clusters indicated that only the typical F-ATPase gene cluster most likely encoded a functional ATPase under these cultivation conditions. Immunogold localization showed that the typical F-ATPase was predominantly located on both the outermost and anammoxosome membrane and to a lesser extent on the middle membrane. This is consistent with the anammox physiology model, and confirms the status of the outermost cell membrane as cytoplasmic membrane. The occurrence of ATPase in the anammoxosome membrane suggests that anammox bacteria have evolved a prokaryotic organelle; a membrane-bounded compartment with a specific cellular function: energy metabolism.

Trichomonas vaginalis is the most common sexually transmitted protozoan in the world and its resistance to metronidazole is increasing. The purpose of this study was to demonstrate that clinical metronidazole resistance in T. vaginalis does not occur via the same mechanism as laboratory-induced metronidazole resistance--that is, via hydrogenosome down sizing. Ultrathin sections of this parasite were examined using transmission electron microscopy and the size and area of the cell and hydrogenosomes were compared between drug-resistant laboratory lines and clinically resistant isolates. Clinical metronidazole-resistant T. vaginalis had similar-sized hydrogenosomes as a metronidazole-sensitive isolate. Inducing metronidazole resistance in both of these isolates caused down sizing of hydrogenosomes. Inducing toyocamycin resistance did not cause any ultrastructural changes to the cell or to the hydrogenosome. No correlation between hydrogenosome number and the drug-resistant status of T. vaginalis isolates and lines was observed. This report demonstrates that clinical metronidazole resistance is not associated with down-sized hydrogenosomes, thus indicating that an alternative resistance mechanism is used by T. vaginalis.

Quambalaria spp. are eucalypt leaf and shoot pathogens of growing global importance, yet virtually nothing is known regarding the manner in which they infect and colonize their hosts. A study of the infection process of Q. pitereka and Q. eucalypti on Corymbia and Eucalyptus species was thus undertaken using light, scanning and transmission electron microscopy after artificial inoculation. Conidial germination was triggered when relative humidity levels exceeded 90% and commenced within 2 h in the presence of free water. Light reduced germination but did not prevent germination from occurring. Conidial germination and hyphal growth occurred on the upper and lower leaf surfaces with penetration occurring via the stomata or wounds on the leaf surface or juvenile stems. There was no evidence of direct penetration of the host. Following penetration through the stomata, Q. pitereka and Q. eucalypti hyphae grew only intercellularly without the formation of haustoria or interaction apparatus, which is characteristic of the order Microstromatales. Instead, the presence of an interaction zone is demonstrated in this paper. Conidiophores arose through stomatal openings producing conidia 7 days after infection.

Marine sponges often harbour communities of symbiotic microorganisms that fulfil necessary functions for the well-being of their hosts. Microbial communities associated with the sponge Rhopaloeides odorabile were used as bioindicators for sublethal cupric ion (Cu2+) stress. A combined strategy incorporating molecular, cultivation and electron microscopy techniques was adopted to monitor changes in microbial diversity. The total density of sponge-associated bacteria and counts of the predominant cultivated symbiont (alpha-proteobacterium strain NW001) were significantly reduced in response to Cu2+ concentrations of 1.7 microg l(-1) and above after 14 days of exposure. The number of operational taxonomic units (OTUs) detected by restriction fragment length polymorphism (RFLP) decreased by 64% in sponges exposed to 223 microg l(-1) Cu2+ for 48 h and by 46% in sponges exposed to 19.4 microg l(-1) Cu2+ for 14 days. Electron microscopy was used to identify 17 predominant bacterial morphotypes, composing 47% of the total observed cells in control sponges. A reduction in the proportion of these morphotypes to 25% of observed cells was evident in sponges exposed to a Cu2+ concentration of 19.4 microg l(-1). Although the abundance of most morphotypes decreased under Cu2+ stress, three morphotypes were not reduced in numbers and a single morpho-type actually increased in abundance. Bacterial numbers, as detected using fluorescence in situ hybridization (FISH), decreased significantly after 48 h exposure to 19.4 microg l(-1) Cu2+. Archaea, which are normally prolific in R. odorabile, were not detected after exposure to a Cu2+ concentration of 19.4 microg l(-1) for 14 days, indicating that many of the microorganisms associated with R. odorabile are sensitive to free copper. Sponges exposed to a Cu2+ concentration of 223 microg l(-1) became highly necrosed after 48 h and accumulated 142 +/- 18 mg kg(-1) copper, whereas sponges exposed to 19.4 microg l(-1) Cu2+ accumulated 306 +/- 15 mg kg(-1) copper after 14 days without apoptosis or mortality. Not only do sponges have potential for monitoring elevated concentrations of heavy metals but also examining changes in their microbial symbionts is a novel and sensitive bioindicator for the assessment of pollution on important microbial communities.

In the process of vitrifying aqueous suspensions for cryotransmission electron microscopy, water is solidified without crystallization. Vitrification can be achieved by rapidly plunging an aqueous thin film into a liquid cryogen. The preparation of aqueous thin films prior to vitrification must be performed in an environmental cabinet at controlled temperature and humidity in order to prevent evaporation and temperature‐induced phase changes in the thin film. The device described here incorporates several important features which make the apparatus simpler and more convenient to use than similar devices described in the literature. One of these features includes the use of a totally enclosed environmental cabinet in which the grid, sample, micropipette and absorbent paper are equilibrated before thin‐film preparation. Other features include a cryogen dewar on a swing arm for easy refilling, a guillotine shutter which is used to trigger the plunger electrically and a semiautomatic system which facilitates rapid transfer of the vitrified specimen from liquid propane to liquid nitrogen for storage and reduces handling of the specimen. To demonstrate the utility of the device, results showing the influence of temperature on the morphology of phospholipid vesicles are presented. A commercial cryotransfer apparatus (which is used for transportation of the vitrified specimen to the electron microscope cold‐stage) has been modified to reduce the possibility of reversion of the vitreous phase to the crystalline ice phases. 1994 Blackwell Science Ltd

Seedlings of Brassica juncea (L.) Czern. were grown in solution culture for 14 d prior to exposure to Pb(2+) at an activity of 31 mu M for 72 h. Electron-dense deposits found within the apoplast and symplast were analyzed using scanning transmission electron microscopy/energy dispersive spectroscopy to determine the chemical identity of the deposits and potential toxicity resistance mechanisms. Irrespective of the cellular compartment in which they were found, the deposits contained Pb, O, P, and Cl. For the extracellular deposits, the average Pb: P: O atomic ratio was 1:0.54:3.0, which together with the hexagonal crystal system suggests that Pb is present as chloropyromorphite (Pb(5)(PO(4))(3)Cl). A weak Ca signal also was detected in approximately half of the spectra, possibly indicating the presence of small concentrations of phosphohedyphane (Pb(3)Ca(2)(PO(4))(3)Cl). The evidence suggests that B. juncea resists Pb toxicity by storing precipitated Pb in the vacuole.