In Vitro UDP-Sugar:Undecaprenyl-Phosphate Sugar-1-Phosphate Transferase Assay and Product Detection by Thin Layer Chromatography

In vitro assays are invaluable for the biochemical characterization of UDP-sugar:undecaprenyl-phosphate sugar-1-phosphate transferases. These assays typically involve the use of a radiolabeled substrate and subsequent extraction of the product, which resides in a lipid environment. Here, we describe the preparation of bacterial membranes containing these enzymes, a standard in vitro transferase assay with solvents containing chloroform and methanol, and two methods to measure product formation: scintillation counting and thin layer chromatography.

Microbiology of sugar-rich environments: Diversity, ecology and system constraints

Microbial habitats that contain an excess of carbohydrate in the form of sugar are widespread in the microbial biosphere. Depending on the type of sugar, prevailing water activity and other substances present, sugar-rich environments can be highly dynamic or relatively stable, osmotically stressful, and/or destabilizing for macromolecular systems, and can thereby strongly impact the microbial ecology. Here, we review the microbiology of different high-sugar habitats, including their microbial diversity and physicochemical parameters, which act to impact microbial community assembly and constrain the ecosystem. Saturated sugar beet juice and floral nectar are used as case studies to explore the differences between the microbial ecologies of low and higher water-activity habitats respectively. Nectar is a paradigm of an open, dynamic and biodiverse habitat populated by many microbial taxa, often yeasts and bacteria such as, amongst many others, Metschnikowia spp. and Acinetobacter spp., respectively. By contrast, thick juice is a relatively stable, species-poor habitat and is typically dominated by a single, xerotolerant bacterium (Tetragenococcus halophilus). A number of high-sugar habitats contain chaotropic solutes (e.g. ethyl acetate, phenols, ethanol, fructose and glycerol) and hydrophobic stressors (e.g. ethyl octanoate, hexane, octanol and isoamyl acetate), all of which can induce chaotropicity-mediated stresses that inhibit or prevent multiplication of microbes. Additionally, temperature, pH, nutrition, microbial dispersion and habitat history can determine or constrain the microbiology of high-sugar milieux. Findings are discussed in relation to a number of unanswered scientific questions.

Effects of KCl concentration on accumulation of acyclic sugar alcohols and trehalose in conidia of three entomopathogenic fungi

Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus were grown on Sabouraud Dextrose Agar (SDA) modified with KCl to give a range of water activity (a(w)) from 0.938 to 0.998. Growth of all three species was optimal at 0.983 a(w) and growth occurred over the a(w) range tested. Acyclic sugar alcohol (polyol) and trehalose content of conidia was determined by HPLC and found to vary with species and a(w). Conidia of B. bassiana and P. farinosus were found to contain totals of 1.5% and 2.3% polyols respectively at 0.998 a(w), and double these amounts at <0.950 a(w). Conidia of M. anisopliae contained from 5.7% to 6.8% polyols at each a(w) tested. In conidia of all three species the predominant polyol was mannitol. The lower molecular weight polyols, arabitol and erythritol, were found to accumulate at reduced a(w). Small amounts of glycerol were present in conidia of each species; <15% total polyols. Conidia of B. bassiana and M. anisopliae contained about 0.5% trehalose from 0.970 to 0.998 a(w), but only trace amounts below 0.950 a(w). Conidia of P. farinosus contained 2.1% trehalose at 0.998 a(w) and this decreased to <0.1% below 0.950 a(w). Potential to manipulate the endogenous reserves of conidia of these biological control agents to enhance viability and desiccation tolerance is discussed.

The extended phenotype of Scots pine Pinus sylvestris structures the understorey assemblage

Heritable variation in plant secondary compounds in dominant species has been hypothesised to effect ecosystem function and the structure of associated assemblages of plants, microbes and animals. The functioning of this extended phenotype in relation to the understorey vegetation composition was tested within a boreal forest system dominated by Pinus sylvestris which contains a range of monoterpenes, the composition of which is largely under genetic control. A variance partitioning approach was adopted to identify the relative importance of tree chemistry, environment, spatial location and tree architecture in controlling the distribution of species in the ground flora under individual trees. The monoterpene composition of the pine needles appeared to contribute significantly to controlling understorey vegetation composition, but was less important than environmental factors, though similar to spatial factors. Thus there appears to be a link between variation in the chemical composition of the single, dominant tree species within this system and the pattern of occurrence and abundance in other species at the same trophic level.

Does chemical composition of individual Scots pine trees determine the biodiversity of their associated ground vegetation?

Despite plant secondary metabolites being major determinants of species interactions and ecosystem processes, their role in the maintenance of biodiversity has received little attention. In order to investigate the relationship between chemical and biological diversity in a natural ecosystem, we considered the impact of chemical diversity in individual Scots pine trees (Pinus sylvestris) on species richness of associated ground vegetation. Scots pine trees show substantial genetically determined constitutive variation between individuals in concentrations of a group of secondary metabolites, the monoterpenes. When the monoterpenes of particular trees were assessed individually, there was no relationship with species richness of associated ground flora. However, the chemical diversity of monoterpenes of individual trees was significantly positively associated with the species richness of the ground vegetation beneath each tree, mainly the result of an effect among the non-woody vascular plants. This correlation suggests that the chemical diversity of the ecosystem dominant species has an important role in shaping the biodiversity of the associated plant community. The extent and significance of this effect, and its underlying processes require further investigation.

The fate of 14C-naphthalene in soil microcosms containing Scots pine seedlings and enchytraeids

The fate of freshly spiked and aged ¹⁴C-naphthalene associated residues as investigated in the presence and absence of ectomycorrhizal Scots pine seedlings and enchytraeid worms, in a factorial experimental design. Microcosms were used which enabled the ¹⁴C-labelled naphthalene associated residues to be quantified, including plant lipids which acted as an additional naphthalene sink within the microcosms. The presence of plant roots altered the availability of the ¹⁴C-naphthalene and associated residues to degrading microbes. Mineralisation and volatilisation of ¹⁴C naphthalene in freshly spiked soil were lower in the presence of Scots pine. Conversely, in soil aged for 180 d, Scots pine increased mineralisation, and bioavailability of naphthalene. Root-mediated processes, microbial activity and enchytraeids interact with desorption, bioavailability and mineralisation of naphthalene. © 2006 Elsevier Ltd. All rights reserved.

Degradation of the polycyclic aromatic hydrocarbon (PAH) fluorene is retarded in a Scots pine ectomycorrhizosphere

Polycyclic aromatic hydrocarbons (PAHs) are an important class of persistent organic pollutants (POPs) in the environment and accumulate in forest soils. These soils are often dominated by ectomycorrhizal (EcM) roots, but little is known about how EcM fungi degrade PAHs, or the overall effect of field colonized EcM roots on the fate of PAHs. The ability of eight EcM fungi to degrade PAHs in liquid culture spiked with ¹⁴C labelled PAHs was investigated. Microcosms were used to determine the impact of naturally colonized mycorrhizal pine seedlings on PAH mineralization and volatilization. Only two EcM fungi (Thelephora terrestris and Laccaria laccata) degraded at least one PAH and none were able to mineralize the PAHs in pure culture. Where degradation occurred, the compounds were only mono-oxygenated. EcM pine seedlings did not alter naphthalene mineralization or volatilization but retarded fluorene mineralization by 35% compared with unplanted, ectomycorrhizosphere soil inoculated, microcosms. The EcM fungi possessed limited PAH degrading abilities, which may explain why EcM dominated microcosms retarded fluorene mineralization. This observation is considered in relation to the 'Gadgil-effect', where retarded litter decomposition has been observed in the presence of EcM roots. © New Phytologist (2004).

Sensitivity to Cd or Zn of host and symbiont of ectomycorrhizal Pinus sylvestris L. (Scots pine) seedlings

Pinus sylvestris seedlings infected with either the ectomycorrhizal (ECM) fungus Paxillus involutus or Suillus variegatus were exposed to a range of Cd or Zn concentrations. This was done to investigate the relationship between the sensitivity of ECM fungi and their host plants over a wide range of concentrations. P involutus ameliorated the toxicity of Cd and Zn to P. sylvestris with respect to root length, despite significant inhibition of ECM infection levels by Cd (Cd EC₅₀ [effective concentration which inhibits ECM infection by 50%] values were: P. involutus 3.7 μg g^-1 Cd; S. variegatus 2.3 μg g^-1 Cd). ECM infection by P. involutus also decreased Cd and Zn transport to the plant shoots at potentially toxic concentrations and also influenced the proportion of Zn transported to the roots and shoots, with a higher proportion retained in the roots of the seedlings. ECM infection did increase host biomass production, but this was not affected by the presence of Cd or Zn. Root and shoot biomass production by P. sylvestris, in both the presence and absence of ECM fungi, was unaffected by Cd and Zn at all concentrations tested.

Toxic effects of cadmium and zinc on ectomycorrhizal colonization of scots pine (Pinus sylvestris L.) from soil inoculum

Scots pine seedlings colonized by ectomycorrhizal (ECM) fungi from natural soil inoculum were exposed to a range of Cd or Zn concentrations to investigate the effects of metals on ECM fungi-Scots pine associations in a realistic soil environment. Experiments focused on the relationship between the sensitivity of ECM fungi and their host plants, the influence of metals on ECM community dynamics on Scots pine roots, and the effects of metal exposure on ECM colonization from soil-borne propagules. Ectomycorrhizal colonization was inhibited by Cd and Zn, with a decrease in the proportion of ECM-colonized root tips. Shoot and root biomass, total root length, and total root-tip density, however, were unaffected by Cd or Zn. A decrease in the diversity of ECM morphotypes also occurred, which could have a negative effect on tree vigor. Overall, colonization by ECM fungi was more sensitive than seedling growth to Cd and Zn, and this could have serious implications for successful tree establishment on metal-contaminated soils.

Cross-colonization of scots pine (Pinus sylvestris) seedlings by the ectomycorrhizal fungus Paxillus involutus in the presence of inhibitory levels of Cd and Zn

The effects of Cd and Zn on cross-colonization by Paxillus involutus of Scots pine seedlings was examined by using pairs of ectomycorrhizal (ECM) and non-mycorrhizal (NM) seedlings grown in the same vessel. This was done to assess, first, the ability of P. involutus to colonize NM Scots pine seedlings by growth from colonized roots of other Scots pine seedlings in the presence of Cd or Zn, and, second whether ECM colonization of Scots pine by P. involutus provided a competitive advantage over NM seedlings. Ectomycorrhizal colonization of Scots pine was shown to be more sensitive than Scots pine itself to Cd and Zn, but prior colonization did provide a competitive advantage with respect to biomass production. This beneficial effect over NM seedlings was, however, equal in the control, Cd and Zn treatments, and was due simply to growth stimulation in the presence of ECM colonization. Cross-colonization from an ECM to a NM seedling was reduced but not prevented by Cd and Zn. Cd had a more negative effect on cross-colonization than on initial colonization of seedlings, whereas Zn had an equally inhibitory effect on both parameters. These results have important implications for plant establishment on metal-contaminated sites. If cross-colonization between plants is reduced by toxic metals, plant establishment on contaminated sites might be retarded.

Mineralization of 2,4-dichlorophenol by ectomycorrhizal fungi in axenic culture and in symbiosis with pine

Experiments were conducted to determine if two ectomycorrhizal fungi (Paxillus involutus and Suillus variegatus) could degrade 2,4-dichlorophenol both in axenic liquid culture and during symbiosis with a host tree species Pinus sylvestris. Both fungi readily degraded 2,4- dichlorophenol in batch culture with similar rates of mineralization on a biomass basis. Up to 17% of the 2,4-dichlorophenol was mineralized over a 17 day period. Growth of the fungi in symbiosis with P. sylvestris stimulated greater mineralization than when fungi were grown in absence of the host. S. variegatus was more efficient than P. involutus (in the presence of P. sylveslris) at mineralizing 2,4- dichlorophenol. Mineralization in vermiculite culture was greatly reduced compared to liquid culture. Only 3% of the 2,4-dichlorophenol was mineralized after 13 days in vermiculite culture for the most efficient degrading treatment.

Does climate change information affect stated risks of pine beetle impacts on forests? An application of the exchangeability method

Risks are an essential feature of future climate change impacts. We explore whether knowledge that climate change might be the source of increasing pine beetle impacts on public or private forests affects stated risk estimates of damage, elicited using the exchangeability method. We find that across subjects the difference between public and private forest status does not influence stated risks, but the group told that global warming is the cause of pine beetle damage has significantly higher risk perceptions than the group not given this information.

Exogenous RNA interference exposes contrasting roles for sugar exudation in host-finding by plant pathogens

Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNA interference (RNAi)-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, STP1 and STP2, in tomato seedlings triggered corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponded directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observed no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato and potentially other crop plants.

A rapid staining-assisted wood sampling method for PCR-based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR-based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining-assisted wood sampling method aiding PCR-based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5-mg wood cross-sections, of 0.5 · 0.5 · 0.015 cm dimension, that were cut from a wood sample of 0.5 · 0.5 · 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5-mg wood cross-sections (that adjoined the three 5-mg wood cross-sections, mentioned above) were cut. Nematode-staining-spots (NSSs) in each of the two stained sections were counted under a microscope at 100· magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus – specific amplicon of 403 bp (DQ855275) was generated by PCR assay from 100.00% of 5-mg wood cross-sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx-detection methods such as PCR assay.