Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function

Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases.

A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cells

An optimized protocol has been developed for the efficient and rapid genetic modification of sugar beet (Beta vulgaris L.). A polyethylene glycol-mediated DNA transformation technique could be applied to protoplast populations enriched specifically for a single totipotent cell type derived from stomatal guard cells, to achieve high transformation frequencies. Bialaphos resistance, conferred by the pat gene, produced a highly efficient selection system. The majority of plants were obtained within 8 to 9 weeks and were appropriate for plant breeding purposes. All were resistant to glufosinate-ammonium-based herbicides. Detailed genomic characterization has verified transgene integration, and progeny analysis showed Mendelian inheritance.

Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax). (c) 2007 Elsevier Inc. All rights reserved.

Antifungal activity of Bacillus subtilis filtrate to control Fusarium oxysporum f.sp. lentis, the causal organism of lentil vascular wilt

In dual cultures, the supernatant filtrate of the biological control agent Bacillus subtilis was evaluated against (Fusarium oxysporum f.sp. lentis) the causal organism of lentil vascular wilt. The antagonistic activity was evaluated as percent reduction of fungal growth (certainly due, in part, to the antifungal metabolites produced by the antagonistic bacterium). The in-vitro experiments showed that B. subtilis filtrate, whether solid or liquid media, had a strong inhibiting activity on the spore germination and mycelial growth of F. oxysporum f. sp. lentis. In a glasshouse experiment, soil was drenched with B. subtilis filtrate at 30 ml/kg (vol/wt) around seedlings of a susceptible lentil line (ILL 4605). In this treatment there was only 31% mortality compared with 100% kill of plants in the control treatment (P≤0.05).

Formulation and delivery of the bacterial antagonist Bacillus subtilis for management of lentil vascular wilt caused by Fusarium oxysporum f. sp lentis

Different formulations of Bacillus subtilis were prepared using standard laboratory protocols. Bacillus subtilis survived in glucose and talc powders at 8.6 and 7.8 log(10) CFU/g, respectively, for 1 year of storage at room temperature compared with 3.5 log(10) CFU/g on a peat formulation. Glasshouse experiments using soil and seed treatments were conducted to test the efficacy of B. subtilis for protecting lentil against the wilt disease caused by Fusariumoxysporum f. sp. lentis. Seed treatments with formulations of B. subtilis on glucose, talc and peat significantly enhanced its biocontrol activity against Fusarium compared with a treatment in which spores were applied directly to seed. The formulations decreased disease severity by reducing colonization of plants by the pathogen, promoting their growth and increased the dry weight of lentil plants. Of these treatments the glucose and talc-based powder formulations were more effective than the peat formulation and the spore application without a carrier. It was shown that the B. subtilis spores applied with glucose were viable for longer than those applied with other carriers. Seed treatment with these formulated spores is an effective delivery system that can provide a conducive environment for B. subtilis to suppress vascular wilt disease on lentil and has the potential for utilization in commercial field application.

Comparative effect of root-knot nematode on severity of Verticillium and Fusarium wilt in cotton

The effect of root-knot nematode (RKN) (Meloidogyne incognita) on Verticillium dahliae and Fusarium oxysporum f.sp. vasinfectum in cotton (Gossypium hirsutum) was investigated. Two different inoculation methods were used, one in which inoculum was added to the soil, so that nematode and fungal inoculum were in close proximity; the other, inoculation into the stem, whereby the two inocula were spatially separated. Invasion of the roots by RKN enhanced disease severity, as measured by the height of vascular browning in the stem, following inoculation with either wilt pathogen. The effect of RKN on Fusarium wilt was more pronounced than that on Verticillium wilt. Nematode-enhanced infection by F. oxysporum is a well known effect but there are few reports of enhanced infection by Verticillium due to RKN. Relative resistance of a number of cotton cultivars to both wilt diseases, as measured by height of vascular browning, was similar to the known field performance of the cultivars. The use of vascular browning as an estimate of disease severity was therefore validated.

Biological control of black vine weevil (Otiorhynchus sulcatus, Coleoptera: Curculionidae) by entomopathogenic bacteria and their cell-free toxic metabolites

Biocontrol agents such as Xeiwrhabduf, nemalophilci and X. nematophila ssp. bovienii and their cell-free protein toxin complexes were lethal to larvae of O. sulcatus when applied to potting compost in the absence of plants. Similarly, strawberry plants infected with 0. sulcaitfi larvae were protected from damage by applications of both cell suspensions of the bacteria and solutions of their cell-free toxic metabolites, indicating that it is the protein toxins, which are responsible for the lethal effects observed. These toxic metabolites were found more effective against 0. sulccitus larvae when treated in soil microflora. Insect mortality is increased by increasing temperature and bacterial concentration. The toxins remained pathogenic for several months when stored in potting soil either at 15 or 20°C, however, bacterial cells were not as persistent as the toxins. It is therefore suggested that these bacteria and their toxic metabolites can he applied in soil for insect pest control.

Are polyphagous geometrid moths with flightless females adapted to budburst phenology of local host species?

The majority of studies demonstrating local adaptation of insect herbivores involve sessile species, particularly those with a parthenogentic phase to their life history or endophagous "parasites" of plants. Current arguments suggest the strength of selection determines whether local adaptation can or cannot take place. Therefore local adaptation should not be limited to species with such traits. We studied the ability of three polyphagous geometrid moths with flightless adult females (Erannisdefoliaria, Operophtera brumata and O. fagata) to synchronise their egg hatching with the budburst of a local host species in north east Scotland. A strong selection for hatching time is expected among generalist moths given the large variation in budburst phenology and an inability to hatch in synchrony with budburst decreases moth fitness substantially. In two successive seasons, we trapped emerging females from patches of five host species and recorded the temperature sum needed for 50% egg hatch of each brood laid by the trapped females. The hatching times of broods were compared against the average budburst time of the maternal host species in the study area. In addition, the trapping dates of each female were recorded. Only O. brumata showed synchrony with egg hatch and budburst which suggests local phenological adaptation to different host species. This could be maintained by selection and partial reproductive isolation between populations dwelling on different host species. No phenological adaptation was found in the other common geometrids of the study area

The control of root-knot nematodes (Meloidogyne spp.) by Pseudomonas oryzihabitans and its immunological detection on tomato roots

Pseudomonas oryzihabitans, a bacterium associated with the entomopathogenic nematode Steinernema abbasi, was evaluated for its potential to colonise roots and thereby control a field population of root-knot nematodes. Immunological techniques were developed to detect root colonisation of P. oryzihabitans on tomato roots using a specific polyclonal antibody raised against vegetative bacterial cells. In vitro, bacterial cell filtrates were also shown significantly to inhibit juveniles hatching. In a glasshouse pot experiment, there were 22 and 82% fewer females in roots of plants treated with suspensions containing 10(3) and 10(6) cells ml(-1) of P oryzihabitans, respectively. In addition, there were significantly fewer egg masses produced; however, the numbers of eggs per egg mass did not differ significantly. The relationship between root colonisation and nematode control is discussed.

The effects of seed mix and management on the abundance of desirable and pernicious unsown species in arable buffer strip communities

Buffer strips are refuges for a variety of plants providing resources, such as pollen, nectar and seeds, for higher trophic levels, including invertebrates, mammals and birds. Margins can also harbour plant species that are potentially injurious to the adjacent arable crop (undesirable species). Sowing perennial species in non-cropped buffer strips can reduce weed incidence, but limits the abundance of annuals with the potential to support wider biodiversity (desirable species). We investigated the responses of unsown plant species present in buffer strips established with three different seed mixes managed annually with three contrasting management regimes (cutting, sward scarification and selective graminicide). Sward scarification had the strongest influence on the unsown desirable (e.g. Sonchus spp.) and unsown pernicious (e.g. Elytrigia repens) species, and was generally associated with higher cover values of these species. However, abundances of several desirable weed species, in particular Poa annua, were not promoted by scarification. The treatments of cutting and graminicide tended to have negative impacts on the unsown species, except for Cirsium vulgare, which increased with graminicide application. Differences in unsown species cover between seed mixes were minimal, although the grass-only mix was more susceptible to establishment by C. vulgare and Galium aparine than the two grass and forb mixes. Annual scarification can enable desirable annuals and sown perennials to co-exist, however, this practice can also promote pernicious species, and so is unlikely to be widely adopted as a management tool in its current form.

Effects of grazing management on beetle and plant assemblages during the re-creation of a flood-plain meadow

Flood-plain meadows (Alopecurus-Sanguisorba grassland) are a floristically rich community of conservation importance throughout Europe. Declines in their distribution due in part to modern farming practices mean they now cover less than 1500 ha in the UK. To investigate the effect of grazing regime during the re-creation of this grassland type, target plant species were sown onto ex-arable land during 1985. Traditional management, based on a July hay cut followed by aftermath grazing was subsequently instigated, and the site was divided into replicated grazing regimes of cattle, sheep and an un-grazed control. Plant and beetle assemblages were sampled and compared to those of target flood-plain meadows and improved grassland communities. Within the re-creation treatments the absence of aftermath grazing reduced beetle abundances and species richness. Assemblages of plants were closest to that of the target flood-plain meadow under sheep grazing, although this differed little from cattle grazing. Beetle species assemblages and functional group structure were, however, closest to the target grassland under cattle grazing. For all taxa the greatest resilience to succession to the target flood-plain meadow occurred when grazing was not part of the management prescription. Although successful re-creation had not been achieved for either the plants or beetles, cutting followed by aftermath cattle grazing has provided the best management to date. (c) 2006 Elsevier B.V. All rights reserved.

The potential of grass field margin management for enhancing beetle diversity in intensive livestock farms

1. Declining populations of UK grassland flora and fauna have been attributed to intensification of agricultural management practices, including changes in cutting, fertilizer, grazing and drainage regimes. We aimed to develop field margin management practices that could reverse declines in intensively managed grassland biodiversity that would have application in the UK and Europe. Here we focus on one aspect of grassland biodiversity, the beetles. 2. In four intensively managed livestock farms in south-west England, 10-m wide field margins in existing grasslands were managed to create seven treatments of increasing sward architectural complexity. This was achieved through combinations of inorganic (NPK) fertilizer, cattle grazing, and timing and height of cutting. To examine the potential influence of complexity on faunal diversity, beetles were identified to species level from suction samples taken between 2003 and 2005, and their assemblage structure was related to margin management, floral assemblages and sward architecture. 3. Beetle abundance, and species richness and evenness were influenced by margin management treatment and its interaction with year. Correlations with sward architecture and the percentage cover of dominant forbs and grasses were also found. Functional groups of the beetles showed different responses to the management treatments. In particular, higher proportional abundances of seed/flower-feeding guilds were found in treatments not receiving NPK fertilizer. 4. The assemblage structure was shown to respond to margin management treatments, sward architecture and the percentage cover of dominant forbs and grasses. The most extensively managed treatments were characterized by distinct successional trajectories from the control treatment. 5. Synthesis and applications. This study provides management options suitable for use within agri-environment schemes intended to improve faunal diversity associated with intensively managed lowland grasslands. Field margins receiving either no management or a single July silage cut were shown to support greater abundances and species richness of beetles, although subtler modifications of conventional management may also be beneficial, for example the absence of NPK fertilizer while maintaining grazing and silage cutting systems.

Use of airborne remote sensing to detect riverside Brassica rapa to aid in risk assessment of transgenic crops

High resolution descriptions of plant distribution have utility for many ecological applications but are especially useful for predictive modeling of gene flow from transgenic crops. Difficulty lies in the extrapolation errors that occur when limited ground survey data are scaled up to the landscape or national level. This problem is epitomized by the wide confidence limits generated in a previous attempt to describe the national abundance of riverside Brassica rapa (a wild relative of cultivated rapeseed) across the United Kingdom. Here, we assess the value of airborne remote sensing to locate B. rapa over large areas and so reduce the need for extrapolation. We describe results from flights over the river Nene in England acquired using Airborne Thematic Mapper (ATM) and Compact Airborne Spectrographic Imager (CASI) imagery, together with ground truth data. It proved possible to detect 97% of flowering B. rapa on the basis of spectral profiles. This included all stands of plants that occupied >2m square (>5 plants), which were detected using single-pixel classification. It also included very small populations (<5 flowering plants, 1-2m square) that generated mixed pixels, which were detected using spectral unmixing. The high detection accuracy for flowering B. rapa was coupled with a rather large false positive rate (43%). The latter could be reduced by using the image detections to target fieldwork to confirm species identity, or by acquiring additional remote sensing data such as laser altimetry or multitemporal imagery.

The ecological niche of Daphnia magna characterized using population growth rate

The concept of an organism's niche is central to ecological theory, but an operational definition is needed that allows both its experimental delineation and interpretation of field distributions of the species. Here we use population growth rate (hereafter, pgr) to de. ne the niche as the set of points in niche space where pgr. 0. If there are just two axes to the niche space, their relationship to pgr can be pictured as a contour map in which pgr varies along the axes in the same way that the height of land above sea level varies with latitude and longitude. In laboratory experiments we measured the pgr of Daphnia magna over a grid of values of pH and Ca2+, and so defined its "laboratory niche'' in pH-Ca2+ space. The position of the laboratory niche boundary suggests that population persistence is only possible above 0.5 mg Ca2+/L and between pH 5.75 and pH 9, though more Ca2+ is needed at lower pH values. To see how well the measured niche predicts the field distribution of D. magna, we examined relevant field data from 422 sites in England and Wales. Of the 58 colonized water bodies, 56 lay within the laboratory niche. Very few of the sites near the niche boundary were colonized, probably because pgr there is so low that populations are vulnerable to extinction by other factors. Our study shows how the niche can be quantified and used to predict field distributions successfully.

Shoot dieback in clipped young Golden Leyland (Cupressocyparis leylandii) trees - a physiological mechanism?

Shoot dieback is a problem in frequently trimmed Leyland hedges and is increasingly affecting gardeners’ choice of hedge trees, having a negative effect on a conifer nursery industry. Some damage can be attributed to the feeding by aphids, but it is unclear if there are also underlying physiological causes. In this study, we tested the hypothesis that shoot-clipping of conifer trees during adverse growing conditions (i.e. high air temperature and low soil moisture) could be leading to shoot ‘dieback’. Three-year-old Golden Leyland Cypress (x Cupressocyparis leylandii ‘Excalibur Gold’) plants were subjected to either a well-watered or droughted irrigation regime and placed in either a ‘hot’ (average day temperature = 40°C) or a ‘cool’ (average day temperature = 27°C) glasshouse compartment. Half of the plants from each glasshouse were clipped on Day 14 and again on Day 50. Measurements of soil moisture content (SMC), net CO2 assimilation rate (A), stomatal conductance (gs), branchlet xylem water potential (XWP), plant height and foliage colour were made. Within the clipped and unclipped treatments of both glasshouse compartments, plants from the droughted regime had significantly lower values for A, gs and XWP than those from the well-watered regime. However, there was no difference in these parameters between the hot and cool glasshouse compartments. The trends seen for A, gs and XWP of all treatments generally mirrored changes in SMC indicating a direct effect of water supply on these parameters. By the end of the experiment the overall foliage colour of plants from the hot glasshouse was darker than that of plants from the cool glasshouse and the overall foliage colour was also darker following shoot clipping. In general, shoot clipping led to increases in A, gs XWP and SMC. This may be due to the reduction in total leaf area leading to a greater supply of water for the remaining leaves. No shoot ‘dieback’ was observed in any treatment in response to drought stress or shoot-clipping.