Molecular typing of Mycoplasma agalactiae: tracing European-wide genetic diversity and an endemic clonal population

Mycoplasma agalactiae causes chronic infections in small ruminants and remains endemic in many regions of the world, despite intensive and costly eradication programs. In this study, the innate genomic plasticity of M. agalactiae was exploited to design and assess a combination of molecular epidemiological tools to trace the pathogen in different geographic locations and to understand its emergence or re-emergence after eradication campaigns. For this purpose, two collections of M. agalactiae isolates, representing European outbreaks or localized endemic disease in a single region of France, were subjected to RFLP (Restriction Fragment Length Polymorphism) analyses using two sets of DNA probes (distributed across the genome and specific for the vpma gene locus), and a previously described VNTR (Variable Number Tandem Repeats) analysis. A combination of four genome-specific DNA probes and two VNTRs gave the highest discriminative power. Molecular typing revealed that, while isolates from diverse geographical origins fell into clearly different groups, the endemic disease repeatedly observed in the Western Pyrenees region over the past 30 years has been caused by a unique subtype of M. agalactiae. This indicates that the re-emergence of the pathogen after seemingly successful eradication programs is not due to the importation of exotic strains, but to the persistence of local reservoirs of infection.     

Basic principles of muscle development and growth in meat-producing mammals as affected by the insulin-like growth factor (IGF) system

This presentation aims to describe how the basic events in prenatal muscle development and postnatal muscle growth are controlled by the insulin-like growth factor system (IGF). The prenatal events (myogenesis) cover the rate of proliferation, the rate and extent of fusion, and the differentiation of three myoblast populations, giving rise to primary fibers, secondary fibers, and a satellite cell population, respectively. The number of muscle fibers, a key determinant of the postnatal growth rate, is fixed late in gestation. The postnatal events contributing to myofiber hypertrophy comprise satellite cell proliferation and differentiation, and protein turnover. Muscle cell cultures produce IGFs and IGF binding proteins (IGFBPs) in various degrees depending on the origin (species, muscle type) and state of development of these cells, suggesting an autocrine/paracrine mode of action of IGF-related factors. In vivo studies and results based on cell lines or primary cell cultures show that IGF-I and IGF-II stimulate both proliferation and differentiation of myoblasts and satellite cells in a time and concentration-dependent way, via interaction with type I IGF receptors. However, IGF binding proteins (IGFBP) may either inhibit or potentiate the stimulating effects of IGFs on proliferation or differentiation. During postnatal growth in vivo or in fully differentiated muscle cells in culture, IGF-I stimulates the rate of protein synthesis and inhibits the rate of protein degradation, thereby enhancing myofiber hypertrophy. The possible roles and actions of the IGF system in regulating and determining muscle growth as affected by developmental stage and age, muscle type, feeding levels, treatment with growth hormone and selection for growth performance are discussed.     

Relationships between biochemical attributes (non-structural carbohydrates and phenolics) and natural durability against fungi in dry teak wood (Tectona grandis L. f.)

• Introduction   

Non-structural carbohydrates and phenolic compounds are implicated in the natural durability of wood. In order to find the chemical traits of natural durability in teak wood, the radial distribution of phenolics compounds and non-structural carbohydrates were studied in trees ranked by contrasting natural durability class against Antrodia sp.     

Assessing the impact of interfering organic matter on soil metaproteomic workflow

Soil organic matter (SOM) is biologically, chemically, and physically complex. As a major store of nutrients within the soil, it plays an important role in nutrient provision to plants. An enhanced understanding of SOM utilisation processes could underpin better fertiliser management for plant growth, with reduced environmental losses. Metaproteomics can allow the characterisation of protein profiles and could help gain insights into SOM microbial decomposition mechanisms. Here, we applied three different extraction methods to two soil types to recover SOM with different characteristics. Specifically, water-extractable organic matter, mineral-associated organic matter and protein-bound organic matter were targeted with the aim to investigate the metaproteome enriched in those extractions. As a proof-of-concept, replicated extracts from one soil were further analysed for peptide identification using liquid chromatography followed by tandem mass spectrometry. We employed a framework for mining mass spectra for both peptide assignment and fragmentation pattern characterisation. Different extracts were found to exhibit contrasting total protein and humic substance content for the two soils investigated. Overall, water extracts displayed the lowest humic substance content (in both soils) and the highest number of peptide identifications (in the soil investigated) with the most frequent peptide hits associated with diverse substrate/ligand binding proteins of Proteobacteria and derived taxa. Our framework also highlighted a strong peptidic signal in unassigned and unmatched spectra, information that is currently not captured by the pipelines employed in this study. Taken together, this work points to specific areas for optimisation in chromatography and mass spectrometry to adequately characterise SOM-associated metaproteomes.     

Attributes of People’s Organisations and the institutional viability of selected Community-Based Forest Management projects in northern Luzon

An assessment was made of the attributes of People’s Organisations (POs) and of the institutional viability of Community-Based Forest Management (CBFM) programs in three project sites in the Philippines. In CBFM, the capacity of People’s Organisations, as de facto managers of forest resources, is being developed by non-government organisations under the guidance of local government units (LGUs) and the Department of Environment and Natural Resources (DENR). A survey was conducted of community members (case informants) and members of the various agencies (key informants) to ascertain the characteristics of successful POs and the relationship between these characteristics and the viability of CBFM. Although commendable contributions have been made to the forest management capability of POs, their standard of living and environmental conditions, there are also many instances in which institutional conflict among stakeholders is apparent. The great challenge for the various stakeholders is to orchestrate their differing interests so as to address successfully the critical issues of the low socioeconomic status of PO members, corruption, lack of transparency and accountability of the organisations involved, loss of interest of PO members in project activities, lack of technical assistance, loss of credibility of the DENR, and lack of equitable benefit sharing.     

Effect of planting stocktype and cultivation treatment on the establishment of Norway spruce on cutaway peatlands

In order to determine the effect of stocktype and cultivation treatment on the field performance (survival and growth) and physiological status of Picea abies in cutaway peatlands, small bare-root, large bare-root and containerised seedlings were planted in a deep ploughed and a control site. Survival after 2 years was good across all treatment (>90%) except for the large bare-root seedlings growing in the control site (84%). For all the morphological characteristics assessed in this study, there was no significant interaction between stocktype and cultivation treatment indicating that the growth response to site cultivation was not stocktype dependent. After two growing seasons, all Norway spruce seedlings performed better in the deep ploughed site and displayed also better nutritional and physiological status. Regardless of cultivation treatment, mean height, diameter and volume increment were significantly smaller for the large bare-root seedlings while the small bare-root seedlings displayed the greatest growth rates. In order to promote early height growth in container and small bare-root stock, large diameter is important. Other initial characteristics such as foliar nitrogen content may also have a strong influence on first year field performance. The physiological status of the seedlings during the first year after outplanting was assessed using chlorophyll fluorescence (CF) measurements. CF measurements detected a higher level of stress for the large-bare root stock (low Fv/Fm). On the other hand, small bare-root stock displayed highest maximum potential photochemical activity which corresponded to greatest growth rates. Container seedlings demonstrated higher capacity for photosynthetic electron transport during the first five months after planting suggesting that they recovered from planting stress quicker and optimised better light interception and utilization than bare-root stock. It can be concluded that intensive management systems including deployment of best-adapted stocktype and site cultivation can be used to enhance early height growth of Norway spruce on cutaway peatlands.     

Mechanisms of resistance to fungicides in field strains of Botrytis cinerea

Field strains of Botrytis cinerea Pers ex Fr, the causal agent of grey mould diseases, were collected from French vineyards between 1993 and 2000. Several phenotypes have been characterized according to the inhibitory effects of fungicides towards germ-tube elongation and mycelial growth. Two types of benzimidazole-resistant strains (Ben R1 and Ben R2) could be detected; negative cross-resistance to phenylcarbamates (e.g. diethofencarb) was only found in Ben R1. Benzimidazole resistance was related to point mutations at codon 198 (Ben R1) or 200 (Ben R2) of the beta-tubulin gene. Most dicarboximide-resistant strains were also weakly resistant to aromatic hydrocarbon fungicides (e.g. dicloran) but remained sensitive to phenylpyrroles (e.g. fludioxonil). These resistant field strains (Imi R1) contained a single base pair mutation at position 365 in a two-component histidine kinase gene, probably involved in the fungal osmoregulation. Three anilinopyrimidine-resistant phenotypes have been identified. In the most resistant one (Ani R1), resistance was restricted to anilinopyrimidines, but no differences were observed in the amino-acid sequences of cystathionine beta-lyase (the potential target site of these fungicides) from Ani R1 or wild-type strains. In the two other phenotypes (Ani R2 and Ani R3), resistance extended to various other groups of fungicide, including dicarboximides, phenylpyrroles and sterol biosynthesis inhibitors. This multi-drug resistance was probably determined by over-production of ATP-binding cassette transporters. The hydroxyanilide fenhexamid is a novel botryticide whose primary target site is the 3-keto reductase involved in sterol C-4 demethylations. Apart from the multi-drug-resistant strain Ani R3, three other fenhexamid-resistant phenotypes have been recognized. For two of them (Hyd R1 and Hyd R2) fenhexamid-resistance seemed to result from P450-mediated detoxification. Reduced sensitivity of the target site could be the putative resistance mechanism operating in the third resistant phenotype (Hyd R3). Increased sensitivity to inhibitors of sterol 14 alpha-demethylase recorded in Hyd R1 strains was related to two amino-acid changes at positions 15 and 105 of this enzyme.     

Seasonal patterns of cadmium accumulation in Arrhenatherum elatius (Poaceae): Influence of mycorrhizal and endophytic fungal colonisation

We investigated the influence of arbuscular mycorrhizae (AM) and dark septate fungi (DSF) colonisation on cadmium (Cd) accumulation in Arrhenatherum elatius from heavy metal-contaminated sites. AM colonisation disappeared when Cd concentrations in soil increased, while DSF infection was weak but constant throughout the experiment indicating that soil heavy metals are toxic to AM but not to DSF. AM colonisation was greatest when plant Cd concentrations were highest providing evidence that AM colonisation may influence Cd accumulation. In addition, the disappearance of AM and the concomitant reduction of Cd in shoots during seed maturation result in our suggestion that seasonal variation in AM may play a role in protecting developing seeds from soil pollution.     

Interseeded legumes with loblolly pine. I. Effect of phosphorus and legume variety on pine seedling establishment and mortality

A study was conducted to determine the effects of legume companion crops and phosphorus (P) fertilizer on the growth and survival characteristics of newly established loblolly pine (Pinus taeda L.) seedlings. At 12 months post‐establishment, there was no legume effect (P>0.05) on root lateral development or ropting depth for pine seedlings. Likewise, there was no legume effect (P>0.05) on aboveground biomass production of pine seedlings. Partridge pea (Cassia fasciculata Michx.) had a negative effect (P<0.05) on pine seedling total root biomass compared to other treatments. Pine seedlings grown with legumes allocated less resources to root development compared to pine seedlings grown alone. Pine seedlings grown alone or with cowpea [Vigna unguiculata (L.) Walp.] were subject to less mortality (P<.05) than seedlings grown with alyceclover [Alysicarpus vaginalis (L.) DC] or partridge pea. Phosphorus fertilization enhanced dry matter (DM) yield of legumes but had no effect on rooting depth of pine seedlings during the first 12 months of growth. After 12 months post‐establishment, the most pronounced effect of P fertilization was that of increased nitrogen (N) content of leaf, stem, and roots of pine seedlings. Native, annual herbaceous grass biomass in the control plots (no legume) reduced the amount of soil N to below pre‐planting levels, while soil N levels in all legume plots exceeded pre‐trial levels.     

Simultaneous determination of ethidimuron, methabenzthiazuron, and their two major degradation products in soil

An analytical method has been developed for the quantification of two herbicides (ethidimuron and methabenzthiazuron) and their two main soil derivatives. This method involves fluidized-bed extraction (FBE) prior to cleanup and analysis by reverse-phase liquid chromatography with UV detection at 282 nm. FBE conditions were established to provide efficient extraction without degradation of the four analytes. (14)C-labeled compounds were used for the optimization of extraction and purification steps and for the determination of related efficiencies. Extraction was optimal using a fexIKA extractor operating at 110 degrees C for three cycles (total time = 95 min) with 75 g of soil and 150 mL of a 60:40 v/v acetone/water mixture. Extracts were further purified on a 500 mg silica SPE cartridge. Separation was performed on a C18 Purosphere column (250 mm x 4 mm i.d.), at 0.8 mL min(-1) and 30 degrees C with an elution gradient made up of phosphoric acid aqueous solution (pH 2.2) and acetonitrile. Calibration curves were found to be linear in the 0.5-50 mg L(-1) concentration range. Besides freshly spiked soil samples, method validation included the analysis of samples with aged residues. Recovery values, determined from spiked samples, were close to 100%. Limits of detection ranged between 2 and 3 microg kg(-1) of dry soil and limits of quantification between 8 and 10 microg kg(-1) of dry soil. An attempt to improve these performances by using fluorescence detection following postcolumn derivatization by orthophthalaldehyde-mercaptoethanol reagent was unsuccessful.