Leaf morphology, functional trait and altitude response in perennial vetch (Vicia unijuga A. Braun), alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.).

MAIN CONCLUSION: Species have plasticity across altitude gradients in leaf morphology and function, and their response to high altitude conditions was mainly reflected in leaf cell metabolism and gas exchange. Leaf morphological and functional adaptation to altitude has received research attention in recent years, but there are no studies for forage legumes. Here we report differences in 39 leaf morphology and functional traits of three leguminous forages (alfalfa, sainfoin and perennial vetch) at three sites in Gansu Province, China, ranging from 1768 to 3074 m altitude to provide information for potential use in breeding programmes. With increasing altitude, plant water status increased, reflecting increase in soil water content and decreased average temperature, which lead to leaf intercellular CO2 concentration. Stomatal conductance and evapotranspiration increased significantly but water-use efficiency decreased. At high altitude, ΦPSII decreased but non-photochemical quenching and chlorophyll a:b ratio increased while spongy mesophyll tissue and leaf thickness increased. These changes may be due to UV or low-temperature damage of leaf protein and metabolic cost of plant protection or defence responses. Contrary to many other studies, leaf mass per area decreased significantly at higher altitude. This was consistent with predictions under the worldwide leaf economic spectrum on the basis that soil nutrients increased with increasing altitude. The key species differences were more irregularly shaped epidermal cells and larger stomatal size in perennial vetch compared to alfalfa or sainfoin that enhanced gas exchange and photosynthesis by generating mechanical force, increasing guard cell turgor, and promoting stomatal operation. The lower adaxial stomatal density also enhanced water-use efficiency. These adaptations might confer perennial vetch an advantage in environments with extreme diurnal temperature fluctuation or in frigid conditions.

Arbuscular mycorrhizal fungus changes alfalfa (Medicago sativa) metabolites in response to leaf spot (Phoma medicaginis) infection, with subsequent effects on pea aphid (Acyrthosiphon pisum) behavior.

Plant disease occurs simultaneously with insect attack. Arbuscular mycorrhizal fungi (AMF) modify plant biotic stress response. Arbuscular mycorrhizal fungi and pathogens may modify plant volatile organic compound (VOC) production and insect behavior. Nevertheless, such effects are rarely studied, particularly for mesocosms where component organisms interact with each other. Plant-mediated effects of leaf pathogen (Phoma medicaginis) infection on aphid (Acyrthosiphon pisum) infestation, and role of AMF (Rhizophagus intraradices) in modifying these interactions were elucidated in a glasshouse experiment. We evaluated alfalfa disease occurrence, photosynthesis, phytohormones, trypsin inhibitor (TI) and total phenol response to pathogen and aphid attack, with or without AMF, and aphid behavior towards VOCs from AMF inoculated and non-mycorrhizal alfalfa, with or without pathogen infection. AM fungus enhanced alfalfa resistance to pathogen and aphid infestation. Plant biomass, root : shoot ratio, net photosynthetic rate, transpiration rate, stomatal conductance, salicylic acid, and TI were significantly increased in AM-inoculated alfalfa. Arbuscular mycorrhizal fungi and pathogen significantly changed alfalfa VOCs. Aphids preferred VOCs of AM-inoculated and nonpathogen-infected to nonmycorrhizal and pathogen-infected alfalfa. We propose that AMF alter plant response to multiple biotic stresses in ways both beneficial and harmful to the plant host, providing a basis for strategies to manage pathogens and herbivore pests.

Pathogen Identification and Factors Influencing Infection Frequency and Severity of Fungal Rust in Four Native Grasses in Hulunber Grassland, China.

A serious rust infection present in 2014 and 2015 on the dominant grass species (Leymus chinensis) in the Hulunber grassland of Inner Mongolia, China, and also present on three other grass species (Agropyron cristatum [wheat grass], Bromus inermis, and Festuca ovina) was investigated. Field surveys, laboratory determination of morphological characteristics, pathogenicity tests, and molecular identification methods were integrated to identify two rust-causing pathogens on L. chinensis. It was found that Puccinia elymi was the major pathogen of L. chinensis, and also infected A. cristatum and F. ovina. This is the first report of P. elymi on A. cristatum in China. P. striiformis caused stripe rust on L. chinensis and B. inermis. The incidence and severity of rust infection increased through the growing season, presumably from asexual spread by urediniospores, and was higher on grass species phylogenetically more closely related to common crop hosts of the pathogens. High host grass density and presence of a potential alternate host for P. elymi, Thalictrum squarrosum, were two further factors promoting rust incidence. These results provide insight into ecological factors linked to the rust epidemic and provide a theoretical basis for the formulation of control strategies.

Transcriptome-Wide Gene Expression Plasticity in Stipa grandis in Response to Grazing Intensity Differences.

Organisms have evolved effective and distinct adaptive strategies to survive. Stipa grandis is a representative species for studying the grazing effect on typical steppe plants in the Inner Mongolia Plateau. Although phenotypic (morphological and physiological) variations in S. grandis in response to long-term grazing have been identified, the molecular mechanisms underlying adaptations and plastic responses remain largely unknown. Here, we performed a transcriptomic analysis to investigate changes in gene expression of S. grandis under four different grazing intensities. As a result, a total of 2357 differentially expressed genes (DEGs) were identified among the tested grazing intensities, suggesting long-term grazing resulted in gene expression plasticity that affected diverse biological processes and metabolic pathways in S. grandis. DEGs were identified in RNA-Seq and qRT-PCR analyses that indicated the modulation of the Calvin-Benson cycle and photorespiration metabolic pathways. The key gene expression profiles encoding various proteins (e.g., ribulose-1,5-bisphosphate carboxylase/oxygenase, fructose-1,6-bisphosphate aldolase, glycolate oxidase, etc.) involved in these pathways suggest that they may synergistically respond to grazing to increase the resilience and stress tolerance of S. grandis. Our findings provide scientific clues for improving grassland use and protection and identifying important questions to address in future transcriptome studies.

Fungal Alkaloid Occurrence in Endophyte-Infected Perennial Ryegrass during Seedling Establishment.

The symbiotic Epichloë festucae var. lolii endophyte produces alkaloids which can provide its host grass, perennial ryegrass (Lolium perenne L), with a selective advantage in both natural and agricultural managed ecosystems. This study focuses on understanding the alkaloid concentrations that occur in endophyte-infected perennial ryegrass during the early establishment phase. In a glasshouse experiment fungal alkaloid concentrations (peramine, lolitrem B, ergovaline, and epoxy-janthitrems) were measured in perennial ryegrass seedlings infected with E. festucae var. lolii proprietary strains AR1, AR37, NEA2, and NZ common toxic for 69 days after sowing. The endophyte becomes metabolically active, starting alkaloid production, as early as 6 days after sowing. Alkaloid concentrations peaked in 8- to 10- day-old seedlings due to a seedling growth slowdown. This study provides data showing that the loss of insect protection in endophyte-infected seedlings is linked to a reduction in chemical defence after seed-stored, maternally synthesised alkaloids are diluted by seedling dry matter accumulation.

Correction: Fungal Alkaloid Occurrence in Endophyte-Infected Perennial Ryegrass during Seedling Establishment.

The original version of this article unfortunately contained a mistake. There is a mistake in the affiliation section and Figure 6. The correct affiliation and Figure 6 are shown here.

Morphological fractions, chemical composition and in vitro digestibility of stover of four common vetch varieties grown on the Tibetan plateau.

This study evaluated the varietal variability and interrelationship of the grain and stover yields, and stover quality characteristics of four common vetch varieties (three improved varieties and one local variety) used on the Tibetan Plateau. Stover quality attributes determined included chemical composition, in vitro true digestibility of dry matter (IVTD) and neutral detergent fibre (dNDF). The improved varieties were superior to the local variety in grain yield, harvest index and potential utility index (PUI). The wide ranges in the proportions of leaf (24.3%-34.6%), pod (22.0%-45.6%) and stem (29.5%-44.7%) fractions were observed. Significant varietal differences (p < .001) were observed for the whole-stover content of crude protein [CP; 78.1-101 g/kg dry matter (DM)], neutral detergent fibre (aNDF; 520-564 g/kg DM), acid detergent fibre (ADF; 520-564 g/kg DM), IVTD (597-658 g/kg DM) and dNDF (305-384 g/kg aNDF). The leaf fraction had the highest levels of CP, IVTD and dNDF, while the highest contents of aNDF and ADF were observed in the stem fraction. Canonical correlation analysis showed that significant (p < .001) correlations between the quality attributes of whole stover and proportions and quality attributes of stover morphological fractions. Pearson correlation between the grain and stover yield was negative and significant (r = -.295; p = .001), while the correlations between the grain/stover yield and stover quality traits were positive or non-significant. Ranking of the varieties differed when grain yield, PUI and stover quality scores were investigated. Of the varieties tested, Lanjian No.2 has the best potential for use as a ruminant feed, as indicated by PUI and stover quality.

Drought-induced senescence of Medicago truncatula nodules involves serpin and ferritin to control proteolytic activity and iron levels.

Drought is a major constraint for legume growth and yield. Senescence of nitrogen-fixing nodules is one of the early drought responses and may cause nutrient stress in addition to water stress in legumes. For nodule senescence to function as part of a drought-survival strategy, we propose that the intrinsically destructive senescence process must be tightly regulated. Medicago truncatula protease inhibitor and iron scavenger-encoding genes, possibly involved in controlling nodule senescence, were identified. RNA interference (RNAi) lines were constructed in which expression of a serpin or ferritins was knocked down. Both wild-type and RNAi lines were subjected to drought stress and nodule activity and plant physiological responses were measured. Drought caused M. truncatula to initiate nodule senescence before plant growth was affected and before an increase in papain-like proteolytic activity and free iron levels was apparent. Knock-down expression of serpin6 and ferritins caused increased protease activity, free iron levels, early nodule senescence and reduced plant growth. The results suggest that M. truncatula nodule-expressed serpin6 and ferritins mediate ordered drought-induced senescence by regulating papain-like cysteine protease activity and free iron levels. This strategy may allow the drought-stressed plants to benefit maximally from residual nitrogen fixation and nutrient recovery resulting from break down of macromolecules.

Defining the pathways of symbiotic Epichloë colonization in grass embryos with confocal microscopy.

Asexual cool-season grass endophytes of the genus Epichloë (Ascomycota: Clavicipitaceae) are strictly vertically disseminated. The hosts of these mutualistic fungi express no symptoms during the fungal lifecycle that takes place entirely within the plant, while their hosts receive beneficial outcomes. These fungi are distributed in two major locations within the mature seeds of their hosts; namely, within the embryo (including the scutellum, coleoptile, plumule, radicle, and coleorhiza tissues) and between the aleurone and pericarp layers, with the latter hyphae playing no role in transmission of the fungus to the next plant generation. Conflicting evidence remains in the literature on the timing of embryo colonization. In a detailed investigation, utilizing confocal microscopy to observe the distribution of Epichloë coenophiala strain AR601 in tall fescue (Lolium arundinaceum), we tracked endophyte hyphal colonization in the ovary (pre-fertilization) through to the fully mature seed stage. Confocal microscopy images revealed that at the early and mature developmental stages of the embryo sac, before host grass fertilization, there were large quantities of endophyte mycelium present, especially around the antipodal cells, indicating that this endophyte enters the embryo sac before the fertilization stage. After host fertilization, fungal hyphae could be seen in the true embryo and early nonstarchy endosperm. Understanding the mechanisms of transmission to the seed is important for commercial seed producers and end users.

Salinity-induced reduction in root surface area and changes in major root and shoot traits at the phytomer level in wheat.

The aim of this study was to investigate the effect of salinity stress on root growth at the phytomer level in wheat to provide novel site-specific understanding of salinity damage in roots. Seedlings of 13 wheat varieties were grown hydroponically. Plants were exposed to three concentrations of NaCl, 0 (control), 50 and 100mM, from 47 days after sowing. In a destructive harvest 12 days later we determined the number of live leaves, adventitious roots, seminal roots and newly formed roots at the youngest phytomer; length and diameter of main axes; and length and diameter of root hairs and their number per millimetre of root axis. Elongation rate of main axes and root hair density were then derived. Root surface area at each root-bearing phytomer (Pr) was mechanistically modelled. New root formation was increased by salt exposure, while number of live leaves per plant decreased. The greatest salinity effect on root axis elongation was observed at the youngest roots at Pr1 and Pr2. Both the 50mM and the 100mM levels of salinity reduced root hair length by approximately 25% and root hair density by 40% compared with the control whereas root hairs alone contributed around 93% of the estimated total root surface area of an individual tiller. Decrease in main axis length of new roots, root hair density and root hair length combined to reduce estimated root surface area by 36-66% at the higher NaCl concentration. The varietal response towards the three salinity levels was found to be trait-specific. The data highlight reduction in root surface area as a major but previously largely unrecognized component of salinity damage. Salinity resistance is trait-specific. Selection for retention of root surface area at a specific phytomer position following salt exposure might be useful in development of salinity-tolerant crop varieties.

Leaf Rubisco turnover in a perennial ryegrass (Lolium perenne L.) mapping population: genetic variation, identification of associated QTL, and correlation with plant morphology and yield.

This study tested the hypotheses that: (i) genetic variation in Rubisco turnover may exist in perennial ryegrass (Lolium perenne L.); (ii) such variation might affect nitrogen use efficiency and plant yield; and (iii) genetic control of Rubisco turnover might be amenable to identification by quantitative trait loci (QTL) mapping. A set of 135 full-sib F1 perennial ryegrass plants derived from a pair cross between genotypes from the cultivars 'Grasslands Impact' and 'Grasslands Samson' was studied to test these hypotheses. Leaf Rubisco concentration at different leaf ages was measured and modelled as a log-normal curve described by three mathematical parameters: D (peak Rubisco concentration), G (time of D), and F (curve standard deviation). Herbage dry matter (DM) yield and morphological traits (tiller weight (TW), tiller number (TN), leaf lamina length (LL), and an index of competitive ability (PI)) were also measured. The progeny exhibited continuous variation for all traits. Simple correlation and principal component analyses indicated that plant productivity was associated with peak Rubisco concentration and not Rubisco turnover. Lower DM was associated with higher leaf Rubisco concentration indicating that Rubisco turnover effects on plant productivity may relate to energy cost of Rubisco synthesis rather than photosynthetic capacity. QTL detection by a multiple QTL model identified seven significant QTL for Rubisco turnover and nine QTL for DM and morphological traits. An indication of the genetic interdependence of DM and the measures of Rubisco turnover was the support interval overlap involving QTL for D and QTL for TN on linkage group 5 in a cluster involving QTL for DM and PI. In this region, alleles associated with increased TN, DM, and PI were associated with decreased D, indicating that this region may regulate Rubisco concentration and plant productivity via increased tillering. A second cluster involving QTL for LL, TN, PI and DM was found on linkage group 2. The two clusters represent marker-trait associations that might be useful for marker-assisted plant breeding applications. In silico comparative analysis indicated conservation of the genetic loci controlling Rubisco concentration in perennial ryegrass and rice.

Seasonal Variation in Soil and Herbage CO2 Efflux for a Sheep-Grazed Alpine Meadow on the North-East Qinghai-Tibetan Plateau and Estimated Net Annual CO2 Exchange.

The Qinghai-Tibetan Plateau is a vast geographic area currently subject to climate warming. Improved knowledge of the CO2 respiration dynamics of the Plateau alpine meadows and of the impact of grazing on CO2 fluxes is highly desirable. Such information will assist land use planning. We measured soil and vegetation CO2 efflux of alpine meadows using a closed chamber technique over diurnal cycles in winter, spring and summer. The annual, combined soil and plant respiration on ungrazed plots was 28.0 t CO2 ha-1 a-1, of which 3.7 t ha-1 a-1occurred in winter, when plant respiration was undetectable. This suggests winter respiration was driven mainly by microbial oxidation of soil organic matter. The winter respiration observed in this study was sufficient to offset the growing season CO2 sink reported for similar alpine meadows in other studies. Grazing increased herbage respiration in summer, presumably through stimulation of gross photosynthesis. From limited herbage production data, we estimate the sustainable yield of these meadows for grazing purposes to be about 500 kg herbage dry matter ha-1 a-1. Addition of photosynthesis data and understanding of factors affecting soil carbon sequestration to more precisely determine the CO2 balance of these grasslands is recommended.

Epichloë seed transmission efficiency is influenced by plant defense response mechanisms.

Asexual Epichloë are endophytic fungi that form mutualistic symbioses with cool-season grasses, conferring to their hosts protection against biotic and abiotic stresses. Symbioses are maintained between grass generations as hyphae are vertically transmitted from parent to progeny plants through seed. However, endophyte transmission to the seed is an imperfect process where not all seeds become infected. The mechanisms underpinning the varying efficiencies of seed transmission are poorly understood. Host gene expression in response to Epichloë sp. LpTG-3 strain AR37 was examined within inflorescence primordia and ovaries of high and low endophyte transmission genotypes within a single population of perennial ryegrass. A genome-wide association study was conducted to identify population-level single nucleotide polymorphisms (SNPs) and associated genes correlated with vertical transmission efficiency. For low transmitters of AR37, upregulation of perennial ryegrass receptor-like kinases and resistance genes, typically associated with phytopathogen detection, comprised the largest group of differentially expressed genes (DEGs) in both inflorescence primordia and ovaries. DEGs involved in signaling and plant defense responses, such as cell wall modification, secondary metabolism, and reactive oxygen activities were also abundant. Transmission-associated SNPs were associated with genes for which gene ontology analysis identified "response to fungus" as the most significantly enriched term. Moreover, endophyte biomass as measured by quantitative PCR of Epichloë non-ribosomal peptide synthetase genes, was significantly lower in reproductive tissues of low-transmission hosts compared to high-transmission hosts. Endophyte seed-transmission efficiency appears to be influenced primarily by plant defense responses which reduce endophyte colonization of host reproductive tissues.

Time Course of Root Axis Elongation and Lateral Root Formation in Perennial Ryegrass (Lolium perenne L.).

Grasses have a segmental morphology. Compared to leaf development, data on root development at the phytomer level are scarce. Leaf appearance interval was recorded over time to allow inference about the age of segmental sites that later form roots. Hydroponically grown Lolium perenne cv. Aberdart tillers were studied in both spring and autumn in increasing and decreasing day length conditions, respectively, and dissected to define the development status of roots of known age on successive phytomers basipetally on the tiller axis. Over a 90-day observation period spring and autumn tillers produced 10.4 and 18.1 root bearing phytomers (Pr), respectively. Four stages of root development were identified: (0) main axis elongation (~0-10 days), (1) primary branching (~10-18 days), (2) secondary branching (~18-25 days), and (3) tertiary and quaternary branching without further increase in root dry weight. The individual spring roots achieved significantly greater dry weight (35%) than autumn roots, and a mechanism for seasonal shift in substrate supply to roots is proposed. Our data define a root turnover pattern likely also occurring in field swards and provide insight for modelling the turnover of grass root systems for developing nutrient efficient or stress tolerant ryegrass swards.

The effects of salinity and osmotic stress on barley germination rate: sodium as an osmotic regulator.

BACKGROUND AND AIMS: Seed germination is negatively affected by salinity, which is thought to be due to both osmotic and ion-toxicity effects. We hypothesize that salt is absorbed by seeds, allowing them to generate additional osmotic potential, and to germinate in conditions under which they would otherwise not be able to germinate. METHODS: Seeds of barley, Hordeum vulgare, were germinated in the presence of either pure water or one of five iso-osmotic solutions of polyethylene-glycol (PEG) or NaCl at 5, 12, 20 or 27 °C. Germination time courses were recorded and germination indices were calculated. Dry mass, water content and sodium concentration of germinating and non-germinating seeds in the NaCl treatments at 12 °C were measured. Fifty supplemental seeds were used to evaluate the changes in seed properties with time. KEY RESULTS: Seeds incubated in saline conditions were able to germinate at lower osmotic potentials than those incubated in iso-osmotic PEG solutions and generally germinated faster. A positive correlation existed between external salinity and seed salt content in the saline-incubated seeds. Water content and sodium concentration increased with time for seeds incubated in NaCl. At higher temperatures, germination percentage and dry mass decreased whereas germination index and sodium concentration increased. CONCLUSIONS: The results suggest that barley seeds can take up sodium, allowing them to generate additional osmotic potential, absorb more water and germinate more rapidly in environments of lower water potential. This may have ecological implications, allowing halophytic species and varieties to out-compete glycophytes in saline soils.

Comparative Effects of Stovers of Four Varieties of Common Vetch on Growth Performance, Ruminal Fermentation, and Nutrient Digestibility of Growing Lambs.

This study evaluated common vetch stover as a feed in mixed rations for growing lambs. Four common vetch varieties were compared with alfalfa (control) for their effects on growth performance, ruminal fermentation, nutrient digestibility, and nitrogen retention. Male Hu lambs (n = 50) aged 3 months, with a mean body weight of 17.5 ± 0.34 kg were allocated randomly to one of the five dietary treatments, making 10 lambs per treatment. The experiment lasted 67 days with a 10-day adaptation period and a 50-day fattening period, and with the final 7 days used for a nutrient digestibility and nitrogen balance trial. All diets contained 30.0% maize straw and 50.0% concentrate, with different forage sources (on a fed basis): 20.0% alfalfa hay (control), 20.0% local common vetch variety 333A (C333A) stover, or 20.0% stover of one of three improved common vetch varieties: Lanjian No. 1 (CLJ1), Lanjian No. 2 (CLJ2), or Lanjian No. 3 (CLJ3). For stover quality, CLJ1 stover had the greatest crude protein (CP), in vitro organic matter digestibility (IVOMD), and metabolizable energy (ME) content and the least cell wall contents, while C333A stover had the least CP, IVOMD, and ME contents and the greatest cell wall contents. Sheep fed the control diet had a greater average daily gain (ADG), apparent digestibility of organic matter (DOM), neutral detergent fiber, acid detergent fiber, and nitrogen retention, and greater ruminal total volatile fatty acids concentration than lambs fed the C333A or CLJ3 diet, but similar performance to lambs fed the CLJ1 and CLJ2 diets. The feed conversion ratio and predicted CH4 emission per unit of DOM intake and ADG of the control, CLJ1, and CLJ2 diets was significantly lower (p < 0.05) than for the other diets. Based on these results, stovers of varieties CLJ1 and CLJ2 can be recommended as an alternative to alfalfa hay and for use in a legume crop rotation with cereals on the Tibetan plateau.

Rebuilding soil organic C stocks in degraded grassland by grazing exclusion: a linked decline in soil inorganic C.

BACKGROUND: Our study evaluated how soil organic carbon (SOC) and soil inorganic carbon (SIC) recovered over time in deep loessial soil as overgrazed grassland was fenced and restored. METHODS: The study was conducted in the Yunwu Mountain Nature Reserve in the Ningxia Autonomous Region of China. In it we compared soil data from three grazed grassland (G) sites, three sites that were fenced for 15 years (F15), and three sites that were fenced for 30 years (F30) as a so-called 'space for time series'. RESULTS AND DISCUSSION: We compared SOC accumulation in soil up to 200 cm below the surface in G, F15, and F30 plots. An increase in SOC correlated with a decrease in soil pH, and decreased soil bulk density. However, SOC sequestration in fenced plots was largely offset by a decrease in SIC, which was closely correlated (r = 0.713, p = 0.001) with SOC-driven soil pH decline. We observed no significant increase in soil total carbon in the F15 or F30 sites after comparing them to G. CONCLUSIONS: Our data indicate that fencing causes the slow diffusion processes to intensify the soil property changes from increased litter return, and this slow diffusion process is still active 30 years after fencing at 100-200 cm soil depths in the studied deep loessial soil. These findings are likely applicable to similar sites.

The Impact of Endophyte Infection, Seed Aging, and Imbibition on Selected Sugar Metabolite Concentrations in Seed.

This study investigated effects of seed aging and imbibition on sugar metabolite concentrations in Epichloë endophyte-infected and endophyte-free seed of tall fescue ( Festuca arundinacea Schreb.). Two treatments, namely, accelerated aging and imbibition, were applied to the seeds, with embryo and endosperm tissues analyzed separately. Gas chromatography with flame ionization detection was employed for analysis of sugar metabolites within the seed tissues. Mannitol, ribitol, and trehalose were more abundant in embryo than endosperm tissues and were identified at consistently higher concentrations within endophyte-infected compared to endophyte-free seeds. The ratio of raffinose to sucrose significantly increased with seed aging in both endophyte-free and endophyte-infected embryo tissues, while significantly lower concentrations of trehalose were detected in tissues dissected from aged-seed regardless of endophyte status. This research provides fundamental insight into the metabolic details of endophyte survival in seed and provides a first evaluation of key carbohydrates present in the fungal-plant symbiosis.

Effect of enrofloxacin on the proteome of earthworms.

The environmental and human health risks of veterinary drugs are becoming public health issues. Enrofloxacin (EF) is an extensively used animal-specific antibacterial agent that leaves drug residues in the environment. This study investigated the proteomic response of the earthworm Eisenia fetida to EF exposure. Earthworms were exposed to EF in soil at 1-500mg·kg-1, and samples were collected at intervals during a 28 day period. The extracted proteins were separated by two dimensional electrophoresis to detect differentially expressed proteins (DEPs) in EF-exposed earthworms. In total, 35 unique DEPs were found. These proteins were subjected to MALDI-TOF/TOF-MS analysis and identified through comparison of their mass spectra with those in protein databases. The DEPs were grouped on the basis of their function, into metabolism, stress-related, transport, transcription, and predicted/hypothetical protein categories. Knowledge of proteins that are induced or repressed by EF in earthworms could provide insight into mechanisms of sub-clinical physiological effects of xenobiotic residues in the environment, and may also help understand synergy between pollutants. As several DEPs in E. fetida showed similarity to human protein sequences, E. fetida has potential as an indicator species to assess the environmental and biological risks of drug residues.

Key sources and seasonal dynamics of greenhouse gas fluxes from yak grazing systems on the Qinghai-Tibetan Plateau.

Greenhouse gas (GHG) emissions from livestock grazing systems are contributing to global warming. To examine the influence of yak grazing systems on GHG fluxes and relationships between GHG fluxes and environmental factors, we measured carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O) fluxes over three key seasons in 2012 and 2013 from a range of potential sources, including: alpine meadows, dung patches, manure heaps and yak night pens, on the Qinghai-Tibetan Plateau. We also estimated the total annual global warming potential (GWP, CO2-equivalents) from family farm grazing yaks using our measured results and other published data. In this study, GHG fluxes per unit area from night pens and composting manure heaps were higher than from dung patches and alpine meadows. Increased moisture content and surface temperature of soil and manure were major factors increasing CO2 and CH4 fluxes. High contributions of CH4 and N2O (21.1% and 44.8%, respectively) to the annual total GWP budget (334.2 tonnes) strongly suggest these GHG other than CO2 should not be ignored when estimating GWP from the family farm grazing yaks on the Qinghai-Tibetan Plateau for the purposes of determining national and regional land use policies or compiling global GHG inventories.