High-affinity iron uptake is required for optimal Epichloë festucae colonization of Lolium perenne and seed transmission.

Epichloë festucae uses a siderophore-mediated system to acquire iron, which is important to maintain endophyte-grass symbioses. Here we investigate the roles of the alternative iron acquisition system, reductive iron assimilation (RIA), via disruption of the fetC gene, which encodes a multicopper ferroxidase, either alone (i.e., ΔfetC) or in combination with disruption of the gene sidA, which encodes a siderophore biosynthesis enzyme (i.e., ΔfetC/ΔsidA). The phenotypic characteristics of these mutants were compared to ΔsidA and wild-type (WT) strains during growth under axenic culture conditions (in culture) and in symbiosis with the host grass, perennial ryegrass (in planta). Under iron deficiency, the colony growth rate of ΔfetC was slightly slower than that of WT, while the growth of ΔsidA and ΔfetC/ΔsidA mutants was severely suppressed. Siderophore analyses indicated that ΔfetC mutants hyperaccumulate ferriepichloënin A (FEA) at low iron concentrations and ferricrocin and FEA at higher iron concentrations. When compared to WT, all mutant strains displayed hyperbranching hyphal structures and a reduced ratio of Epichloë DNA to total DNA in planta. Furthermore, host colonization and vertical transmission through infection of the host seed were significantly reduced in the ΔfetC/ΔsidA mutants, confirming that high-affinity iron uptake is a critical process for Epichloë transmission. Thus, RIA and siderophore iron uptake are complementary systems required for the maintenance of iron metabolism, fungal growth, and symbiosis between E. festucae and perennial ryegrass.

Effects of endophyte-infected tall fescue seed and protein supplementation on stocker steers: I. Growth performance and hemodynamic responses.

Fescue toxicosis is a multifaceted syndrome common in cattle grazing endophyte-infected tall fescue and is detrimental to growth and performance. Recent research has shown that supplementing protein has the potential to enhance growth performance in weaned steers. Therefore, the objective of this study was to evaluate the effect of supplemental CP on physiological parameters in stocker steers experiencing fescue toxicosis. Thirty-six weaned Angus steers (6 mo of age) stratified by weight (196.1 ± 3.6 kg) were assigned to a 2 × 2 factorial arrangement for 56 d: endophyte-free (EF) seed and 14% CP (EF-14; n = 9), EF seed and 18% CP (EF-18; n = 9), endophyte-infected (EI) seed and 14% CP (EI-14; n = 9), and EI seed and 18% CP (EI-18; n = 9). Steer growth and hemodynamic responses were collected weekly during ergot alkaloid exposure. On day 14 of the trial, iButton temperature data loggers were subcutaneously inserted in the lateral neck region to record hourly body temperature for 42 d. Data were analyzed using PROC MIXED of SAS with repeated measures. No differences were observed in DMI, BW, ADG, F:G, or BCS during the treatment period (P > 0.05). Hair shedding scores, rectal temperatures, surface temperatures, and respiration rates were greater in EI steers compared to EF steers regardless of supplemental CP (P < 0.05). However, subcutaneous body temperature was greater in EI-14 steers (37.94 °C) compared to other steer groups (37.60, 37.68, 37.72 ± 0.04 °C for EF-14, EF-18, and EI-18, respectively; P < 0.05). Prolactin concentrations tended to be greater in EF steers when compared to EI steers (P = 0.07). Heart rate and hematocrit were reduced for EI-18 steers compared to other steer groups (P < 0.05). Caudal artery diameter was reduced in EI-18 steers compared to EI-14 steers (2.60 vs. 2.75 ± 0.05 mm, respectively; P < 0.05) and caudal vein diameter was reduced in EI-18 steers (3.20 mm) compared to all other steer groups (3.36, 3.39, 3.50 mm for EF-14, EF-18, and EI-14, respectively; P < 0.05). However, there was no difference observed in systolic or diastolic blood pressure during the treatment period (P > 0.05). Based on the data, exposure to low to moderate levels of ergot alkaloids during the stocker phase had a negative impact on hemodynamic responses and supplemental CP had minimal impact to alleviate symptoms. Therefore, feeding additional protein above established requirements is not expected to help alleviate fescue toxicosis.

Interactions between electrokinetics and rhizoremediation on the remediation of crude oil-contaminated soil.

An electrokinetics (EK)-enhanced phytoremediation system with ryegrass was constructed to remediate crude oil-polluted soil. The four treatments employed in this study included (1) without EK or ryegrass (CK-NR), (2) EK only (EK-NR), (3) ryegrass only (CK-R), and (4) EK and ryegrass (EK-R). After 30d of ryegrass growth, EK at 1.0 V·cm-1 with polarity reversal (PR-EK) was supplied for another 30 d. The electric current was recorded during remediation. The pH, electrical conductivity, total petroleum hydrocarbon content (TPH), 16S rDNA, functional genes of AlkB, Nah, and Phe, DGGE, and dehydrogenase activity in soil were measured. The physical-chemical indexes of the plant included the length, dry mass, and chlorophyll contents of the ryegrass. Results showed that EK-R removed 18.53 ±â€¯0.53% of TPH, which was higher than that of other treatments (13.34-14.31%). Meanwhile, the values of 16S rDNA, AlkB, Nah, Phe, and dehydrogenase activity in the bulk soil of EK-R all increased. Further clustering analysis with numbers of genes and DGGE demonstrated that EK-R was similar to the ryegrass rhizosphere soils in both EK-R and CK-R, while the EK treatment of EK-NR was similar to that of CK-NR without EK and ryegrass. These results indicate that the PR-EK treatment used in this experiment successfully enlarged the existing scale of the rhizosphere microorganisms, improved microbial activity and enhanced degradation of TPH.

Development of plant-microbe phytoremediation system for petroleum hydrocarbon degradation: An insight from alkb gene expression and phytotoxicity analysis.

Aim of present work was to assess in-planta association potential of isolated endophytic bacterial strain Pseudomonas sp. (J10) (KY608252) with two cultivars of Lolium perenne L. (small & jumbo) and Arabidopsis thaliana L. for total petroleum hydrocarbon (TPH) degradation, alkane monooxygenase (alkb) gene expression and phytotoxicity analysis. A plant-microbe phytoremediation system was established to investigate the bacteria's ability to colonize the plant body and quantification of alkb gene to help withstand TPH stress in soil as well as in hydroponics. A real-time PCR method was developed to analyze bacterial colonization and survival within the plant body. Analysis revealed that J10 efficiently colonized all the tested plant species and expressed alkb gene under hydrocarbon stress ranging between 3.7 × 102-3.9 × 106 in A. thaliana and L. perenne (small), respectively. The colonization was more pronounced in soil as compared to hydroponic system. J10 inoculation reduced phytotoxicity and suggested that inoculation had a positive effect on plant growth under stress conditions as compared to control. L. perenne (small) showed significant TPH removal efficiency (45.6%) followed by L. perenne jumbo (24.5%) and A. thaliana (6.2%). In hydroponics, L. perenne (small) degraded about 28.2% TPH followed by L. perenne (jumbo) as 24.4%. Potential of the indigenously isolated plant endophytes may be exploited further for phytoremediation efficiency and industrial applications.

Increasing the Size of the Microbial Biomass Altered Bacterial Community Structure which Enhances Plant Phosphorus Uptake.

Agricultural production can be limited by low phosphorus (P) availability, with soil P being constrained by sorption and precipitation reactions making it less available for plant uptake. There are strong links between carbon (C) and nitrogen (N) availability and P cycling within soil P pools, with microorganisms being an integral component of soil P cycling mediating the availability of P to plants. Here we tested a conceptual model that proposes (i) the addition of readily-available organic substrates would increase the size of the microbial biomass thus exhausting the pool of easily-available P and (ii) this would cause the microbial biomass to access P from more recalcitrant pools. In this model it is hypothesised that the size of the microbial population is regulating access to less available P rather than the diversity of organisms contained within this biomass. To test this hypothesis we added mixtures of simple organic compounds that reflect typical root exudates at different C:N ratios to a soil microcosm experiment and assessed changes in soil P pools, microbial biomass and bacterial diversity measures. We report that low C:N ratio (C:N = 12.5:1) artificial root exudates increased the size of the microbial biomass while high C:N ratio (C:N = 50:1) artificial root exudates did not result in a similar increase in microbial biomass. Interestingly, addition of the root exudates did not alter bacterial diversity (measured via univariate diversity indices) but did alter bacterial community structure. Where C, N and P supply was sufficient to support plant growth the increase observed in microbial biomass occurred with a concurrent increase in plant yield.

Variation in Alkaloid Production from Genetically Diverse Lolium Accessions Infected with Epichloë Species.

Widespread infection of Epichloë occultans in annual ryegrass in Australia suggests that infection provides its weedy host, Lolium rigidum, some ecological advantage. Initial studies determined the distribution and profiles of known Epichloë alkaloids (epoxy-janthitrems, ergovaline, lolines, lolitrem B, and peramine) in plant extracts using a combination of GC-FID and HPLC techniques utilizing a single accession of Australian L. rigidum. However, the lolines N-acetylnorloline (NANL) and N-formylloline (NFL) were the only alkaloids detected and were highly concentrated in the immature inflorescences of mature plants. Additional glasshouse studies subjected a wide range of Australian L. rigidum haplotypes and international annual Lolium accessions to a suite of analyses to determine alkaloid levels and profiles. Again, NFL and NANL were the key lolines produced, with NFL consistently predominating. Considerable variation in alkaloid production was found both within and between biotypes and accessions evaluated under identical conditions, at the same maturation stage and on the same tissue type. The pyrrolopyrazine alkaloid peramine was also present in 8 out of 17 Australian biotypes of L. rigidum and 7 out of 33 international accessions infected with Epichloë spp.; the highest peramine concentrations were observed in seed extracts from L. rigidum collected from Australia. This study represents the first report of alkaloids from a geographically diverse collection of annual ryegrass germplasm infected with Epichloë spp. when grown under identical controlled conditions.

[Physical and chemical methods for eliminating propagules of indigenous mycorrhizal fungi from soil samples]. / Métodos físicos y químicos para la eliminación de propágulos de hongos formadores de micorrizas nativos de muestras de suelo.

The objective of this work was to evaluate methods to eliminate or reduce the number of indigenous arbuscular mycorrhizal fungi (AMF) from soil samples without affecting their edaphic or microbiological properties. At an early trial we evaluated moist heat (autoclaving), dry heat (oven), sodium hypochlorite (NaClO) and formaldehyde at a range of 100.0-3.3µl/g and 16.7-3.3µl/g respectively. There was no germination in plants of ryegrass (Lolium multiflorum Lam.) sown on substrates receiving NaClO (100.0-33.3µl/g), whereas autoclaving significantly increased the available soil phosphorous content. Both treatments failed to eradicate AMF colonization at 9 weeks; therefore, they were discarded. In a second trial, oven and formaldehyde (10.0µl/g) treatments were analyzed to assess the effects of seed decontamination and AMF reinoculation. Both procedures were effective in reducing or eliminating indigenous AMF at a range of soil P availability of 12-29mg/kg. However, the time between soil treatment and AMF multiplication and safety requirements were greater in the case of formaldehyde application.

Controlled release fertilizer increased phytoremediation of petroleum-contaminated sandy soil.

A greenhouse experiment was conducted to determine the effect of the application of controlled release fertilizer [(CRF) 0, 4,6, or 8 kg m(-3)] on Lolium multiflorum Lam. survival and potential biodegradation of petroleum hydrocarbons (0, 3000, 6000, or 15000 mg kg(-1)) in sandy soil. Plant adaptation, growth, photosynthesis, total chlorophyll, and proline content as well as rhizosphere microbial population (culturable heterotrophic fungal and bacterial populations) and total petroleum hydrocarbon (TPH)-degradation were determined. Petroleum induced-toxicity resulted in reduced plant growth, photosynthesis, and nutrient status. Plant adaptation, growth, photosynthesis, and chlorophyll content were enhanced by the application of CRF in contaminated soil. Proline content showed limited use as a physiological indicator of petroleum induced-stress in plants. Bacterial and filamentous fungi populations were stimulated by the petroleum concentrations. Bacterial populations were stimulated by CRF application. At low petroleum contamination, CRF did not enhance TPH-degradation. However, petroleum degradation in the rhizosphere was enhanced by the application of medium rates of CRF, especially when plants were exposed to intermediate and high petroleum contamination. Application of CRF allowed plants to overcome the growth impairment induced by the presence of petroleum hydrocarbons in soils.

The role of sulfur- and phosphorus-mobilizing bacteria in biochar-induced growth promotion of Lolium perenne.

Plants rely on microorganisms to mobilize organically and inorganically bound sulfur (S) and phosphorus (P) in which the plant can then readily utilize. The aim of this study was to investigate the role of S- and P-mobilizing bacteria in plant growth promotion in biochar-amended soil, which has been rarely investigated so far. Pot experiments of Lolium perenne were established on S and P limited soil with 1% or 2% biochar (Miscanthus × giganteus) or without biochar (control) for a period of 126 days. Both biochar amendments resulted in significant plant growth promotion. Rhizobacteria capable of growing with (1) S from aromatic sulfonates, (2) P from phosphate esters, (3) P from phosphonates, and (4) P from tri-calcium phosphates as sole source of S or P, respectively, were significantly more abundant in the biochar treatments. 16S rRNA gene-based rhizobacteria community analysis revealed a significant biochar treatment effect. Abundance of nematodes feeding on bacteria was also significantly increased in the biochar treatments. Diversity analysis of rhizospheric asfA and phnJ genes revealed broad sequence diversities in bacterial sulfonate and phosphonate-mineralizing capabilities. These findings suggest that biochar amendment enhances microbially mediated nutrient mobilization of S and P resulting in improved plant growth.

Diversity of endophytic bacteria in Lolium perenne and their potential to degrade petroleum hydrocarbons and promote plant growth.

The aim of this study was to assess the ability of twenty-nine endophytic bacteria isolated from the tissues of ryegrass (Lolium perenne L.) to promote plant growth and the degradation of hydrocarbon. Most of the isolates belonged to the genus Pseudomonas and showed multiple plant growth-promoting abilities. All of the bacteria that were tested exhibited the ability to produce indole-3-acetic acid and were sensitive to streptomycin. These strains were capable of phosphate solubilization (62%), cellulolytic enzyme production (62%), a capacity for motility (55%) as well as for the production of siderophore (45%), ammonium (41%) and hydrogen cyanide (38%). Only five endophytes had the emulsification ability that results from the production of biosurfactants. The 1-aminocyclopropane-1-carboxylate deaminase (ACCD) gene (acdS) was found in ten strains. These bacteria exhibited ACCD activities in the range from 1.8 to 56.6 µmol of α-ketobutyrate mg(-1)h(-1), which suggests that these strains may be able to modulate ethylene levels and enhance plant growth. The potential for hydrocarbon degradation was assessed by PCR amplification on the following genes: alkH, alkB, C23O, P450 and pah. The thirteen strains that were tested had the P450 gene but the alkH and pah genes were found only in the Rhodococcus fascians strain (L11). Four endophytic bacteria belonging to Microbacterium sp. and Rhodococcus sp. (L7, S12, S23, S25) showed positive results for the alkB gene.

Annual ryegrass-associated bacteria with potential for plant growth promotion.

Annual ryegrass is a fast-growing cool-season grass broadly present in the Portuguese "montado", a typically Mediterranean agro-forestry-pastoral ecosystem. A culture-dependent approach was used to investigate natural associations of this crop with potentially beneficial bacteria, aiming to identify strains suitable for biofertilization purposes. Annual ryegrass seedlings were used to trap bacteria from three different soils in laboratory conditions. Using a nitrogen-free microaerophilic medium, 147 isolates were recovered from the rhizosphere, rhizoplane, and surface-sterilized plant tissues, which were assigned to 12 genera in classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli and Actinobacteria. All isolates were able to grow in the absence of nitrogen and several of them were able to perform in vitro activities related to plant growth promotion. Isolates of the genera Sphingomonas and Achromobacter were found to be the most effective stimulators of annual ryegrass growth under nitrogen limitation (47-92% biomass increases). Major enhancements were obtained with isolates G3Dc4 (Achromobacter sp.) and G2Ac10 (Sphingomonas sp.). The latest isolate was also able to increment plant growth in nitrogen-supplemented medium, as well as the phosphate solubilizer and siderophore producer, G1Dc10 (Pseudomonas sp.), and the cellulose/pectin hydrolyser, G3Ac9 (Paenibacillus sp.). This study represents the first survey of annual ryegrass-associated bacteria in the "montado" ecosystem and unveiled a set of strains with potential for use as inoculants.

Métodos físicos y químicos para la eliminación de propágulos de hongos formadores de micorrizas nativos de muestras de suelo. / [Physical and chemical methods for eliminating propagules of indigenous mycorrhizal fungi from soil samples].

The objective of this work was to evaluate methods to eliminate or reduce the number of indigenous arbuscular mycorrhizal fungi (AMF) from soil samples without affecting their edaphic or microbiological properties. At an early trial we evaluated moist heat (autoclaving), dry heat (oven), sodium hypochlorite (NaClO) and formaldehyde at a range of 100.0-3.3Al/g and 16.7-3.3Al/g respectively. There was no germination in plants of ryegrass (Lolium multiflorum Lam.) sown on substrates receiving NaClO (100.0-33.3Al/g), whereas autoclaving significantly increased the available soil phosphorous content. Both treatments failed to eradicate AMF colonization at 9 weeks; therefore, they were discarded. In a second trial, oven and formaldehyde (10.0Al/g) treatments were analyzed to assess the effects of seed decontamination and AMF reinoculation. Both procedures were effective in reducing or eliminating indigenous AMF at a range of soil P availability of 12-29mg/kg. However, the time between soil treatment and AMF multiplication and safety requirements were greater in the case of formaldehyde application.

Mycorrhizal colonisation and P-supplement effects on N uptake and N assimilation in perennial ryegrass under well-watered and drought-stressed conditions.

To compare the effect of arbuscular mycorrhiza (AM) and P-supplement on N uptake and N assimilation under well-watered or drought-stressed conditions, Glomus intraradices-colonised, P-supplemented non-mycorrhizal (P) and non-mycorrhizal (control) plants of Lolium perenne were exposed to 12 days of water treatment. Leaf water potential (Ψ (w)), photosynthetic ability, and N and P nutritional status were measured at the beginning (day 0) and end (day 12) of water treatment. N absorption, amino acid and protein synthesis were quantified using the isotopic tracer (15)N at day 12. Under well-watered conditions, growth response and physiological parameters were similar in AM and P plants, as compared to controls. Drought (10% water) significantly decreased these parameters in all three treatments. As compared to control plants, the negative impact of water deficit on the Ψ (w), photosynthesis, biomass, and N and P content was highly alleviated in AM plants, while only slightly improved or remained the same level in P plants. The effect of AM symbiosis on N absorption and N assimilation was greater than that of the P supplement under well-watered and drought-stressed conditions, and this effect was highly enhanced under drought-stressed conditions. At terminal drought stress on day 12, the effect of AM colonisation on de novo synthesis of amino acids and proteins was 4.4- and 4.8-fold higher than that of the P supplement. These results indicate that the AM symbiosis plays an integrative role in N nutrition by alleviating the negative impacts of drought on N or P uptake and N assimilation, whereas the efficiency of a direct P supplement is very limited under drought-stressed conditions.

Constriction of bovine vasculature caused by endophyte-infected tall fescue seed extract is similar to pure ergovaline.

Ergovaline has been extensively used to study vasoactive effects of endophyte- (Neotyphodium coenophialum) infected tall fescue (Lolium arundinaceum). However, initial results indicated that an extract of toxic tall fescue seed (E+EXT) is more potent than ergovaline alone in a right ruminal artery and vein bioassay. The E+EXT induced a greater contractile response than an equal concentration of ergovaline alone in the ruminal artery of heifers (P = 0.018). This led to a hypothesis that other compounds in the seed extract contribute to vasoconstriction. Thus, experiments were conducted to determine if vasoactivity of an E+EXT is different from a mixture of ergot alkaloids (ALK; ergovaline, ergotamine, ergocristine, ergocryptine, ergocornine, ergonovine, and lysergic acid) of similar concentrations and to determine if the vasoactivity of an E+EXT differs from an endophyte-free tall fescue seed extract (E-EXT). Segments of lateral saphenous vein and right ruminal artery and vein were collected from Holstein steers (n = 6) shortly after slaughter. Vessels were cleaned of excess connective tissue and fat and sliced into segments that were suspended in a multimyograph chamber with 5 mL of continually oxygenated Krebs-Henseleit buffer, equilibrated for 90 min, and exposed to a reference compound (120 mM KCl for ruminal vessels and 0.1 mM norepinephrine for saphenous vein). Increasing concentrations of each treatment (E+EXT, E-EXT, ALK, and ergovaline) were added to the respective chamber every 15 min after buffer replacement. Data were normalized as a percentage of maximal contractile response of the reference compound and fit to a sigmoidal concentration response curve. Ergovaline, ALK, and E+EXT induced similar responses in the saphenous vein, ruminal artery, and ruminal vein. The E+EXT displayed a smaller EC(50) (half maximal effective concentration) than ergovaline or ALK in the saphenous vein and ruminal vein (P < 0.008), but not the ruminal artery (P = 0.31). Extrapolated maximum response was greatest in the saphenous vein for ergovaline, least for E+EXT, and intermediate for ALK (P < 0.0001). The E-EXT did not induce a contractile response in any vessel tested (P > 0.1). Data from this study indicate that ergovaline is largely responsible for the locally induced vasoconstriction of bovine vasculature observed with endophyte-infected tall fescue.

Phylogenetic and functional diversity of alkane degrading bacteria associated with Italian ryegrass (Lolium multiflorum) and Birdsfoot trefoil (Lotus corniculatus) in a petroleum oil-contaminated environment.

Twenty-six different plant species were analyzed regarding their performance in soil contaminated with petroleum oil. Two well-performing species, Italian ryegrass (Lolium multiflorum var. Taurus), Birdsfoot trefoil (Lotus corniculatus var. Leo) and the combination of these two plants were selected to study the ecology of plant-associated, culturable alkane-degrading bacteria. Hydrocarbon degrading bacteria were isolated from the rhizosphere, root interior and shoot interior and subjected to the analysis of 16S rRNA gene, the 16S and 23S rRNA intergenic spacer region and alkane hydroxylase genes. Furthermore, we investigated whether alkane hydroxylase genes are plasmid located. Higher numbers of culturable, alkane-degrading bacteria were associated with Italian ryegrass, which were also characterized by a higher diversity, particularly in the plant interior. Only half of the isolated bacteria hosted known alkane hydroxylase genes (alkB and cytochrome P153-like). Degradation genes were found both on plasmids as well as in the chromosome. In regard to application of plants for rhizodegradation, where support of numerous degrading bacteria is essential for efficient break-down of pollutants, Italian ryegrass seems to be more appropriate than Birdsfoot trefoil.

Movement of newly assimilated 13C carbon in the grass Lolium perenne and its incorporation into rhizosphere microbial DNA.

One of the key processes that drives rhizosphere microbial activity is the exudation of soluble organic carbon (C) by plant roots. We describe an experiment designed to determine the impact of defoliation on the partitioning and movement of C in grass (Lolium perenne L.), soil and grass-sterile sand microcosms, using a (13)CO(2) pulse-labelling method. The pulse-derived (13)C in the shoots declined over time, but that of the roots remained stable throughout the experiment. There were peaks in the atom% (13)C of rhizosphere CO(2) in the first few hours after labelling probably due to root respiration, and again at around 100 h. The second peak was only seen in the soil microcosms and not in those with sterilised sand as the growth medium, indicating possible microbial activity. Incorporation of the (13)C label into the microbial biomass increased at 100 h when incorporation into replicating cells, as indicated by the amounts of the label in the microbial DNA, started to increase. These results indicate that the rhizosphere environment is conducive to bacterial growth and replication. The results also show that defoliation had no impact on the pattern of movement of (13)C from plant roots into the microbial population in the rhizosphere.

Arbuscular mycorrhizas contribute to phytostabilization of uranium in uranium mining tailings.

Uranium (U) tailings pose environmental risks and call for proper remediation. In this paper medic and ryegrass plants were used as host plants to examine whether inoculation with an AM fungus, Glomus intraradices, would help phytostabilization of U tailings. The need of amending with uncontaminated soil for supporting plant survival was also examined by mixing soil with U tailing at different mixing ratios. Soil amendment increased plant growth and P uptake. Ryegrass produced a more extensive root system and a greater biomass than medic plants at all mixing ratios. Medic roots were extensively colonized by G. intraradices whereas ryegrass were more sparsely colonized. Plant growth was not improved by mycorrhizas, which, however, improved P nutrition of medic plants. Medic plants contained higher U concentrations and showed higher specific U uptake efficiency compared to ryegrass. In the presence of U tailing, most U had been retained in plant roots, and this distribution pattern was further enhanced by mycorrhizal colonization. The results suggest a role for AM fungi in phytostabilization of U tailings.

Arbuscular mycorrhiza and petroleum-degrading microorganisms enhance phytoremediation of petroleum-contaminated soil.

While plants can phytoremediate soils that are contaminated with petroleum hydrocarbons, adding microbes to remediate contaminated sites with petroleum-degrading microorganisms and arbuscular mycorrhizal fungi (AMF) is not well understood. The phytoremediation of Arabian medium crude oil (ACO) was done with a Lolium multiflorum system inoculated with an AMF (Glomus intraradices) and a mixture of petroleum-degrading microorganisms--the bacterium, Sphingomonas paucimobilis (Sp) and the filamentous fungus, Cunninghamella echinulata (Ce, SpCe)--or with a combination of microorganisms (AMF + SpCe). Based on an earlier study on screening plants for phytoremediation of ACO, L. multiflorum (Italian ryegrass) was selected for its tolerance and rapid growth response (Alarcón, 2006). The plants were exposed to ACO-contaminated soil (6000 mg kg(-1)) for 80 d under greenhouse conditions. A modified Long Ashton Nutrient Solution (LANS) was supplied to all treatments at 30 microg P mL(-1), except for a second, higher P, control treatment at 44 microg P mL(-1). Inoculation with AMF, SpCe, or AMF + SpCe resulted in significantly increased leaf area as well as leaf and pseudostem dry mass as compared to controls at 30 microg P mL(-1). Populations of bacteria grown on a nitrogen-free medium and filamentous fungi increased with AMF + SpCe and SpCe treatments. The average total colonization and arbuscule formation of AMF-inoculated plants in ACO-contaminated soil were 25% and 8%, respectively. No adverse effects were caused by SpCe on AMF colonization. Most importantly, ACO degradation was significantly enhanced by the addition of petroleum-degrading microorganisms and higher fertility controls, as compared to plants at 30 microg P mL(-1). The highest ACO degradation (59%) was observed with AMF + SpCe. The phytoremediation of ACO was also enhanced by single inoculation of AMF or SpCe. The effect of AMF and petroleum-degrading microorganisms on plant growth and ACOdegradation was not attributable to differences in proline, total phenolics, nitrate reductase levels, or variation in plant-gas exchange.

Hydrolysis and microbial community analyses in two-stage anaerobic digestion of energy crops.

AIMS: The roles of the diverse populations of micro-organisms responsible for biodegradation of organic matter to form methane and carbon dioxide are rudimentarily understood. To expand the knowledge on links between microbial communities and the rate limiting, hydrolytic stage of two-stage biogas production from energy crops, this study was performed. METHODS AND RESULTS: The process performance and microbial communities (as determined by fluorescence in situ hybridization) in two separate two-stage batch digestions of sugar beets and grass/clover were studied. The microbial populations developed in the hydrolytic stage of anaerobic digestion of beets and grass/clover showed very few similarities, despite that the hydrolysis dynamics were similar. In both substrates, the solubilization of organic material was rapid for the first 10 days and accompanied by a build-up of volatile fatty acids (VFAs) and lactate. Between days 10 and 15, VFA and lactate concentrations decreased, as did the solubilization rates. For both substrates, Archaea started to appear in the hydrolytic stage between days 10 and 15, and the fraction of Bacteria decreased. The major bacterial group detected in the leachate fraction for beets was Alphaproteobacteria, whereas for grass/clover it was Firmicutes. The number of cells that bound to probes specifically targeting bacteria with cellulolytic activity was higher in the digestion of grass than in the digestion of beet. CONCLUSIONS: This study allowed the identification of the general bacterial groups involved, and the identification of a clear shift in the microbial population when hydrolysis rate became limiting for each of the substrates investigated. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings from this study could be considered as a first step towards the development of strategies to stimulate hydrolysis further and ultimately increasing the methane production rates and yields from reactor-based digestion of these substrates.

High-throughput direct-infusion ion trap mass spectrometry: a new method for metabolomics.

A fast method was developed to directly infuse raw plant extracts into a linear ion trap mass spectrometer, using the ion trap to isolate and fragment as many ions as possible from the extract. The full mass spectra can be analysed by multivariate statistics to determine discriminating ions, and the fragmentation data allows rapid classification or identification of these ions. The methodology was used to screen a wide range of strains of endophytic fungi in perennial ryegrass seeds for differences in metabolic profiles. The results show that this newly developed methodology is able to determine discriminating ions that can be present in very low concentrations. It also yielded sufficient fragmentation data to classify or identify the discriminating ions.