Pete Millard

Assessing shifts in microbial community structure across a range of grasslands of differing management intensity using CLPP, PLFA and community DNA techniques.

This study aimed to characterise soil microbial community structure and function in temperate upland grassland ecosystems. We compared the use of community level physiological profiles (CLPP), phospholipid fatty acid (PLFA) profiles and community DNA (%G+C base distribution) approaches to quantify soil microbial community structure and potential activity across a gradient of three upland grassland types at 10 geographically distinct sites within the UK. Soil microbial biomass (Cmic) was highest in unimproved (U4a) and lowest in improved (MG6) grasslands. In contrast, potential soil microbial activity (carbon utilisation) was greatest in the improved and lowest in the semi-improved (U4b) grasslands. PLFA and culturing revealed that the soil microbial community shifted from one favouring fungi to one favouring bacteria as grassland improvement increased. Canonical variate analysis (CVA) of the CLPP and PLFA data differentiated microbial communities from the grassland types and sites and the separation between grasslands was greater using PLFA than CLPP. Discrimination between grasslands was mainly due to the presence of higher concentrations of fatty acids typical for Gram −ve bacteria in improved grasslands and actinomycete and fungal fatty acids in the semi and unimproved grasslands. CVA of the %G+C data gave less discrimination of the microbial communities than the other two methods. Correlation analysis of the CVA data for each microbial analysis showed a small, but significant, level of matching between the CLPP and PLFA data suggesting these two analyses may be reporting on similar members of the microbial community. Correlation between microbial community structure and soil physio-chemical properties indicated that PLFA were highly correlated with calcium, phosphorus, sodium, nitrogen and organic matter content and pH. CLPP were highly correlated with sodium and organic matter content and pH, while %G+C content correlated with pH. Correlation between microbial community structure and plant community structure indicated that fatty acids typical for Gram −ve bacteria were highly correlated with the presence of Lolium perenne and Trifolium repens and all microbial PLFA with the presence of Vaccinium myrtillus. Correlation of plant species with CLPP indicated that the presence of a number of rushes, shrubs, herbs and grasses influenced the metabolic profiles of the microbial communities from these grasslands. The presence of herbs were found to be highly correlated with certain %G+C classes within the community DNA.

Spatial structure in soil chemical and microbiological properties in an upland grassland

We characterised the spatial structure of soil microbial communities in an unimproved grazed upland grassland in the Scottish Borders. A range of soil chemical parameters, cultivable microbes, protozoa, nematodes, phospholipid fatty acid (PLFA) profiles, community-level physiological profiles (CLPP), intra-radical arbuscular mycorrhizal community structure, and eubacterial, actinomycete, pseudomonad and ammonia-oxidiser 16S rRNA gene profiles, assessed by denaturing gradient gel electrophoresis (DGGE) were quantified. The botanical composition of the vegetation associated with each soil sample was also determined. Geostatistical analysis of the data revealed a gamut of spatial dependency with diverse semivariograms being apparent, ranging from pure nugget, linear and non-linear forms. Spatial autocorrelation generally accounted for 40-60% of the total variance of those properties where such autocorrelation was apparent, but accounted for 97% in the case of nitrate-N. Geostatistical ranges extending from approximately 0.6-6 m were detected, dispersed throughout both chemical and biological properties. CLPP data tended to be associated with ranges greater than 4.5 m. There was no relationship between physical distance in the field and genetic similarity based on DGGE profiles. However, analysis of samples taken as close as 1 cm apart within a subset of cores suggested some spatial dependency in community DNA-DGGE parameters below an 8 cm scale. Spatial correlation between the properties was generally weak, with some exceptions such as between microbial biomass C and total N and C. There was evidence for scale-dependence in the relationships between properties. PLFA and CLPP profiling showed some association with vegetation composition, but DGGE profiling did not. There was considerably stronger association between notional sheep urine patches, denoted by soil nutrient status, and many of the properties. These data demonstrate extreme spatial variation in community-level microbiological properties in upland grasslands, and that despite considerable numeric ranges in the majority of properties, overarching controlling factors were not apparent.

Measuring the 13C content of soil‐respired CO2 using a novel open chamber system

Carbon dioxide respired by soils comes from both autotrophic and heterotrophic respiration. 13C has proved useful in differentiating between these two sources, but requires the collection and analysis of CO2 efflux from the soil. We have developed a novel, open chamber system which allows for the accurate and precise quantification of the delta13C of soil-respired CO2. The chamber was tested using online analyses, by configuring a GasBench II and continuous flow isotope ratio mass spectrometer, to measure the delta13C of the chamber air every 120 s. CO2 of known delta13C value was passed through a column of sand and, using the chamber, the CO2 concentration stabilized rapidly, but 60 min was required before the delta13C value was stable and identical to the cylinder gas (-33.3 per thousand). Changing the chamber CO2 concentration between 200 and 900 micromol.mol(-1) did not affect the measured delta13C of the efflux. Measuring the delta13C of the CO2 efflux from soil cores in the laboratory gave a spread of +/-2 per thousand, attributed to heterogeneity in the soil organic matter and roots. Lateral air movement through dry sand led to a change in the delta13C of the surface efflux of up to 8 per thousand. The chamber was used to measure small transient changes (+/-2 per thousand) in the delta13C of soil-respired CO2 from a peaty podzol after gradual heating from 12 to 35 degrees C over 12 h. Finally, soil-respired CO2 was partitioned in a labelling study and the contribution of autotrophic and heterotrophic respiration to the total efflux determined. Potential applications for the chamber in the study of soil respiration are discussed.

Browsing affects intra-ring carbon allocation in species with contrasting wood anatomy

Current knowledge on tree carbon (C) allocation to wood is particularly scarce in plants subjected to disturbance factors, such as browsing, which affects forest regeneration worldwide and has an impact on the C balance of trees. Furthermore, quantifying the degree to which tree rings are formed from freshly assimilated vs. stored carbohydrates is highly relevant for our understanding of tree C allocation. We used (13)C labelling to quantify seasonal allocation of stored C to wood formation in two species with contrasting wood anatomy: Betula pubescens Ehrh. (diffuse-porous) and Quercus petraea [Matt.] Liebl. (ring-porous). Clipping treatments (66% shoot removal, and unclipped) were applied to analyse the effect of browsing on C allocation into tree rings, plus the effects on tree growth, architecture, ring width and non-structural carbohydrates (NSCs). The relative contribution of stored C to wood formation was greater in the ring-porous (55-70%) than in the diffuse-porous species (35-60%), although each species followed different seasonal trends. Clipping did not cause a significant depletion of C stores in either species. Nonetheless, a significant increase in the proportion of stored C allocated to earlywood growth was observed in clipped birches, and this could be explained through changes in tree architecture after clipping. The size of C pools across tree species seems to be important in determining the variability of seasonal C allocation patterns to wood and their sensibility to disturbances such as browsing. Our results indicate that the observed changes in C allocation to earlywood in birch were not related to variations in the amount or concentration of NSC stores, but to changes in the seasonal availability of recently assimilated C caused by modifications in tree architecture after browsing.

Effect of P supply upon seasonal growth and internal cycling of P in Sitka spruce (Picea sitchensis (Bong.)Carr.) seedlings

The availability of phosphorus in many UK forest soils limits growth of Sitka spruce (Picea sitchensis (Bong.) Carr.). Efficient cycling of P within such systems is therefore necessary for sustained tree growth. Internal cycling of P is an important component of the overall P cycle in forests and the current work aims to quantify the impact of P nutrition on internal cycling and seasonal growth of Sitka spruce.

Detection of Nitrogen Sufficiency in Potato Plants Using Gene Expression Markers

In-season chemical or optical measures of crop N status can be effective tools in optimizing potato fertilizer N management. The feasibility of using a gene expression as an alternative approach for early detection of potato nitrate deficiency was examined using three potato cultivars (Shepody, Russet Norkotah, and Red Pontiac) with abundant (7.5xa0mM NO3), limited (0.75xa0mM NO3) or deficient (0xa0mM NO3) nitrate supply in nutrient culture over a 7xa0d period. RNA was extracted from the last fully expanded leaf and quantified using realtime RT-qPCR. Reduced nitrate supply had no measurable effect on shoot dry weight or leaf chlorophyll concentration, but decreased petiole nitrate concentration. Under deficit nitrate supply, down-regulation of nitrate reductase and nitrite reductase was measured within 3xa0days for all cultivars, and down-regulation of asparagine synthetase was measured in two cultivars. Nitrate supply had no effect on expression of ammonium transporter. In this experimental system, plant gene expression markers detected a reduction of nitrate supply prior to measureable reductions in plant growth or in N status measured using common chemical or optical methods.ResumenLas mediciones químicas y ópticas de temporada del estado del N en el cultivo, pueden ser herramientas efectivas en la optimización del manejo de la fertilización nitrogenada en papa. Se examinó la factibilidad del uso de la expresión génica como un enfoque alternativo para la detección temprana de deficiencia de nitrato en papa, en tres cultivares (Shepody, Russet Norkotah, y Red Pontiac) con suministro de nitrato abundante (7.5xa0mM NO3), limitado (0.75xa0mM NO3) o deficiente (0xa0mM NO3), en solución nutritiva en un período de 7xa0d. Se extrajo el RNA de la última hoja completamente expandida y se cuantificó usando RT-qPCR de tiempo real. El suministro reducido de nitrato no tuvo efecto medible en el peso seco del tallo o en la concentración de la clorofila foliar, pero disminuyó la concentración de nitrato en el pecíolo. Bajo el suministro deficitario de nitrato, se midió la regulación de nitrato reductasa y nitrito reductasa en los primeros tres días para todos los cultivares, y la regulación de asparagina sintetasa en dos cultivares. No tuvo efecto el suministro de nitrato en la expresión del transportador de amonio. En este sistema experimental, los marcadores de la expresión génica detectaron una reducción en el suministro de nitrato antes que las reducciones medibles en el crecimiento de la planta o en el estado del N medido usando los métodos comunes químicos u ópticos.

The impact of defoliation on nitrogen translocation patterns in the woody invasive plant, Buddleia davidii

The influence of defoliation on nitrogen (N) re-translocation and the source for N remobilisation by the invasive shrub, Buddleia davidii Franch. (buddleia) was determined. Eighty plants were grown over two growing seasons, and half were repeatedly defoliated by removing 66% of their leaf area. During the second season, the N supply was labelled with 15N (10u2009atom% enrichment), to distinguish the use of stored N (unlabelled) from N taken up by roots (labelled) for growth. Defoliation significantly decreased root (39%) and total biomass (26%). Old leaves were the main source of N for remobilisation which was accelerated and increased (by 50% in the second season) in response to defoliation. In spring, root uptake of N increased by 57% in defoliated plants. Thus, defoliation induced changes in N remobilisation and uptake as compensatory growth increased the demand for N. Continued leaf removal decreased the pool of stored N and caused a significant decline in biomass production, especially in roots (39%) and flowers (31%). This has important implications for the efficacy of defoliation as a control measure, as smaller roots suggest a reduced capacity for uptake of nutrients from the soil and reduced flower production may assist in reducing the invasive spread of the species. These findings clearly show that, although the success of B. davidii is associated, in part, with efficient remobilisation of N from storage, this advantage can be overcome by continued defoliation.

Sample Collection Protocol Effects on Quantification of Gene Expression in Potato Leaf Tissue

New platforms allow quantification of gene expression from large, replicated experiments but current sampling protocols for plant tissue using immediate flash freezing in liquid nitrogen are a barrier to these high-throughput studies. In this study, we compared four sampling methods for RNA extraction for gene expression analysis: (1) the standard sampling method of flash freezing whole leaves in liquid nitrogen immediately upon removal from the plant; (2) incubation of excised leaf disks for 2xa0min at field temperature followed by flash freezing; (3) incubation of excised leaf disks for 1xa0h on ice followed by flash freezing; and (4) incubation of excised leaf disks for 1xa0h at field temperature followed by flash freezing. Gene expression analysis was done for 23 genes using nCounter, and normalization of the data was done using the geometric mean of five housekeeping genes. Quality of RNA was highest for protocol A and lowest for protocol D. Despite some differences in RNA quality, gene expression was not significantly different among protocols A, B, and C for any of the 23 genes. Expression of some genes was significantly different between protocol D and the other protocols. This study demonstrates that when sampling leaf disks for gene expression analysis, the time between tissue removal from the plant and flash freezing in liquid nitrogen can be extended. This increase in time allowable during sampling provides greater flexibility in sampling large replicated field experiments for statistical analysis of gene expression data.

Differential gene expression as an indicator of nitrogen sufficiency in field-grown potato plants

Use of an in-season measure of crop N sufficiency to guide fertilizer management is one approach to match the supply of N to the crop N demand. This study examined use of gene expression in leaf tissue of field-grown potatoes for use in assessment of potato N sufficiency. Potato cultivar ‘Shepody’ was grown with six fertilizer N rates (0–250xa0kgxa0N ha–1). Leaf disks were collected weekly for quantification of the expression of N uptake/transport, N assimilation, and amino acid metabolism genes in leaf tissue by nCounter. Many of the genes evaluated were responsive to crop N supply, but the response varied widely among sampling dates. The exception was an ammonium transporter gene (AT1) which was highly expressed, was relatively consistent across sampling dates, was closely related to root zone soil nitrate concentration across N rates and sampling dates, and was highly negatively correlated with total tuber yield. The level of expression of AT1 in leaf tissue was as good as or better than conventional chemical or optical measures of potato N sufficiency in the current study.

Effect of Nitrogen Form on Gene Expression in Leaf Tissue of Greenhouse Grown Potatoes During Three Stages of Growth

This study examined the expression of genes in leaf tissue of greenhouse-grown potatoes to the form of N supplied in the context of development of a diagnostic tool for detection of N status. Potato cultivar ‘Shepody’ was grown over a two-week period at three stages of growth (tuber initiation, flowering and early tuber bulking) with three forms of N (NO3− only, NH4+ only, or 1:1 NO3−:NH4+) at two levels of N supply (0.75 or 7.5xa0mM). Leaf disks were collected and extracted for RNA, and 28 genes examined for expression using nCounter. Expression of ammonium transporter AT1 was increased when the N supply was reduced, regardless of N form, and was generally independent of growth stage. Expression of AT1 was less influenced by N form than currently used chemical or optical measures of plant N status. Expression of AT1 can be used as a quantitative indicator for plant N status of potato regardless of nitrogen form and crop growth stage.ResumenEn este estudio se examinó la expresión de genes en tejido foliar de papas de invernadero respecto a la forma de suministro de N en el contexto de desarrollo de una herramienta de diagnóstico para la detección de la situación del N. Se cultivó la variedad “Shepody” en un período de dos semanas y en tres etapas de crecimiento (iniciación de la tuberización, floración e inicio del llenado de tubérculo) con tres formas de suministro de N (NO3−solamente, NH4+ solamente, o 1:1 NO3−:NH4+), a dos niveles de suministro (0.75 o7.5xa0mM). Se colectaron discos de hoja y se les extrajo el ARN, examinándose la expresión de 28 genes mediante el uso de un nContador. Se incrementó la expresión de un transportador de amonio AT1 cuando se reducía el suministro de N, independientemente de su forma, y fue generalmente independiente del estado de crecimiento. La expresión de AT1 estuvo menos influenciada por la forma de N que las medidas químicas u ópticas comúnmente usadas para medir el estado de N en la planta. La expresión de AT1 puede usarse como un indicador cuantitativo para la situación del N de la planta de papa, independientemente de la forma de nitrógeno y etapa de crecimiento del cultivo.