Characterization of culturable bacterial endophytes of switchgrass (Panicum virgatum L.) and their capacity to influence plant growth

Switchgrass (Panicum virgatum L.) is a perennial warm season grass that is native to the plains of North America and is widely grown as a forage, bioenergy or groundcover crop. Despite its importance, a bottleneck in switchgrass production is poor seedling vigor, which as a perennial crop represents an important time for management. Herein, data identify a suite of culturable bacterial microflora extracted from switchgrass, and show their capability to influence host plant growth and development. A total of 307 bacterial isolates were cultured and isolated from surface sterilized switchgrass biomass and sequence identified into 76 strains (subspecies classification), 36 species and 5 phyla. Approximately 58% of bacterial strains, when reintroduced into surface‐sterilized switchgrass seeds, were documented to increase lamina length (cm from base to tip after 60 days growth) relative to uninoculated controls. Ecologically, Phylum Firmicutes was the most abundant bacterial classification and encompassed 75% of all isolates. Although the culturable bacterial community studies herein represent an unknown and assumedly minor proportion of the total microbiome, by focusing on culturable bacteria, we delineate functional feedback between the presence of isolated bacteria and switchgrass seedling growth.     

Overexpression of miR156 in switchgrass (Panicum virgatum L.) results in various morphological alterations and leads to improved biomass production

Switchgrass (Panicum virgatum L.) has been developed into a dedicated herbaceous bioenergy crop. Biomass yield is a major target trait for genetic improvement of switchgrass. microRNAs have emerged as a prominent class of gene regulatory factors that has the potential to improve complex traits such as biomass yield. A miR156b precursor was overexpressed in switchgrass. The effects of miR156 overexpression on SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes were revealed by microarray and quantitative RT-PCR analyses. Morphological alterations, biomass yield, saccharification efficiency and forage digestibility of the transgenic plants were characterized. miR156 controls apical dominance and floral transition in switchgrass by suppressing its target SPL genes. Relatively low levels of miR156 overexpression were sufficient to increase biomass yield while producing plants with normal flowering time. Moderate levels of miR156 led to improved biomass but the plants were non-flowering. These two groups of plants produced 58%-101% more biomass yield compared with the control. However, high miR156 levels resulted in severely stunted growth. The degree of morphological alterations of the transgenic switchgrass depends on miR156 level. Compared with floral transition, a lower miR156 level is required to disrupt apical dominance. The improvement in biomass yield was mainly because of the increase in tiller number. Targeted overexpression of miR156 also improved solubilized sugar yield and forage digestibility, and offered an effective approach for transgene containment.     

Effects of ecotypes and morphotypes in feedstock composition of switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) is a C4 grass with high biomass yield potential and is now a model species for the Bioenergy Feedstock Development Program. Two distinct ecotypes (e.g., upland and lowland) and a range of plant morphotypes (e.g., leafy and stemmy) have been observed in switchgrass. The objective of this study was to determine the influence of ecotype and morphotype on biomass feedstock quality. Leaf and stem tissues of leafy and stemmy morphotypes from both lowland and upland ecotypes were analyzed for key feedstock traits. The leaf : stem ratio of leafy morphotype was more than 40% higher than the stemmy morphotype in both upland and lowland ecotypes. Therefore, the stemmy morphotype has significant advantages over leafy morphotype during harvesting, storage, transportation and finally the feedstock quality. Remarkable differences in feedstock quality and mineral composition were observed in switchgrass genotypes with distinct ecotypic origins and variable plant morphotypes. Lignin, hemicelluloses and cellulose concentrations were higher in stems than in the leaves, while ash content was notably high in leaves. A higher concentration of potassium was found in the stems compared to the leaves. In contrast, calcium was higher and magnesium was generally higher in the leaves compared to stems. The upland genotypes demonstrated considerably higher lignin (14.4%) compared with lowland genotypes (12.4%), while hemicellulose was higher in lowland compared with upland. The stemmy type demonstrated slightly higher lignin compared with leafy types (P < 0.1). Differences between the ecotypes and morphotypes for key quality traits demonstrated the potential for improving feedstock composition of switchgrass through selection in breeding programs.     

Physiological and growth responses of switchgrass (Panicum virgatum L.) in native stands under passive air temperature manipulation

In the central Great Plains of North America, climate change predictions include increases in mean annual temperature of 1.5–5.5 °C by 2100. Ecosystem responses to increased temperatures are likely to be regulated by dominant plant species, such as the potential biofuel species Panicum virgatum (switchgrass) in the tallgrass prairie. To describe the potential physiological and whole‐plant responses of this species to future changes in air temperatures, we used louvered open‐sided chambers (louvered OSC; 1 × 1 m, adjustable height) to passively alter canopy temperature in native stands of P. virgatum growing in tallgrass prairie at varying topographic positions (upland/lowland). The altered temperature treatment decreased daily mean temperatures by 1 °C and maximum temperatures by 4 °C in May and June, lowered daytime stomatal conductance and transpiration, decreased tiller density, increased specific leaf area, and delayed flowering. Among topographic contrasts, aboveground biomass, flowering tiller density, and tiller weight were greater in lowland sites compared to upland sites, with no temperature treatment interactions. Differences in biomass production responded more to topography than the altered temperature treatment, as soil water status varied considerably between topographic positions. These results indicate that while water availability as a function of topography was a strong driver of plant biomass, many leaf‐level physiological processes were responsive to the small decreases in daily mean and maximum temperature, irrespective of landscape position. The varying responses of leaf‐level gas exchange and whole‐plant growth of P. virgatum in native stands to altered air temperature or topographic position illustrate that accurately forecasting yields for P. virgatum in mixed communities will require greater integration of physiological responses to simulated climate change (increased temperature) and resource availability over natural environmental gradients (soil moisture).     

Gateway-compatible vectors for high-throughput gene functional analysis in switchgrass (Panicum virgatum L.) and other monocot species

Switchgrass (Panicum virgatum L.) is a C4 perennial grass and has been identified as a potential bioenergy crop for cellulosic ethanol because of its rapid growth rate, nutrient use efficiency and widespread distribution throughout North America. The improvement of bioenergy feedstocks is needed to make cellulosic ethanol economically feasible, and genetic engineering of switchgrass is a promising approach towards this goal. A crucial component of creating transgenic switchgrass is having the capability of transforming the explants with DNA sequences of interest using vector constructs. However, there are limited options with the monocot plant vectors currently available. With this in mind, a versatile set of Gateway-compatible destination vectors (termed pANIC) was constructed to be used in monocot plants for transgenic crop improvement. The pANIC vectors can be used for transgene overexpression or RNAi-mediated gene suppression. The pANIC vector set includes vectors that can be utilized for particle bombardment or Agrobacterium-mediated transformation. All the vectors contain (i) a Gateway cassette for overexpression or silencing of the target sequence, (ii) a plant selection cassette and (iii) a visual reporter cassette. The pANIC vector set was functionally validated in switchgrass and rice and allows for high-throughput screening of sequences of interest in other monocot species as well.     

Enhancement of switchgrass (Panicum virgatum L.) biomass production under drought conditions by the ectomycorrhizal fungus Sebacina vermifera

Experiments were conducted to examine the effects of cocultivating the important bioenergy crop switchgrass with the ectomycorrhizal fungus Sebacina vermifera under severe drought conditions. Plants cocultivated with the fungus produced significantly higher biomass and had a higher macronutrient content than uninoculated control plants under both adequately watered and drought conditions.     

Modeled spatial assessment of biomass productivity and technical potential of Miscanthus × giganteus,Panicum virgatum L., and Jatropha on marginal land in China

This article identifies marginal land technically available for the production of energy crops in China, compares three models of yield prediction for Miscanthus × giganteus, Panicum virgatum L. (switchgrass), and Jatropha, and estimates their spatially specific yields and technical potential for 2017. Geographic Information System (GIS) analysis of land use maps estimated that 185 Mha of marginal land was technically available for energy crops in China without using areas currently used for food production. Modeled yields were projected for Miscanthus × giganteus, a GIS‐based Environmental Policy Integrated Climate model for switchgrass and Global Agro‐Ecological Zone model for Jatropha. GIS analysis and MiscanFor estimated more than 120 Mha marginal land was technically available for Miscanthus with a total potential of 1,761 dry weight metric million tonne (DW Mt)/year. A total of 284 DW Mt/year of switchgrass could be obtained from 30 Mha marginal land, with an average yield of 9.5 DW t ha^?1 year^?1. More than 35 Mha marginal land was technically available for Jatropha, delivering 9.7 Mt/year of Jatropha seed. The total technical potential from available marginal land was calculated as 31.7 EJ/year for Miscanthus, 5.1 EJ/year for switchgrass, and 0.13 EJ/year for Jatropha. A total technical bioenergy potential of 34.4 EJ/year was calculated by identifying best suited crop for each 1 km² grid cell based on the highest energy value among the three crops. The results indicate that the technical potential per hectare of Jatropha is unable to compete with that of the other two crops in each grid cell. This modeling study provides planners with spatial overviews that demonstrate the potential of these crops and where biomass production could be potentially distributed in China which needs field trials to test model assumptions and build experience necessary to translate into practicality.     

Evaluation of Agricultural Production Systems Simulator as yield predictor of Panicum virgatum and Miscanthus x giganteus in several US environments

Simulation models for perennial energy crops such as switchgrass (Panicum virgatum L.) and Miscanthus (Miscanthus x giganteus) can be useful tools to design management strategies for biomass productivity improvement in US environments. The Agricultural Production Systems Simulator (APSIM) is a biophysical model with the potential to simulate the growth of perennial crops. APSIM crop modules do not exist for switchgrass and Miscanthus, however, re‐parameterization of existing APSIM modules could be used to simulate the growth of these perennials. Our aim was to evaluate the ability of APSIM to predict the dry matter (DM) yield of switchgrass and Miscanthus at several US locations. The Lucerne (for switchgrass) and Sugarcane (for Miscanthus) APSIM modules were calibrated using data from four locations in Indiana. A sensitivity analysis informed the relative impact of changes in plant and soil parameters of APSIM Lucerne and APSIM Sugarcane modules. An independent dataset of switchgrass and Miscanthus DM yields from several US environments was used to validate these re‐parameterized APSIM modules. The re‐parameterized modules simulated DM yields of switchgrass [0.95 for CCC (concordance correlation coefficient) and 0 for SB (bias of the simulation from the measurement)] and Miscanthus (0.65 and 0% for CCC and SB, respectively) accurately at most locations with the exception of switchgrass at southern US sites (0.01 for CCC and 2% for SB). Therefore, the APSIM model is a promising tool for simulating DM yields for switchgrass and Miscanthus while accounting for environmental variability. Given our study was strictly based on APSIM calibrations at Indiana locations, additional research using more extensive calibration data may enhance APSIM robustness.     

Soil microbial community structure and enzymatic activity responses to nitrogen management and landscape positions in switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) is usually grown on marginal land for biofuel system, in which nitrogen (N) is an essential management practice, and landscape position is a key topographical factor in impacting the production. However, limited information is available regarding how the N application and landscape positions affect soil microbial communities and enzyme activities under switchgrass. Thus, the specific objective of this study was to evaluate the responses of N rate (high, 112 kg N/ha; medium, 56 kg N/ha; and low, 0 kg N/ha) and landscape positions (shoulder and footslope) on soil biological health under switchgrass field. Data showed that N addition significantly influenced carbon and N fractions. The hot water‐soluble organic carbon (HWC) and nitrogen (HWN) fractions were significantly higher at footslope position than the shoulder position. The amount of total phospholipid fatty acid (PLFA), total bacterial, actinomycetes, gram‐negative and gram‐positive bacteria, total fungi, arbuscular mycorrhizal (AM) fungi, and saprophytes PLFAs were highest with medium and high N rates and footslope position. The N addition increased total PLFAs in N fertilizer treatments, viz. medium (5,946 ng PLFA‐C/g soil) and high N rates (5,871 ng PLFA‐C/g soil). Microbial biomass carbon and nitrogen and enzyme activities (urease, β‐glucosidase, acid phosphatase and arylsulfatase) were significantly enhanced by N fertilization (medium and high N rates) compared to control (low N rates) under footslope position. The urease activity under medium (36.3 µmol N‐NH₄⁺ g^?1 soil hr^?1) and high N rates (31.4 µmol N‐NH₄⁺ g^?1 soil hr^?1) was 42.9% and 23.6% higher than low N rates, respectively. This study suggests that the application of medium N rate in footslope position to switchgrass can enhance the soil biological properties and hence can protect the environment from the excessive use of N fertilizer.     

Field‐grown transgenic switchgrass (Panicum virgatum L.) with altered lignin does not affect soil chemistry,microbiology, and carbon storage potential

Cell wall recalcitrance poses a major challenge on cellulosic biofuel production from feedstocks such as switchgrass (Panicum virgatum L.). As lignin is a known contributor of recalcitrance, transgenic switchgrass plants with altered lignin have been produced by downregulation of caffeic acid O‐methyltransferase (COMT). Field trials of COMT‐downregulated plants previously demonstrated improved ethanol conversion with no adverse agronomic effects. However, the rhizosphere impacts of altering lignin in plants are unknown. We hypothesized that changing plant lignin composition may affect residue degradation in soils, ultimately altering soil processes. The objective of this study was to evaluate effects of two independent lines of COMT‐downregulated switchgrass plants on soils in terms of chemistry, microbiology, and carbon cycling when grown in the field. Over the first two years of establishment, we observed no significant differences between transgenic and control plants in terms of soil pH or the total concentrations of 19 elements. An analysis of soil bacterial communities via high‐throughput 16S rRNA gene amplicon sequencing revealed no effects of transgenic plants on bacterial diversity, richness, or community composition. We also did not observe a change in the capacity for soil carbon storage: There was no significant effect on soil respiration or soil organic matter. After five years of establishment, δ¹³C of plant roots, leaves, and soils was measured and an isotopic mixing model used to estimate that 11.2 to 14.5% of soil carbon originated from switchgrass. Switchgrass‐contributed carbon was not significantly different between transgenic and control plants. Overall, our results indicate that over the short term (two and five years), lignin modification in switchgrass through manipulation of COMT expression does not have an adverse effect on soils in terms of total elemental composition, bacterial community structure and diversity, and capacity for carbon storage.     

Five nuclear loci resolve the polyploid history of switchgrass (Panicum virgatum L.) and relatives

Polyploidy poses challenges for phylogenetic reconstruction because of the need to identify and distinguish between homoeologous loci. This can be addressed by use of low copy nuclear markers. Panicum s.s. is a genus of about 100 species in the grass tribe Paniceae, subfamily Panicoideae, and is divided into five sections. Many of the species are known to be polyploids. The most well-known of the Panicum polyploids are switchgrass (Panicum virgatum) and common or Proso millet (P. miliaceum). Switchgrass is in section Virgata, along with P. tricholaenoides, P. amarum, and P. amarulum, whereas P. miliaceum is in sect. Panicum. We have generated sequence data from five low copy nuclear loci and two chloroplast loci and have clarified the origin of P. virgatum. We find that all members of sects. Virgata and Urvilleana are the result of diversification after a single allopolyploidy event. The closest diploid relatives of switchgrass are in sect. Rudgeana, native to Central and South America. Within sections Virgata and Urvilleana, P. tricholaenoides is sister to the remaining species. Panicum racemosum and P. urvilleanum form a clade, which may be sister to P. chloroleucum. Panicum amarum, P. amarulum, and the lowland and upland ecotypes of P. virgatum together form a clade, within which relationships are complex. Hexaploid and octoploid plants are likely allopolyploids, with P. amarum and P. amarulum sharing genomes with P. virgatum. Octoploid P. virgatum plants are formed via hybridization between disparate tetraploids. We show that polyploidy precedes diversification in a complex set of polyploids; our data thus suggest that polyploidy could provide the raw material for diversification. In addition, we show two rounds of allopolyploidization in the ancestry of switchgrass, and identify additional species that may be part of its broader gene pool. This may be relevant for development of the crop for biofuels.     

Hydrological responses of land use change from cotton (Gossypium hirsutum L.) to cellulosic bioenergy crops in the Southern High Plains of Texas,USA

The Southern High Plains (SHP) region of Texas in the United States, where cotton is grown in a vast acreage, has the potential to grow cellulosic bioenergy crops such as perennial grasses and biomass sorghum (Sorghum bicolor). Evaluation of hydrological responses and biofuel production potential of hypothetical land use change from cotton (Gossypium hirsutum L.) to cellulosic bioenergy crops enables better understanding of the associated key agroecosystem processes and provides for the feasibility assessment of the targeted land use change in the SHP. The Soil and Water Assessment Tool (SWAT) was used to assess the impacts of replacing cotton with perennial Alamo switchgrass (Panicum virgatum L.), Miscanthus × giganteus (Miscanthus sinensis Anderss. [Poaceae]), big bluestem (Andropogon gerardii) and annual biomass sorghum on water balances, water use efficiency and biofuel production potential in the Double Mountain Fork Brazos watershed. Under perennial grass scenarios, the average (1994–2009) annual surface runoff from the entire watershed decreased by 6–8% relative to the baseline cotton scenario. In contrast, surface runoff increased by about 5% under the biomass sorghum scenario. Perennial grass land use change scenarios suggested an increase in average annual percolation within a range of 3–22% and maintenance of a higher soil water content during August to April compared to the baseline cotton scenario. About 19.1, 11.1, 3.2 and 8.8 Mg ha^?1 of biomass could potentially be produced if cotton area in the watershed would hypothetically be replaced by Miscanthus, switchgrass, big bluestem and biomass sorghum, respectively. Finally, Miscanthus and switchgrass were found to be ideal bioenergy crops for the dryland and irrigated systems, respectively, in the study watershed due to their higher water use efficiency, better water conservation effects, greater biomass and biofuel production potential, and minimum crop management requirements.     

Developing xylem‐preferential expression of PdGA20ox1, a gibberellin 20‐oxidase 1 from Pinus densiflora,improves woody biomass production in a hybrid poplar

Woody biomass has gained popularity as an environmentally friendly, renewable and sustainable resource for liquid fuel production. Here, we demonstrate biotechnological improvement of the quantity and quality of woody biomass by employing developing xylem (DX)‐preferential production of gibberellin (GA), a phytohormone that positively regulates stem growth. First, for the proof of concept experiment, we produced transgenic Arabidopsis plants expressing GA20‐oxidase, a key enzyme in the production of bioactive GAs, from Pinus densiflora (PdGA20ox1) under the control of either a constitutive 35S promoter, designated 35S::PdGA20ox1, or a DX‐specific promoter (originated from poplar), designated DX15::PdGA20ox1. As we hypothesized, both transgenic Arabidopsis plants (35S::PdGA20ox1 and DX15::PdGA20ox1) exhibited an accelerated stem growth that resulted in a large increase of biomass, up to 300% compared to wild‐type control plants, together with increased secondary wall thickening and elongation of fibre cells. Next, we applied our concept to the production of transgenic poplar trees. Both transgenic poplar trees (35S::PdGA20ox1 and DX15::PdGA20ox1) showed dramatic increases in biomass, up to 300%, with accelerated stem growth and xylem differentiation. Cell wall monosaccharide composition analysis revealed that in both Arabidopsis and poplar, glucose and xylose contents were significantly increased. However, undesirable phenotypes of 35S::PdGA20ox1 poplar, including poor root growth and leaf development, were found. Interestingly, DX15::PdGA20ox1 poplar resulted in a reduction of undesirable phenotypes. Our results indicate that the controlled production of GAs through a tissue‐specific promoter can be utilized as an efficient biotechnological tool for producing enhanced plant biomass, minimizing unwanted effects.     

Phytoremediation of biosolids from an end-of-life municipal lagoon using cattail (Typha latifolia L.) and switchgrass (Panicum virgatum L.)

Land spreading of biosolids as a disposal option is expensive and can disperse pathogens and contaminants in the environment. This growth room study examined phytoremediation using switchgrass (Panicum virgatum L.) and cattail (Typha latifolia L.) as an alternative to land spreading of biosolids. Seedlings were transplanted into pots containing 3.9 kg of biosolids (dry wt.). Aboveground biomass (AGB) was harvested either once or twice during each 90-day growth period. Switchgrass AGB yield was greater with two harvests than with one harvest during the first 90-day growth period, whereas cattail yield was not affected by harvest frequency. In the second growth period, harvesting frequency did not affect the yield of either plant species. However, repeated harvesting significantly improved nitrogen (N) and phosphorus (P) uptake by both plants in the first period. Phytoextraction of P was significantly greater for switchgrass (3.9% of initial biosolids P content) than for cattail (2.8%), while plant species did not have a significant effect on N phytoextraction. The trace element accumulation in the AGB of both plant species was negligible. Phytoextraction rates attained in this study suggest that phytoremediation can effectively remove P from biosolids and offers a potentially viable alternative to the disposal of biosolids on agricultural land.     

Global simulation of bioenergy crop productivity: analytical framework and case study for switchgrass

A global energy crop productivity model that provides geospatially explicit quantitative details on biomass potential and factors affecting sustainability would be useful, but does not exist now. This study describes a modeling platform capable of meeting many challenges associated with global‐scale agro‐ecosystem modeling. We designed an analytical framework for bioenergy crops consisting of six major components: (i) standardized natural resources datasets, (ii) global field‐trial data and crop management practices, (iii) simulation units and management scenarios, (iv) model calibration and validation, (v) high‐performance computing (HPC) simulation, and (vi) simulation output processing and analysis. The HPC‐Environmental Policy Integrated Climate (HPC‐EPIC) model simulated a perennial bioenergy crop, switchgrass (Panicum virgatum L.), estimating feedstock production potentials and effects across the globe. This modeling platform can assess soil C sequestration, net greenhouse gas (GHG) emissions, nonpoint source pollution (e.g., nutrient and pesticide loss), and energy exchange with the atmosphere. It can be expanded to include additional bioenergy crops (e.g., miscanthus, energy cane, and agave) and food crops under different management scenarios. The platform and switchgrass field‐trial dataset are available to support global analysis of biomass feedstock production potential and corresponding metrics of sustainability.