Evaluation and optimization of lysis method for microbial DNA extraction from epiphytic phyllosphere samples.

Analysis of microbial communities in the epiphytic phyllosphere can be challenging, especially when applying sequencing-based techniques, owing to the interference of plant-derived biomolecules such as nucleic acids. A review of recent studies on the epiphytic microbiome revealed that both mechanical and enzymatic lysis methods are widely used. Here, we evaluated the effects of the two lysis methods on DNA extraction yield, purity, integrity, and microbial 16S rRNA gene copy number per ng of template genomic DNA under different extraction conditions. Furthermore, the effect on bacterial community composition, diversity, and reproducibility was examined using 16S rRNA gene amplicon sequencing. The enzymatic lysis method yielded one to two orders of magnitude more DNA, but the DNA quality was suboptimal. Conversely, the samples prepared using the mechanical method showed high DNA purity albeit lower yield. Unexpectedly, mechanical lysis showed a higher DNA integrity number (DIN) than enzymatic lysis. The 16S rRNA amplicon sequencing results demonstrated that the samples prepared via mechanical disruption exhibited reproducibly similar microbial community compositions regardless of the extraction conditions. In contrast, the enzymatic lysis method resulted in inconsistent taxonomic compositions under different extraction conditions. This study demonstrates that mechanical DNA disruption is more suitable for epiphytic phyllosphere samples than enzymatic disruption.

Yield gap analysis of rainfed alfalfa in the United States.

The United States (US) is the largest alfalfa (Medicago sativa L.) producer in the world. More than 44% of the US alfalfa is produced under rainfed conditions, although it requires a relatively high amount of water compared to major field crops. Considering that yield and production of rainfed alfalfa have been relatively stagnant in the country for decades, there is a need to better understand the magnitude of yield loss due to water limitation and how far from yield potential current yields are. In this context, the main objective of this study was to estimate the current yield gap of rainfed alfalfa in the US. We collected 10 year (2009-2018) county-level government-reported yield and weather data from 393 counties within 12 major US rainfed alfalfa producing states and delineated alfalfa growing season using probabilistic approaches based on temperature thresholds for crop development. We then calculated county-level growing season rainfall (GSR), which was plotted against county-level yield to determine attainable yield (Ya) using frontier function analysis, and water-limited potential yield (Yw) using boundary function analysis. Average and potential water use efficiencies (WUE) were estimated, and associated yield gap referring to attainable (YGa) or water-limited yields (YGw) were calculated. Finally, we used conditional inference trees (CIT) to identify major weather-related yield-limiting factors to alfalfa forage yield. The frontier model predicted a mean Ya of 9.6 ± 1.5 Mg ha-1 and an associated optimum GSR of 670 mm, resulting in a mean YGa of 34%. The boundary function suggested a mean Yw of 15.3 ± 3 Mg ha-1 at the mean GSR of 672 ± 153 mm, resulting in a mean yield gap of 58%. The potential alfalfa WUE was 30 kg ha-1 mm-1 with associated minimum water losses of 24% of mean GSR, which was three times greater than the mean WUE of 10 kg ha-1 mm-1. The CIT suggested that GSR and minimum temperature in the season were the main yield-limiting weather variables in rainfed alfalfa production in the US. Our study also revealed that alfalfa was only limited by water availability in 21% of the environments. Thus, future research on management practices to narrow yield gaps at current levels of water supply is necessary.

Harvesting schedule effects on forage yield and nutritive values in low-lignin alfalfa.

Under the four-cut system, low-lignin alfalfa (Medicago sativa L.) may extend harvesting intervals improving harvest management flexibility and producing forage products with higher nutritive values. The objective of this study was to compare forage yield and nutritive values of low-lignin and conventional alfalfa varieties when applied to six different harvest schedules in the first (2016) and second (2017) production years. There were 12 treatments of two alfalfa varieties as whole plots and six harvest schedules as subplots. Across harvest schedules, there were four cuttings in two production years. Three harvest intervals including "Standard" (high quality, HQ), "Standard+5-day" (medium quality, MQ), and "Standard+10-day" (high yield, HY) were chosen for the first cutting, and 30-day (HQ) and 35-day (HY) for the second cuttings. The third and fourth cuttings in 2016 were timed near final harvest date and in 2017 occurred at 35-day (MQ) and 40-day (HY). Variety by harvest schedule interaction was not significant, but the whole plot and sub-plot effects were significant. Hi-Gest 360 was consistently higher in nutritive value and with a similar yield as Gunner. Harvest schedules did not consistently differ in forage yield and nutritive values. HS-1 ("Standard" + 35-day + Medium Quality + High Yield) with shorter first two cutting intervals provided lower acid detergent fiber (ADF), neutral detergent fiber (NDF), higher relative feed value (RFV), and similar forage yield compared to other schedules. HS-1 had the highest economic incomes when considering RFV and yield among the six different harvest schedules.

Omics Path to Increasing Productivity in Less-Studied Crops Under Changing Climate-Lentil a Case Study.

Conventional breeding techniques for crop improvement have reached their full potential, and hence, alternative routes are required to ensure a sustained genetic gain in lentils. Although high-throughput omics technologies have been effectively employed in major crops, less-studied crops such as lentils have primarily relied on conventional breeding. Application of genomics and transcriptomics in lentils has resulted in linkage maps and identification of QTLs and candidate genes related to agronomically relevant traits and biotic and abiotic stress tolerance. Next-generation sequencing (NGS) complemented with high-throughput phenotyping (HTP) technologies is shown to provide new opportunities to identify genomic regions and marker-trait associations to increase lentil breeding efficiency. Recent introduction of image-based phenotyping has facilitated to discern lentil responses undergoing biotic and abiotic stresses. In lentil, proteomics has been performed using conventional methods such as 2-D gel electrophoresis, leading to the identification of seed-specific proteome. Metabolomic studies have led to identifying key metabolites that help differentiate genotypic responses to drought and salinity stresses. Independent analysis of differentially expressed genes from publicly available transcriptomic studies in lentils identified 329 common transcripts between heat and biotic stresses. Similarly, 19 metabolites were common across legumes, while 31 were common in genotypes exposed to drought and salinity stress. These common but differentially expressed genes/proteins/metabolites provide the starting point for developing high-yielding multi-stress-tolerant lentils. Finally, the review summarizes the current findings from omic studies in lentils and provides directions for integrating these findings into a systems approach to increase lentil productivity and enhance resilience to biotic and abiotic stresses under changing climate.

Cultivated and wild pearl millet display contrasting patterns of abundance and co-occurrence in their root mycobiome.

Fungal communities associated with roots play a key role in nutrient uptake and in mitigating the abiotic and biotic stress of their host. In this study, we characterized the roots mycobiome of wild and cultivated pearl millet [Pennisetum glaucum (L.) R. Br., synonym: Cenchrus americanus (L.) Morrone] in three agro-ecological areas of Senegal following a rainfall gradient. We hypothesized that wild pearl millet could serve as a reservoir of endophytes for cultivated pearl millet. We therefore analyzed the soil factors influencing fungal community structure and whether cultivated and wild millet shared the same fungal communities. The fungal communities associated with pearl millet were significantly structured according to sites and plant type (wild vs cultivated). Besides, soil pH and phosphorus were the main factors influencing the fungal community structure. We observed a higher fungal diversity in cultivated compared to wild pearl millet. Interestingly, we detected higher relative abundance of putative pathotrophs, especially plant pathogen, in cultivated than in wild millet in semi-arid and semi-humid zones, and higher relative abundance of saprotrophs in wild millet in arid and semi-humid zones. A network analysis based on taxa co-occurrence patterns in the core mycobiome revealed that cultivated millet and wild relatives had dissimilar groups of hub taxa. The identification of the core mycobiome and hub taxa of cultivated and wild pearl millet could be an important step in developing microbiome engineering approaches for more sustainable management practices in pearl millet agroecosystems.

Genetic and molecular mechanisms underlying root architecture and function under heat stress-A hidden story.

Heat stress events are resulting in a significant negative impact on global food production. The dynamics of cellular, molecular and physiological homoeostasis in aboveground parts under heat stress are extensively deciphered. However, root responses to higher soil/air temperature or stress signalling from shoot to root are limited. Therefore, this review presents a holistic view of root physio-morphological and molecular responses to adapt under hotter environments. Heat stress reprogrammes root cellular machinery, including crosstalk between genes, phytohormones, reactive oxygen species (ROS) and antioxidants. Spatio-temporal regulation and long-distance transport of phytohormones, such as auxin, cytokinin and abscisic acid (ABA) determine the root growth and development under heat stress. ABA cardinally integrates a signalling pathway involving heat shock factors, heat shock proteins and ROS to govern heat stress responses. Additionally, epigenetic modifications by transposable elements, DNA methylation and acetylation also regulate root growth under heat stress. Exogenous application of chemical compounds or biological agents such as ascorbic acid, metal ion chelators, fungi and bacteria can alleviate heat stress-induced reduction in root biomass. Future research should focus on the systemic effect of heat stress from shoot to root with more detailed investigations to decipher the molecular cues underlying the roots architecture and function.

Effects of harvest intervals and seeding rates on dry matter yield and nutritive value of alfalfa cultivars.

Maturation process of alfalfa (Medicago sativa L.) could be prevented by the reduction of lignin content in terms of conventional breeding or transgenic technology. Alfalfa could exhibit higher leaf/stem ratio, with a concern of yield loss. The objective of this study was to compare forage yield and nutritive value of low lignin alfalfa and two reference varieties subjecting to two harvest intervals and three seeding rates. The experimental design was a randomized complete block in a split-split plot arrangement with four replicates, where harvest intervals (28-day and 35-day) were assigned to whole plots, seeding rates were subplots, and varieties were sub-subplots. The weighted mean nutritive value was applied to two production years of 2016 and 2017. Hi-Gest 360 (low lignin alfalfa) provided similar yield potential and increased nutritive value compared to two reference varieties. Over a two-year production period, alfalfa harvested at every 28-day interval provided more economic returns than those at 35-day interval. For the seeding year and first production year, five cuts made by the 28-day interval produced more yield than four cuts by the 35-day interval. Due to limited rainfall in May 2017, a sharp drop of the first cutting overturned the advantage of the five-cut system. Shorter intervals between harvests generally increased crude protein (CP) concentrations. The differences of relative feed value (RFV) between two harvest intervals tended to be great during the first and second cuttings. Overall, harvest interval had a large effect on nutritive value and a more significant effect on alfalfa dry matter yield than variety selection. Seeding rate did not affect alfalfa yield and nutritive value.

Effect of a fall cut on dry matter yield, nutritive value, and stand persistence of alfalfa.

Information is lacking about the effect of date of a fall cut of alfalfa (Medicago sativa L.) on dry matter yield (DMY), forage nutritive value, and stands persistence. The objective of this study was to determine the effect of timing of a fall cut on DMY, forage nutritive value and stand persistence of three alfalfa varieties: low-lignin Hi-Gest 360, Roundup Ready Tonica, and conventional Gunner in Northeastern Kansas in the United States. The field study was carried out by splitting plot in randomized complete block design with four replications. The harvesting data of different maturity stages were collected in each year from 2015 to 2018. Three cuts were harvested based on the stage of maturity, and the last (fourth) cut was done on September 15, September 30, October 15, and October 30 of each year. The persistence of the alfalfa stands was determined each fall after the last cut, and each spring after the first cut, by counting the number of live plants in a randomly placed quadrat in each plot. Alfalfa cut on September 15 and September 30 had a higher stand persistence compared to alfalfa cut on October 15 and October 30. The DMY of the first cut in 2016 was significantly higher in roundup ready than the low-lignin alfalfa variety. In the second cut, DMY was significantly higher in conventional alfalfa than the roundup ready. There were no significant differences in DMY between alfalfa varieties in the rest of seasonal cuttings in 2016 and 2017 and annual total yield in both years. In general, low lignin alfalfa variety had higher crude protein and relative feed value and lower acid detergent fiber and neutral detergent fiber contents than those in roundup ready and conventional alfalfa varieties. On average, nutritive value of alfalfa was generally affected by last cutting dates in 2017. Based on 3-year data the last cutting of alfalfa in the fall could be done by September 30-October 15 without harmful effect on DMY.

Effects of cultivar and harvest days after planting on dry matter yield and nutritive value of teff.

One of the most pressing issues facing the dairy industry is drought. In areas where annual precipitation is low, irrigation for growing feed presents the greatest water-utilization challenge for dairy producers. Here, we investigated the effects of cultivar and harvest days after planting (DAP) on dry matter (DM) yield and nutritive value of teff (Eragrostis tef), a warm-season annual grass native to Ethiopia that is well adapted to drought conditions. Eighty pots were blocked by location in a greenhouse and randomly assigned to four teff cultivars (Tiffany, Moxie, Corvallis, and Dessie) and to five harvest times (40, 45, 50, 55, or 60 DAP). Cultivars had no effect on DM yield and nutritive value. As harvest time increased from 40 to 60 DAP, DM yield and ash-free neutral detergent fiber (aNDFom) concentrations increased, while crude protein (CP) concentrations and in vitro NDF digestibility decreased. To assess carryover effects of time of harvest on yield and nutritive value, two additional cuttings were taken from each pot. Increasing first-cutting harvest time decreased CP concentrations in the second cutting and reduced DM yields in the second and third cutting. Harvesting teff between 45 and 50 DAP best optimized forage yield and nutritive value in the first and subsequent cuttings.

Effects of alfalfa and alfalfa-grass mixtures with nitrogen fertilization on dry matter yield and forage nutritive value.

Alfalfa (Medicago sativa L.) is an important forage legume grown in Kansas, USA and its productivity with cool-season grasses however is unknown. The objective of this study was to determine the dry matter yield (DMY) and forage nutritive value of alfalfa-grass mixtures compared to those of alfalfa and grasses grown in monoculture with and without nitrogen fertilization. Three different alfalfa varieties were planted (reduced-lignin alfalfa, Roundup Ready, and conventional alfalfa) and two kinds of cool-season grasses (smooth brome, Bromus inermis Leyss, and tall fescue, Festuca arundinacea Schreb) were planted as a monoculture or in alfalfa-grass mixtures. Nitrogen fertilizer (urea) was applied at green-up at a rate of 56 kg/ha and after the second cutting at a rate of 56 kg/ha in 2016 and 2017, respectively. and control treatments received no nitrogen. DMY was significantly higher in monoculture alfalfa and alfalfa-grass mixtures than in grass monocultures. Between alfalfa monoculture and alfalfa-grass mixtures, no significant differences in DMY were found. For all treatments, nitrogen application significantly increased DMY compared to the control. In 2016 and 2017, the low-lignin alfalfa monoculture had the lowest acid detergent fiber (ADF) and the grass monocultures had the highest ADF. In 2016 and 2017, neutral detergent fiber (NDF) in smooth bromegrass and tall fescue was higher than in other species treatments. A low-lignin alfalfa monoculture had significantly lower NDF concentration compared to alfalfa-grass mixtures. When averaged over 2016 and 2017, relative feed value (RFV) was highest in low-lignin alfalfa and lowest in the grass monocultures. In both years, nitrogen fertilizer application did not affect nutritive values.

Development of a model estimating root length density from root impacts on a soil profile in pearl millet (Pennisetum glaucum (L.) R. Br). Application to measure root system response to water stress in field conditions.

Pearl millet is able to withstand dry and hot conditions and plays an important role for food security in arid and semi-arid areas of Africa and India. However, low soil fertility and drought constrain pearl millet yield. One target to address these constraints through agricultural practices or breeding is root system architecture. In this study, in order to easily phenotype the root system in field conditions, we developed a model to predict root length density (RLD) of pearl millet plants from root intersection densities (RID) counted on a trench profile in field conditions. We identified root orientation as an important parameter to improve the relationship between RID and RLD. Root orientation was notably found to depend on soil depth and to differ between thick roots (more anisotropic with depth) and fine roots (isotropic at all depths). We used our model to study pearl millet root system response to drought and showed that pearl millet reorients its root growth toward deeper soil layers that retain more water in these conditions. Overall, this model opens ways for the characterization of the impact of environmental factors and management practices on pearl millet root system development.

Impact of Nitrogen Application Rate on Switchgrass Yield, Production Costs, and Nitrous Oxide Emissions.

Switchgrass ( L.) has been promoted as a potential feedstock for cellulosic biofuel in the United States. Switchgrass is known to respond to N fertilizer, but optimal rates remain unclear. Given the potential nonlinear response of nitrous oxide (NO) emissions to N inputs, N additions to switchgrass above optimal levels could have large impacts on the greenhouse gas balance of switchgrass-based biofuel. Additionally, N additions are likely to have large impacts on switchgrass production costs. Yield, N removal, and net returns were measured in switchgrass receiving 0 to 200 kg N ha in Manhattan, KS, from 2012 to 2014. Emissions of NO were measured in the 0- to 150-kg N ha treatments. Total emissions of NO increased from 0.2 to 3.0 kg NO-N ha as N inputs increased from 0 to 150 kg N ha. The 3-yr averages of fertilizer-induced emission factors were 0.7, 2.1, and 2.6% at 50, 100, and 150 kg N ha, respectively. Removal of N at harvest increased linearly with increasing N rate. Switchgrass yields increased with N inputs up to 100 to 150 kg N ha, but the critical N level for maximum yields decreased each year, suggesting that N was being applied in excess at higher N rates. Net returns were maximized at 100 kg N ha at both a high and low urea cost (US$394.71 and $945.91 ha, respectively). These results demonstrate that N inputs were necessary to increase switchgrass productivity, but rates exceeding optimal levels resulted in excessive NO emissions and increased costs for producers.

Production of free fatty acids from switchgrass using recombinant Escherichia coli.

Switchgrass is a promising feedstock to generate fermentable sugars required for the sustainable operation of biorefineries because of their abundant availability, easy cropping system, and high cellulosic content. The objective of this study was to investigate the potentiality of switchgrass as an alternative sugar supplier for free fatty acid (FFA) production using engineered Escherichia coli strains. Recombinant E. coli strains successfully produced FFAs using switchgrass hydrolysates. A total of about 3 g/L FFAs were attained from switchgrass hydrolysates by engineered E. coli strains. Furthermore, overall yield assessments of our bioconversion process showed that 88 and 46% of the theoretical maximal yields of glucose and xylose were attained from raw switchgrass during sugar generation. Additionally, 72% of the theoretical maximum yield of FFAs were achieved from switchgrass hydrolysates by recombinant E. coli during fermentation. These shake-flask results were successfully scaled up to a laboratory scale bioreactor with a 4 L working volume. This study demonstrated an efficient bioconversion process of switchgrass-based FFAs using an engineered microbial system for targeting fatty acid production that are secreted into the fermentation broth with associated lower downstream processing costs, which is pertinent to develop an integrated bioconversion process using lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:91-98, 2018.

Self-Assembled Dendron-Cyclodextrin Nanotubes with a Polyethylenimine Surface and Their Gene Delivery Capability.

Self-assembled dendron-cyclodextrin nanotubes (Den-CD-NTs) with selected surface functionalities can serve as templates for the formation of complexes with polymers, and the resulting nanotube-polymer complexes can be utilized as gene carriers. The negatively charged surfaces of Den-CD-NTs were covered with a positively charged polyethylenimine (PEI) layer using electrostatic interactions, and the resulting nanotube-PEI complex, having a positively charged surface exhibited intracellular uptake. Furthermore, the nanotube-PEI complex exhibited the capability for DNA complexation with reduced enzymatic degradation, and also showed higher transfection efficiency and lower cytotoxicity than PEI. Therefore, Den-CD-NTs, in simple complexation with a surface PEI layer, are potentially useful gene delivery vectors.