Long-term Crop Rotation Study for Greenhouse gas Reduction through Agricultural Carbon Enhancement network in Lubbock, Texas
Lubbock TX, 2. Material and Methods 2.1. Cropping Systems and Tillage Treatments This dryland research study was established at the USDA-ARS farm near Lubbock, TX at latitude 33.68° and longitude -101.77°. Prior to the initiation of this study, the land (4 ha) was fallow during fall of 2001, cotton was planted in summer 2002, and rye was grown from December 2002 to April 2003. The soil is an Olton sandy loam (Fine, mixed, superactive, thermic Aridic Paleustolls) with an average of 16.4% clay, 67.6% sand, and 0.65 g kg-1 of organic matter (OM) content at the beginning of the study. In summer 2003, the land was divided into three field replicates of a split-plot design experiment with cropping systems as the main treatment and tillage as subplots. Each field replicate was 64.6 m wide and 210 m long. In brief, Srg-Ct represents a rotation of cotton and grain sorghum without a winter cover crop (fallow periods). The Ct-Rye-Srg rotation involves growing either cotton or grain sorghum during the summer with a winter cover crop (rye). The Srf-Rye rotation represents a high biomass cropping system with high residue crops during summer (forage sorghum also known as haygrazer) and winter (rye), which does not include a cotton crop because our purpose was to investigate the maximum impact achievable on the soil properties. All systems were under no-tillage (nt) or conventional tillage (ct). In the no-tillage (nt) treatments, there is no soil disturbance, and the summer crop residues remain on the soil surface. For example, forage sorghum and grain sorghum were layed-down by grain drill and left on the surface; whereas, cotton stalks remained standing. Conventional tillage (ct) was accomplished every fall using a shredder and moldboard plow equipment to incorporate the summer crops residues up to 15 cm before planting the winter cover crops. The moldboard equipment was also used to raise beds (101 cm row spacing) to plant the rye every fall (using a drill at 62 kg ha-1) on the tilled treatments. Every year, depending on precipitation and wind storms, beds may be prepared (bed prep) again before planting (same day) in May for rotations under this tillage treatment. 2.2. Projected General Management and Soil Sampling Crops were planted in May every year. The forage sorghum (also known as haygrazer) variety was Pacesetter, which is typically produced for cattle feed, and planted at a rate of 16.8 kg ha-1 with a drill in 43 cm spacing. The cotton and grain sorghum varieties were Paymaster 23-26rr and K35-Y5, respectively, and were generally planted at 101.6 cm row spacing. Cotton was planted at a rate of 9–11 kg ha-1. Grain sorghum was generally planted at a rate of 3 kg ha-1. Pesticides and fertilizer were applied after precipitation events, which is a typical practice for dryland management. The herbicides used to control weeds were Markman® (2.34 L ha-1) for grain or forage sorghum and Round-up® (2.34 L ha-1) for cotton. Cotton was generally chemically terminated around mid October using 1.17 L Cyclone ha-1. When precipitation was sufficient, the winter cover crop (rye) was planted in December and terminated during April of the next year using Round-up® at 2.3 L ha-1 in Ct-Rye-Srg and Srf-Rye rotations. In general, during the 5 years prior to our sampling, the projected management was not possible every year due to significant climatic variations reflected in certain years with lack of fertilization (i.e., 2003, 2007), summer crop failure (2003, 2006) and only few weeks of winter cover crops (i.e., 2005) for cropping systems that apply. Soil samples were taken from each of three field replicates available for each cropping system (Srg-Ct, Ct-Rye-Srg and Srf-Rye) and tillage (nt and ct) treatment combination. Two composite (0–10 cm depth) soil samples were taken across each field replicate plot (210 m), one from the north side and another from the south side (n = 6 per treatment; 2 samples per treatment plot × 3 field replicate plots). This sampling occurred in November 2007 after harvest of cotton (Ct-Rye-Srg), grain sorghum (Srg-Ct) and forage sorghum (Srf-Rye), which represented the end of the 5 year study. All samples from each treatment (n = 6) were analyzed for FAME and all soil properties evaluated, however, only 3 samples per treatment were analyzed for pyrosequencing. 2.3. Selected Soil Properties Total C, organic C, and total N were determined in air-dried soil samples in a private laboratory (Ward Laboratories, Nebraska) by automated dry combustion (LECO TruSpec CN) [26,27]. Soil pH was measured in the air-dried soil (<5 mm) using a combination glass electrode (soil: water ratio, 1:2.5). Microbial biomass C (MBC) and N (MBN) were determined in field-moist soil (15-g oven-dry equivalent ) by the chloroform-fumigation-extraction method [28,29]. In brief, organic C and N from the fumigated (24 h) and non-fumigated (control) soil were quantified using a CN analyzer (Shimadzu Model TOC-V/CPH-TN, Shimadzu Corporation, Japan). The MBC and MBN (difference between fumigated and non-fumigated values) were calculated using a kEC factor of 0.45  and kEN factor of 0.54 , respectively. Each sample had duplicate analyses and results are expressed on a moisture-free basis. Enzyme activities important for C (ß-glucosidase, a-galactosidase), C and N (ß-glucosaminidase), P (i.e., alkaline phosphatase, phosphodiesterase) and S (arylsulfatase) cycling were evaluated using 1 g of air-dried soil (<5 mm) with their appropriate substrate and incubated for 1 h (37 oC) at their optimal pH as described previously [32,33]. 2.4. Microbial Community according to FAME Profiling The FAME-MIDI method was used to extract fatty acids from the field-moist soil samples (3-g oven-dry equivalent) following the MIDI (Microbial ID, Inc., Newark, DE, USA) protocol as previously applied to soils . In brief, the four steps of the MIDI protocol applied on the are: (1) saponification of fatty acids at 100 °C with 3 mL of 3.75 M NaOH in aqueous methanol [methanol : water ratio = 1:1] for 30 min; (2) methylation (esterification) at 80 °C in 6 mL of 6 M HCl in aqueous methanol [1:0.85] for 10 min; (3) extraction of the FAMEs with 3 mL of 1:1 [vol.:vol.] methyl-tert-butyl ether:hexane; and (4) washing of the solvent extract with 1.2% [wt./vol.] NaOH. The FAME-EL method was performed as described by Schutter and Dick  using also 3 g of fresh soil (oven dried basis) as for the FAME-MIDI method described above. This method also involves 4 steps: (1) saponification and methylation of ester-linked fatty acids by incubation of 3 g of soil in 15 mL of 0.2 M KOH in methanol at 37 oC for 1 h. During that time, the samples are vortexed every 10 min, and addition of 3 mL of 1.0 M acetic acid to neutralize the pH of the mixture at the end of incubation, (2) FAMEs were partitioned into an organic phase by adding 10 mL of hexane followed by centrifugation at 480 × g for 10 min; (3) the hexane layer is transferred to a clean glass test tube and the hexane can be evaporated under a stream of N2 , and (4) In the final step, FAMEs are dissolved in 0.5 mL of 1:1 hexane:methyl-tert butyl ether and transferred to a GC vial for analysis. For both methods, FAMEs were analyzed in a 6890 GC Series II (Hewlett Packard, Wilmington, DE, USA) equipped with a flame ionization detector and a fused silica capillary column (25 m × 0.2 mm) using H2 (ultra high purity) as the carrier gas. The temperature program was ramped from 170 °C to 250 °C at 5 °C min-1. Fatty acids were identified and quantified by comparison of retention times and peak areas to components of MIDI standards. The MIDI software provides FAME relative peak areas (percentage) based on the total FAMEs in a sample (based on the Aerobe method of the MIDI system). FAME concentrations (nmol g-1 soil) were calculated by comparing peak areas to an analytical standard (19:0, Sigma Chemical Co., St. Louis, MO) calibration curve. The FAMEs are described by the number of C atoms, followed by a colon, the number of double bonds and then by the position of the first double bond from the methyl (?) end of the molecule. Cis isomers are indicated by c, and branched fatty acids are indicated by the prefixes i and a for iso and anteiso, respectively. Other notations are Me for methyl, OH for hydroxy and cy for cyclopropane. 2.5. Pyrosequencing DNA was extracted from approximately 0.5 g of soil (oven dry basis of field-moist soil) using the Fast DNA Spin Kit for soil (QBIOgene, Carlsbad, CA, USA) following the manufacturer’s instructions. The DNA extracted (1uL) was quantified using Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). The integrity of the DNA extracted from the soils was confirmed by running DNA extracts on 0.8% agarose gel with 0.5X TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0). All DNA samples were diluted to 100 ng/µL for a 50 µL PCR reaction. The 16S universal Eubacterial primers 530F (5’-GTG CCA GCM GCN GCG G) and 1100R (5’-GGG TTN CGN TCG TTG) were used for amplifying the ~600 bp region of 16S rRNA genes. HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA, USA) was used for PCR under the following conditions: 94 °C for 3 minutes followed by 32 cycles of 94 °C for 30 seconds; 60 °C for 40 seconds and 72 °C for 1 minute; and a final elongation step at 72 °C for 5 minutes. A secondary PCR (6 cycles rather than 32) was performed for FLX Amplicon Sequencing under the same condition by using designed special fusion primers with different tag equences as: LinkerA-Tags-530F and LinkerB-1100R . After secondary PCR, all amplicon products from different samples were mixed in equal volumes, and purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, MA, USA). Pyrosequencing was used to characterize primary predominant bacterial populations. In preparation for FLX sequencing (Roche, Nutley, NJ, USA), the size and concentration of DNA fragments were accurately measured using DNA chips under a Bio-Rad Experion Automated Electrophoresis Station (Bio-Rad Laboratories, CA, USA) and a TBS-380 Fluorometer (Turner Biosystems, CA, USA). A 9.6 × 106 sample of double-stranded DNA molecules/µl with a size of 625 bp were combined with 9.6 million DNA capture beads, and then amplified by emulsion PCR. After bead recovery and bead enrichment, the bead-attached DNAs were denatured with NaOH, and sequencing primers were annealed. A two-region 454 sequencing run was performed on a 70 × 75 GS PicoTiterPlate (PTP) by using a Genome Sequencer FLX System (Roche, Nutley, NJ, USA). It should be noted that 100 total samples were run within this same FLX 2-region sequencing reaction Pico Plate. All FLX related procedures were performed following Genome Sequencer FLX System manufacturers instructions (Roche, Nutley, NJ, USA). Thus, moderate diversity pyrosequencing analysis (2,000–3,000 reads per sample) was performed at the Research and Testing Laboratory (Lubbock, TX, USA). A custom script written in the C# within the Microsoft® .NET development environment (Microsoft Corp, Seattle, WA, USA) was utilized to generate all possible combinations of 10-mer oligonucleotide tags with GC % between 40 and 60% . Individual tags were chosen to label our samples. Custom software developed within the Microsoft® .NET environment (Microsoft Corp, Seattle, WA, USA) was also utilized for all post sequencing processing [22,34]. In-depth discussion of software code is outside the scope of this report; however, a description of the algorithm follows. Quality trimmed sequences obtained from the FLX sequencing run were processed using a custom scripted bioinformatics pipeline as depicted in Acosta-Martinez et al. . In short, each sequence was trimmed back to utilize only high quality sequence information, tags were extracted from the FLX generated multi- FASTA file, while being parsed into individual sample specific files based upon the tag sequence. Tags which did not have 100% homology to the original sample tag designation were not considered as they might be suspect in quality. Sequences which were less than 200 bp after quality trimming were not considered. Samples were then depleted of definite chimeras using B2C2 software, described by Research and Testing Laboratory (Lubbock, TX, USA; www.researchandtesting.com/B2C2.html). The resulting sequences were then evaluated using BLASTn  against a custom database derived from the RDP-II database and GenBank (http://ncbi.nlm.nih.gov) [22,36]. The sequences contained within the curated 16S database were those considered of high quality based upon RDP-II standards  and which had complete taxonomic information within their annotations. Following best-hit processing, a secondary post-processing algorithm was utilized to combine genus and other taxonomic designations generating data with relative abundance of each taxonomic entity within the given sample, and phylogenetic assignments were based upon NCBI taxonomic designations.Cropping systems and tillage treatments This dryland research study was established at the USDAARS farm near New Deal, TX, USA (33°42' N, 101°49' W and average elevation of 960 m above sea level). Prior to this study, the land (4 ha) was fallow during fall of 2001, cotton was planted in summer 2002, and rye was grown from December 2002 to April 2003. The soil is classified as Olton sandy loam (fine, mixed, superactive, thermic Aridic Paleustolls) with an average of 16.4% clay, 67.6% sand, 656 Biol Fertil Soils (2011) 47:655–667 and 0.65 g kg-1 OM. In summer 2003, the field was divided into three replicates of a split-plot design, with cropping systems as the main treatment and tillage as subplots (each plot was 210 m in length). A description of the cropping systems and tillage subplots follows. Three cropping systems in order of increasing cropping intensity (CI) are described as follows: 1. Srg–Ct Rotation of cotton and sorghum without a winter cover crop. This rotation represents about 50% of CI because of the fallow periods without a winter cover crop. 2. Ct–Rye–Srg Rotation of cotton and sorghum every summer with a winter cover crop (rye). This rotation represents about 100% of CI because of using a summer and a winter crop during the year (when possible). 3. Srf–Rye High biomass cropping system with high residue crops during summer and winter, representing about 100% of CI. This system did not include a cotton crop as it has been previously reported that cotton produces less residue per hectare than the other major crops (Unger and Parker 1976; Lal 2004), and thus our objective was to investigate the maximum impact achievable on the soil properties. The two tillage treatments are described as follows: 1. No tillage The summer above-ground crop residues were left undisturbed on the soil surface. Forage sorghum (haygrazer) and grain sorghum were laid down by grain drill and left on the surface, whereas cotton stalks remained standing. 2. Conventional tillage Summer crops were shredded and incorporated while mixing the soil up to 15 cm after harvest every fall. For treatments with a winter cover crop, plots were listed to create beds, 1 m apart, and rye was planted every fall at 62 kg ha-1 on respective treatments. Beds were prepared (bed prep) again before planting (same day) every May for rotations under this tillage treatment. The cropping systems and tillage practices mentioned earlier were replicated three times using a randomized block design. As a baseline, we used the common cropping system in the THP, i.e., tilled cotton monoculture (Ct–Ct) from nearby research plots on the same soil. General crop management Crops were generally planted in May. The forage sorghum (haygrazer) variety Pacesetter1 was planted at a rate of 16.8 kg ha-1 with a drill at 0.4-m spacing. Cotton, variety Paymaster 23-26rr, was planted at a rate of 9–11 kg ha-1 and grain sorghum, variety K35-Y5, was planted at a rate of 3 kg ha- 1, both on 1-m row spacing. Pesticides and fertilizer were applied after rainfall events, which is a typical management practice for dryland crops in the THP. For weed control, Marksman® (Dicamba and Atrazine) herbicide was sprayed on forage and grain sorghum in July (2.34 L ha-1), and Round-up® (Glyphosate) herbicide was sprayed on cotton (2.34 L ha-1). Cotton was generally chemically terminated and defoliated around mid-October using 1.17 L Cyclone® (Paraquat dichloride) ha-1. When rain was sufficient, the winter cover crop (rye) was planted in December and terminated during April of the next year using the herbicide Round-up® (Glyphosate) at 2.3 L ha-1 in Ct–Rye–Srg and Srf–Rye rotations. Climate data and crop measurements Climate data (precipitation and air temperatures) were obtained from the weather station located near these research plots. Crop biomass (cotton and forage and grain sorghum) was determined three times during the year based primarily on the growth stages of cotton since this crop has more definitive growth stages than forage or grain sorghum: first square in mid-June, first bloom the first week of July (when at least 50% of the plants have flowers), and peak bloom in August. The results from August samplings will be reported in this paper. The crop biomass samples were randomly taken from a 1-m2 area from the three field replicates of a treatment plot at three locations (north, center, and south part) in each plot (n=9). Biomass samples were taken from the winter cover crop following the same protocol of the summer crops. Soil sampling and measurements Soil samples at 0–10 cm in depth were collected using a hand auger. For each plot, composite samples were taken from the south and north end of the field, for a total of six samples per cropping system and tillage treatment combination (two soil samples per plot × three field replicates). The samples were taken in November 2003, 2005, and 2007, representing the start and 3 and 5 years of the study, respectively. Additionally, soil samples were taken in July 2005 and these were used to evaluate the changes in MB and EAs during and after the 2005 growing season. Soil samples, used for baseline, were taken from a Ct–Ct under ct from a nearby field (n=3). For soil MB analyses, the soil samples were sieved (<5 mm) and stored at 4°C until analyses were performed within the next 2 weeks. Soil gravimetric water content was determined after drying the samples at 105°C for 48 h. The MBC and MBN contents were determined on fieldmoist soil (15 g oven-dry equivalent) samples by the chloroform fumigation–extraction method using 0.5 M K2SO4 as an extractant (Brookes et al. 1985; Vance et al. 1987). Briefly, organic C and N extracted from the fumigated (24 h) and non-fumigated (control) soil were quantified by a CN analyzer (Shimadzu Model TOCV/CPH-TN, Shimadzu Corp., Kyoto, Japan). The MBC and MBN, difference between fumigated and non-fumigated values, were calculated using a kEC factor of 0.45 (Wu et al. 1990) and kEN factor of 0.54 (Jenkinson 1988), respectively. The EAs, ß-glucosidase, a-galactosidase, ß- glucosaminidase, alkaline phosphomonoesterase, phosphodiesterase, and arylsulfatase, were assayed using 1 g of airdried soil with their appropriate substrate and incubated for 1 h (37°C) at their optimal pH as described by Tabatabai (1994) and Parham and Deng (2000) for ß-glucosaminidase activity. The EAs were assayed in duplicate with one control, to which substrate was added after incubation (product of all reactions is PN= P-nitrophenol). In addition, soil C (organic and total) and total N, P, and “NO3–N” (available N) were determined in air-dried soil samples (Ward Laboratories, Nebraska) by automated dry combustion (LECO TruSpec CN), the Mehlich P-3 method (Mehlich 1984), and 2 N KCl extraction method (Keeney and Nelson 1982), respectively. Soil pH was measured in the air-dried soil (< 5 mm) using a combination glass electrode (soil/water ratio, 1:2.5). Total soil organic matter (OM) content of the soil surface < 2 mm diameter was determined by loss on ignition (LOI) at 400º C as described in NRCS (1996). Bulk density was determined using the core method. Wet aggregate stability was measured on duplicate subsamples of aggregates smaller than 8.0 mm but greater than 4.75 mm in diameter to determine water-stable mean weight diameter (MWD) of the aggregate size distribution and percent > 250 µm diameter by wet sieving according the procedure of Kemper and Rosenau (1986).
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|Spatial / Geographical Coverage Area|
POLYGON ((-101.769359 33.6849, -101.767286 33.6849, -101.767286 33.683784, -101.769359 33.683784))
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License Not Specified
005:037 - Department of Agriculture - Research and Education
005:18 - Agricultural Research Service
|Harvest Source Title||Geodata Harvest|
|Harvest Source URI||https://geodata:NAL2geodata@firstname.lastname@example.org/geonetwork/srv/eng/csw|
|Last Harvest Performed||Tue, 04/10/2018 - 13:22|
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