SOIL MICROBIAL COMMUNITY RESISTANCE TO NPK FERTILIZATION AND RESILIANCE TO FERTILIZATION WITH DICYANDIAMIDE-TREATED SWINE SLURRY

The experiment was carried out in the Federal University of Santa Maria (UFSM), Rio Grande do Sul State, Brazil (29°43’ S, 53°43’ W, altitude 105). The field was cultivated during 2 years before soil sampling (2011 and 2012) with a corn/cereal succession (corn/oat/corn/wheat) under a no-tillage system. a composite soil sample (mixture of 8 random points in each plot to form a single composite sample) was taken of the top soil (0-5 cm) from the triplicate plots. Pre-experiment soil samples were collected one day before application of treatments, when there were 6.1 t ha-1 of wheat residues (Triticum aestivum L.; cv. Quartz) on the soil surface. Four days after treatment application, maize (Zea mays L.; hibrid 32R22 Herculex) was sown manually, with an interrow distance of 0.7 m. After maize was sown, soil samples were taken on days 3, 6, 11, 25 and 50 after maize seedling for molecular analyses. The samples were immediately placed in liquid nitrogen and stored at -80ºC until total RNA extraction. The PowerSoil Total RNA Isolation Kit (MO-BIO Laboratories, Inc., CA) was used to extract the total RNA from two grams of soil per sample (n = 72) by following the manufacturer’s instructions. The V4 region of the 16S rRNA gene was amplified and sequenced using the PGM Ion Torrent (Life Technologies) using archaeal/bacterial primers 515F and 806R. Multiple samples were PCR-amplified using barcoded primers linked with the Ion adapter “A” sequence (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′) and Ion adapter “P1” sequence (5′-CCTCTCTATGGGCAGTCGGTGAT-3′) to obtain a sequence of primer composed for A-barcode-806R and P1-515F adapter and primers. Five runs were performed. The raw sequences and the barcodes used are provided as follow: Chp1.fastq (TCCCTTGTCTCC-Control_0; ACGAGACTGATT-Control_0; GCTGTACGGATT-Control_0; ATCACCAGGTGT-NPK_0; TGGTCAACGATA-NPK_0; ATCGCACAGTAA-NPK_0; GTCGTGTAGCCT-Slurryswine_0; AGCGGAGGTTAG-Slurryswine_0; ATCCTTTGGTTC-Slurryswine_0; TACAGCGCATAC-Slurryswine_DCD_0; ACCGGTATGTAC-Slurryswine_DCD_0; AATTGTGTCGGA-Slurryswine_DCD_0; TGCATACACTGG-Control_3; AGTCGAACGAGG-Control_3; ACCAGTGACTCA-Control_3; GAATACCAAGTC-NPK_3; GTAGATCGTGTA-NPK_3; TAACGTGTGTGC-NPK_3; CATTATGGCGTG-Slurryswine_3; CCAATACGCCTG-Slurryswine_3); Chip2.fastq (TCCCTTGTCTCC-Slurryswine_3; ACGAGACTGATT-Slurryswine_DCD_3; GCTGTACGGATT-Slurryswine_DCD_3; ATCACCAGGTGT-Slurryswine_DCD_3; GTCGTGTAGCCT-Control_6; TACAGCGCATAC-NPK_6; ACCGGTATGTAC-Slurryswine_6; AATTGTGTCGGA-Slurryswine_6; TGCATACACTGG-Slurryswine_6; AGTCGAACGAGG-Slurryswine_DCD_6; ACCAGTGACTCA-Slurryswine_DCD_6; GTAGATCGTGTA-Control_12; TAACGTGTGTGC-Control_12) Chip3.fastq (TCCCTTGTCTCC-NPK_12; ACGAGACTGATT-NPK_12; GCTGTACGGATT-Slurry_swine_12; ATCACCAGGTGT-Slurry_swine_12; TGGTCAACGATA-Slurry_swine_12; ATCGCACAGTAA-Slurry_swine_DCD_12; GTCGTGTAGCCT-Slurry_swine_DCD_12; AGCGGAGGTTAG-Slurry_swine_DCD_12; ATCCTTTGGTTC-Control_25; TACAGCGCATAC-Control_25; ACCGGTATGTAC-Control_25; AATTGTGTCGGA-NPK_25; TGCATACACTGG-NPK_25; AGTCGAACGAGG-NPK_25; ACCAGTGACTCA-Slurry_swine_25; GAATACCAAGTC-Slurry_swine_25; GTAGATCGTGTA-Slurry_swine_25; TAACGTGTGTGC-Slurry_swine_DCD_25; CATTATGGCGTG-Slurry_swine_DCD_25; CCAATACGCCTG-Slurry_swine_DCD_25) Chip4.fastq (TCCCTTGTCTCC-Control_50; ACGAGACTGATT-Control_50; GCTGTACGGATT-Control_50; ATCACCAGGTGT-NPK_50; TGGTCAACGATA-NPK_50; ATCGCACAGTAA-NPK_12; GTCGTGTAGCCT-Slurry swine_50; AGCGGAGGTTAG-Slurry swine_50; ATCCTTTGGTTC-Slurry swine_50; TACAGCGCATAC-Slurry swine _ DCD_50; ACCGGTATGTAC-Slurry swine _ DCD_50; AATTGTGTCGGA-Slurry swine _ DCD_50; TGCATACACTGG-Slurry_input; AGTCGAACGAGG-Slurry_input; ACCAGTGACTCA-Slurry_input; GAATACCAAGTC-Slurry_input_DCD; GTAGATCGTGTA-Slurry_input_DCD; TAACGTGTGTGC-Slurry_input_DCD) Chip5.fastq (TGGTCAACGATA-Control_6; ATCGCACAGTAA-Control_6; AGCGGAGGTTAG-NPK_6; ATCCTTTGGTTC-NPK_6; ACCGGTATGTAC-Slurry swine _ DCD_0; AATTGTGTCGGA-NPK_25; TGCATACACTGG-NPK_25; ACCAGTGACTCA-Slurry swine_25; GAATACCAAGTC-Slurry swine _ DCD_6; TAACGTGTGTGC-Control_12; CATTATGGCGTG-Control_12; CCAATACGCCTG-NPK_12)