Cropping intensity and organic amendments in transitional farming systems

Population levels of fluorescent species of Pseudomonas were used as indicators for disease suppressiveness of soils resulting from three cropping system and three organic amendment treatments as described in the project proposal. The soil samples used for Pseudomonas fluorescens quantification were collected using a 4.8 cm diameter soil corer inserted into the soil to a depth of 30 cm. Each core was fractionated into two depths, 0-15 cm and 15-30 cm. Two soil cores were collected from: 1) planting beds (high intensity system) or 2) approximately the 6th row from the edge of the plot (the intermediate intensity system) and 3) approximately 1.83–2.44 m into the plot, about the same distance into the plot as was sampled in the other two systems (low intensity system). Soil cores from the same split-plot were combined into a composite sample. Sieved, moist soil samples were stored at -20 oC until further process. The 0-15 cm depth samples were used for P fluorescens quantification. The dates for samples used in this study were sampled on June-7-04, May-11-05, June-05-06. DNA was extracted from 1.5 grams of soil samples using a FastDNA® Spin kit following the manufacturer’s procedure, and then cleaned up with hexadecyltrimethylammonium bromide (CTAB) (Ausubel et al., 1995) as mentioned in Riesenfeld et al. (2004). Briefly, adjust the NaCl concentration of DNA extract to 0.7 M, then add 0.1 volume of warm CTAB. Extract with 1 volume of chloroform:isoamyl alcohol (24:1). Precipitate with 2 volumes of cold 100% alcohol, and then washed with 70% alcohol. Resuspended in 100 ml of sterile double distilled water (ddH2O). The DNA concentration then was measure by Nanodrop ND-1000 Spectrophotometer. QPCR standards were made from a pure culture of Pseudomonas fluorescens, pf-5, provided by Dr. Linda Thomashow. Fresh single colony of pf-5 grown on a luria broth plate (LB plate) was transfered to luria broth and allowed to incubate overnight. The culture broth was centrifuged to pellet the cells, which was then washed in PBS. The pellet was resuspended in 750 ml TE buffer, and transfer to bead beating. Following cell disruption in the bead beating apparatus, the resulting solution was mixed using a vortex mixer on the highest speed for 2 min. Extract with equal volume of phenol/chloroform/isoamyl alcohol, then extract again with equal volume of chloroform/isoamyl alcohol. Precipitate DNA with 200 ml volume of 10.5 M sodium acetate) and 900 ml of isopropanol. Let stand 10 minutes at room temperature, then 2 hours at -80 oC. Centrifuge 30 min at 12000 rpm. Wash pellet with 500 ml of 70% ethanol. Resuspend in 200 ml TE buffer. The DNA extract was diluted ten times and measured by Nanodrop to be 140 ng/ml after dilution. 1.5 ml of 2x108 CFU/ml yields 200 ml of 1400 ng/ml. Pseudomonas-specific primer pair, Primer I (5’-GAGTTTGATCCT-GGCTCAG-3’) and primer II (5’-CCTTCCTCCCAACTT-3’) were ordered from Integrated DNA Technologies, Inc. (Coralville, IA, USA). The primers target a 440 bp region of the 16S rRNA gene (Johnsen et al., 1999). Serial dilution of standards in 140 ng/ml, 27 ng/ml, 5.4 ng/ml, 1.08 ng/ml, 0.216 ng/ml, and 0.0432 ng/ml were used. Real time PCR reaction for fluorescens Pseudomonads was performed by DNA engine OpticonTM using Qiagen QuantiTech SYBR green PCR kit following the manufacturer’s protocol. Reaction mixture (50 ml) consisted of 1 ml of serial dilution of standards, 12.5 ml of 2xQuantiTech SYBR green master mix, Primer I and Primer II with final concentration of 300 nM, and H2O to make up 25 ml in total for each reaction. PCR amplification program was set following the manufacturer’s protocol, except the following modification: 30 seconds at 50°C for annealing, and 30 seconds at 72°C for extension. The number of cycles was 41. At the end, the melting curve was generated from 60 – 95 oC read every 0.2 oC increment and hold 2 seconds. The melting curves were checked individually to make sure that the reactions were correct without error. The r2 threshold value of the standards is 98.5%. STATISTICAL ANALYSIS Data of bioassays were compared by mixed models (PROC MIXED) and differences between means were tested with least-squares means (LSMEANS) using SAS 9.0 program (SAS Institute, Inc.). Within each year, the system is treated as split plot design that “block” is the random effect, “cropping system” is the fixed effect for whole plot factor and “organic amendment” is the fixed effect for sub-plot factor (Lochinkohl &Boehm, 2001). To compare the effect of number of years since the beginning of transition on disease suppression, data from each year was normalized to avoid greenhouse condition fluctuation. Normalization was conducted by dividing the data with corresponding average derived from all the autoclaved soil samples at the same time the samples were assayed. The assumption is that bioassays in autoclaved soil provide a baseline for normalization of the greenhouse condition fluctuation. The same PROC MIXED procedure was used to analyze the data.