Fungal and plant materials
The single-spore isolate W01 of B. cinerea used in this study was collected in 2010 from a vineyard of the Vitis interspecific hybrid cv. Vignoles located at the New York State Agricultural Experiment Station near Geneva, NY using the technique that previously described by Saito et al. (2009). The isolate was maintained on potato dextrose agar (Difco, Franklin Lakes, NJ, USA) and fungal mycelium for DNA extraction was obtained by scraping the surface of 7–10-day-old cultures. Extraction of DNA is described later.
Plant material was collected from pea-sized berries of the ‘Pixie’ mutant of V. vinifera cv. Pinot Meunier, grown from green cuttings in a growth chamber. Berries with receptacles were harvested. The receptacles were separated from the berries using sterile secateurs to obtain the desired weight of each tissue type and then frozen in liquid nitrogen. Samples were stored at −80°C until use. The absence of internal latent B. cinerea in the plant material was confirmed by plating some berries and receptacles on potato dextrose agar medium at 25°C for a week, on which no growth was detected.
Yield and purity of DNA
Berry or receptacle tissue (100 mg) previously frozen and stored at −80°C was placed in a 2-mL centrifuge tube with a 5-mm stainless-steel bead (OPS Diagnostics, Lebanon, NJ, USA) and ground by shaking in a TissueLyser (Retsch Inc., Newtown, PA, USA) for 1 min at 30 cycles/s. A preliminary experiment indicated a yield of DNA with the TissueLyser grinding method similar or higher than that obtained by grinding tissues to a fine powder in liquid nitrogen with a mortar and pestle (data not presented). TissueLyser was also selected because it enables a higher sample throughput (up to 48 at a time) than manual grinding.
Three methods were compared for the extraction of DNA from young grape berries. The first (I) was a slight modification of that described by Cadle-Davidson (2008), substituting a 2-mL centrifuge tube for a 96-well plate. The second (II) was described by Celik et al. (2009), and the third (III) was a slight modification of that described by Saito et al. (2009) and was used for both plant and fungal DNA extraction. Briefly, the ground tissue was transferred into a 1.5-mL microcentrifuge tube and was mixed with 500 μL of cetyltrimethylammonium bromide (CTAB) extraction buffer (2% CTAB, 100 mM Tris-HCl pH 8.0, 20 mM ethylenediaminetetraacetic acid (EDTA), 1.4 M NaCl, 1% polyvinylpyrrolidone). The mixture was then shaken vigorously with a vortex mixer and heated for 15 min at 65°C. Five-hundred microlitres of chloroform-isoamyl alcohol (24:1, v:v) was added. After further vigorous shaking, the tube was centrifuged at 12 000 rpm for 5 min, and the supernatant was transferred to a new microcentrifuge tube. The chloroform-isoamyl alcohol centrifugation and supernatant transfer were repeated. The supernatant was transferred to a new microcentrifuge tube, and a 65°C solution of 10% CTAB with 0.7 M NaCl was added at a rate of 1:10 (v/v). A third chloroform-isoamyl alcohol extraction and centrifugation was performed, and the resulting supernatant was transferred to a new microcentrifuge tube, to which was added an equal volume of cold (approximately 0°C) isopropanol and a 10% volume of 3 M sodium acetate, followed by centrifugation at 12 000 rpm for 5 min at 4°C. After centrifugation, the pellet was washed with 70% (v/v) ethanol, air dried and resuspended in 100 μL of sterile distilled water.
Each DNA extraction method was applied to two independently collected tissue samples, and this process was repeated three times resulting in a total of six DNA solutions per extraction method. The yield and purity of extracted DNA were determined spectrophotometrically by a NanoDrop instrument (NanoDrop Technologies, Wilmington, DE, USA) and normalised to the desired concentration.
Absorbance ratios A260/A280 and A260/A230 were calculated to indicate contamination by protein and by polyphenols and carbohydrates, respectively (Manning 1991, Varma et al. 2007). A ratio of 1.8–2.0 for A260/A280 and a ratio >1.8 for A260/A230 indicate DNA with little contamination (Yeates et al. 1998).
The purity of each DNA solution was determined further by testing amplification of V. vinifera resveratrol synthase gene I, using the probe set reported by Valsesia et al. (2005) with the fluorescent reporter 6-carboxyfluorescein (FAM) being substituted for hexachlorofluorescein (HEX). The sequences are: Res F: 5′-CGA GGA ATT TAG AAA CGC TCA AC-3′; Res R: 5′-GCT GTG CCA ATG GCT AGG A-3′; Res P-HEX: 5′-HEX-TGC CAA GGG TCC GGC CAC C-BHQ-2-3′. Vitis vinifera DNA (10 ng/μL) was serially diluted (from 10 ng/Ll to 0.01 ng/μL) in sterile water. The qPCR was conducted using a 10 μL total volume in a well of a 96-well PCR plate which contained 1 unit of HS Taq (Takara Ex Taq DNA Polymerase Hot Start Version; Takara Bio Inc., Shiga, Japan), 1 μL of 10 × Ex Taq Buffer, 0.4 μL of deoxynucleotide triphosphate (dNTP) mixture (2.5 mM each), 1 μL DNA (desired concentration), 150 nM probe and 500 nM each primer. After incubation at 95°C for 3 min, 40 cycles of a two-step amplification were run at 95°C for 15 s and 60°C for 45 s using a Bio-Rad iQ cycler system (Bio-Rad, Hercules, CA, USA). Each DNA sample was assayed twice with qPCR, resulting in two standard curves. The slopes of the efficiency equation of the two standard curves were subjected to an analysis of covariance (ANCOVA) using PROC GLM in SAS (v. 9.3; SAS Institute, Cary, NC, USA). If there was no significant difference between the two slopes, all data were pooled to create one standard curve.
Comparison of HS Taq and a pre-assembled reaction mixture
Pre-assembled reaction mixtures purchased ‘off the shelf’ are convenient; this convenience, however, may correspond to a higher cost per reaction than those prepared from individual reagents. The HS Taq mixture was compared with a widely used, pre-assembled mixture (iQ Supermix, Bio-Rad, Hercules, CA, USA) to check if this cheaper mixture could be used routinely without any loss in the quality of the qPCR. For qPCR with iQ Supermix, 5 μL 2 × iQ Supermix was added to the 10 μL reaction, instead of HS Taq, Ex Taq buffer and dNTP mixture. Cycling conditions were as previously mentioned, and qPCR assays were performed twice.
Effect of grape DNA on amplification of fungal DNA and PC
Concurrent amplification of B. cinerea and the V. vinifera target DNAs in a duplex qPCR was tested using the V. vinifera probe set (Valsesia et al. 2005) and the Bc3 probe set. Botrytis cinerea DNA (10 ng/μL) was serially diluted in sterile water as before. One μL of each B. cinerea DNA dilution was mixed with 1 μL of V. vinifera DNA (20 ng/μL), producing a tenfold dilution series (from 1:2 to 1:20 000 w/w pathogen : grape DNA). This dilution series was established during preparation of the duplex qPCR mixtures as follows: a 10 μL reaction volume contained 1 unit of HS Taq, 1 μL of 10 × Ex Taq Buffer, 0.5 μL of dNTP mixture (2.5 mM each), 1 μL of each normalised grape and pathogen (diluted) DNA, 150 nM V. vinifera probe, 150 nM B. cinerea (Bc3) probe, 100 nM each V. vinifera primer and 500 nM each B. cinerea (Bc3) primer. The mixtures were amplified under the same conditions as described earlier. The qPCR experiment was performed twice with three replicates for each dilution of B. cinerea DNA in V. vinifera DNA. Data for all six replicates for each dilution were pooled if there was no significant difference between the slopes according to ANCOVA as described earlier.
For each duplex assay, the PC is the Ct number for the V. vinifera gene, amplified from a constant amount of grape DNA, divided by that for B. cinerea DNA, which is likely to vary among samples. In short, PC = CtV. vinifera/CtB. cinerea (Valsesia et al. 2005).
Quantification of B. cinerea DNA in the receptacle
Conidia were harvested by flooding 2-week-old cultures of B. cinerea on 9-cm diameter petri dishes with sterile distilled water and suspended in sterile distilled water. The resultant conidial suspension was filtered through autoclaved gauze; the conidial concentration was then quantified microscopically with a haemocytometer and diluted to the desired concentration described later. Conidial suspensions were prepared immediately prior to inoculation and placed on ice until use.
One 2 μL droplet of a conidial suspension, providing a total of either 10, 50, 250 or 1250 conidia, was inoculated onto 50 mg of the receptacle dissected from Pixie berries (derived from approximately three individual berries), and the plant material was then immediately ground as described earlier. DNA was extracted using method (III) for application of the duplex assay and calculation of the PC as described earlier. This test was performed twice.