Soil testing diagnostics

DNA extraction method

5.2.2.1.

Two methods for extracting DNA from soil were tested. The first method used the dilution plating principle and the second method was based on the one described by Yeates et al. (1998). For both methods, a conidium suspension was obtained as described in Section 2.2.1.2 for isolates L1, D2 and M2 (Appendix 1) with final concentrations of 107 conidia /mL. The suspensions were diluted to 105 and 103 conidia /mL with sterile water. Conidia from each species were mixed with 50 g of field soil in deep Petri dishes to achieve a final concentration of 0, 102, 104 or 106 conidia of each species per 10 g of soil.

Method 1

This method was based on the premise of dilution plating whereby it is assumed that when the soil is mixed with water and agar and the soil is allowed to sediment, all (or most) of propagules in the soil will remain suspended in the liquid suspension. For each treatment, 10 g of inoculated soil was placed in a 250 mL bottle and 90 mL of autoclaved distilled water with 0.01% agar was added. The bottles were placed on a wrist action shaker (Griffin and George Ltd., London, UK) and were shaken for 10 min and then left to stand for 3, 6 or 10 min. Two replicates were used for each treatment combination. After standing, approximately 75 mL of each supernatant was placed into two sterile 50 mL centrifuge tubes. The tubes were centrifuged at 3220 × g for 15 min. The supernatant was discarded and the two pellets for each replicate were combined into a single 50 mL centrifuge tube using 5 mL sterile distilled water. The tubes were centrifuged at 3220 × g for a further 15 min and the supernatant was discarded. One third of the pellet was used for DNA extraction with the PowerSoil® DNA isolation kit (MoBio Laboratories, Carlsbad, CA) according to manufacturer‟s instructions. The DNA was suspended in a final volume of 100 µL of TE.

Method 2

diameter glass beads (BioSpec Products, Inc., OK, USA). The bottles were shaken for 8 min on a wrist action shaker (Griffin and George Ltd.) after which, 1 mL of 20% (w/v) sodium dodecyl sulphate (SDS) was added and the bottles were shaken for a further 2 min. The bottles were then incubated at 65ºC for 1 h before the contents were transferred into 50 mL centrifuge tubes. The tubes were centrifuged at 3220 × g for 10 min. The supernatant was transferred to a clean 50 mL centrifuge tube. The pellet was re-extracted with 10 mL of extraction buffer and centrifuged for 10 min at 3220 × g. The supernatant was again transferred into a 50 mL centrifuge tube to which contained half volume of 30% (v/v) polyethylene glycol /1.6 M NaCl. The solutions were incubated at room temperature for 2 h. They were centrifuged at 3220 × g for 25 min, each pellet was suspended in 2 mL of TE (Appendix 2) and sufficient 7.5 M potassium acetate was added to each tube to reach a final concentration of 0.5 M. The solution was placed on ice for 5 min then centrifuged at 3220 × g for 35 min at 4ºC. The aqueous phase was extracted with phenol /chloroform as described by Whiteman (2004). Typically, the supernatant was placed into 15 mL centrifuge tubes and 4 mL each of phenol and chloroform was added and the solutions mixed by inversion five times. The tubes were centrifuged at 600 × g for 4 min. The aqueous phase was placed into a new tube and 4 mL each of phenol and chloroform was again added. The solution was centrifuged at 600 × g for 4 min and the aqueous phase was put into 15 mL centrifuge tubes. To remove residual phenol one volume of chloroform (approximately 2 mL) was added to each tube and they were centrifuged at 600 × g for 4 min. The aqueous phase was placed into a new tube and 1/10 volume of 3 M sodium acetate was added followed by two volumes of ice-cold 100% ethanol. The solution was stored in the freezer (-20ºC) overnight.

Each solution was thawed, separated into two equal portions in 2 mL centrifuge tubes which were centrifuged at -5°C at 20,817 × g for 5 min. Each supernatant was discarded and the pellet was washed twice with 1 mL of 70% ethanol. The tubes were centrifuged at 20,817 × g for 2 min at -5°C. Each pellet was air dried for 15 min and re-suspended overnight in 75 μL of SNW. The DNA solutions derived from the same frozen solution were recombined at this stage. DNA was visualised by electrophoresis on a 1% agarose gel as described in Section 3.2.2.

Nested species specific PCR

5.2.2.2.

An initial PCR using the universal fungal primers ITS4 and ITS1F (Chapter 3, Table 3.1) was conducted to demonstrate that the DNA was suitable for PCR and that it was not inhibited by any contaminants co-purified with the DNA. The PCR reaction mix consisted of 1 × PCR buffer, 200 µM of each dNTP, 0.2 μM of each primer, 1 U of Taq DNA polymerase (Roche), 0.2, 0.5, 1 or 2 μL of the DNA extract and SNW to a final volume of 25 μL.

DNA extracted from soil samples containing no Cylindrocarpon spores at the 3 standing times from Method 1 and the 0 conidium concentration in Method 2 was amplified using either 0.2, 0.5, 1 and 2 µL of DNA to determine whether the DNA amplification was inhibited. A negative control with SNW in place of DNA and a positive control (10 ng of DNA extracted from pure culture of isolates M1, L1 or D2) were used. The PCR conditions were as follows: 94°C for 3 min followed by 30 cycles of 30 s at 94°C, 30 s at 50°C and 30 s at 72°C with a final extension at 72°C for 7 min. PCR products were separated by electrophoresis on a 1.5% agarose gel and visualised under UV light using ethidium bromide staining as described in Section 3.2.2.

The nested PCR developed in Section 3.2.4 was employed using the optimal DNA quantity found above in the first PCR using general tubulin primers and the products were diluted 1/100 for the second (species specific) PCR. The PCR products were sequenced to determine the specificity of the primers and the sequences were aligned on GenBank using BLAST as in Section 3.2.5.

Quantitative PCR

5.2.2.3.

Quantitative PCR was used to increase the detection sensitivity of the PCR and to quantify the numbers of spores in soil.

5.2.2.3.1. DNA extraction from cultures

DNA from isolates L1, L2, D1, D2, M2 and M3 (Appendix 1) was extracted from pure cultures as described in Section 3.2.2. The extracted DNA was visualised by electrophoresis on a 1% agarose gel as described in Section 3.2.2. The extracted DNA was quantified by spectrophotometry using a Nanodrop-ND-1000 spectrometer and was diluted to 30 ng, 3 ng, 0.3 ng, 30 pg, 3 pg and 1 pg of DNA /µL.

5.2.2.3.2. DNA extraction from conidia

A conidium suspension was made as described in Section 2.2.1.2 with a concentration of 106 conidia /mL for isolates L1, L2, M2, M3, D1 and D2. For each isolate, 1 mL of the conidium suspension was placed into a 1.5 mL centrifuge tube and was centrifuged at 20, 817 × g for 15 min. The supernatant was discarded and DNA was extracted using a PowerSoil kit (MO BIO Laboratories Inc.) according to the manufacturer‟s instructions. The resulting DNA sample was serially diluted 10 fold with SNW to achieve solutions of DNA with the equivalent of 101, 102, 103, 104, 105 and 106 conidia per 100 µL.

5.2.2.3.3. DNA extraction from soil over time

A mixed isolate conidium suspension (106 conidia /mL) was obtained as described in Section 2.2.1.2 using a three isolate mixture for each species (D1, D2, D3, L1, L2, L3, M1, M2 and M3) and 500 mL of each conidium suspension was mixed with field soil in 5 L pots to achieve a final concentration of 105 conidia /g of soil. Controls were treated with a similar quantity of water and three replicates were used per treatment. The 5 L pots were sunk into and levelled with the ground in the Lincoln University vineyard in a randomised design. Once a week two 15 g soil samples were taken from each pot, placed individually into 50 mL tubes and stored at -80°C prior to DNA extraction. DNA was extracted from samples at 0, 1, 2, 3 and 6 weeks after set up using Method 1 as described in Section 5.2.2.1.

5.2.2.3.4. Quantitative PCR

Each reaction consisted of 1 × PCR buffer, 200 µM dNTP, 50 pM of each species specific primer (Cyde F1 small /Cyde R2 for C. destructans, Cyma F1 /Cyma R1 for C. macrodidymum and Cyli F1 /Cyli R1 for C. liriodendri; Chapter 3, Table 3.1), 1 U of Taq DNA polymerase (Roche), 0.28 µL diluted Sybr Green I (Invitrogen™, CA, USA; Appendix 2), 0.4 µL Rox (Invitrogen™) and SNW to a final volume of 19 µL. Each reaction mix was placed into a single well on a 96 PCR well plate (Axygen Scientific, CA, USA) on a MicroAmp® splash free 96 well base (Applied Biosystems Inc, CA, USA) and 1 µL of the appropriate sample DNA or in the case of the negative control, SNW, was added to the mix. In addition to sample DNA, standard curves were developed for DNA extracted from cultures (30 ng to 1 pg) and DNA extracted from conidia (106 to 101 conidia per mL). Each PCR reaction was conducted in duplicate. The plate was covered with a PCR plate cover seal and was spun in a centrifuge for 3 min at 400  g. A compression mat was placed onto the plate which was then placed into an ABI Prism 7000 Sequence detection system (Applied Biosystems Inc.). The qPCR optimised in Section 3.2.5 was used for each species. The PCR products were sequenced to determine the specificity of the primers and the sequences were aligned on GenBank using BLAST as in Section 3.2.5.

5.3. Results