2.2.5.1 Amplified ribosomal DNA restriction analysis (ARDRA)
To confirm the identities of all 118 botryosphaeriaceous isolates recovered from the nurseries, the amplified ribosomal DNA restriction analysis (ARDRA) was conducted as described by Alves et al. (2005). DNA was extracted using the REDExtract-N-AmpTM Plant PCR Kit (Sigma Aldrich, Missouri, USA). The isolates were grown on PDA at room
temperature for 3 days and a sterile pipette tip was used to scrape up a tuft of mycelium (approximately 2 mm) from the edge of each culture and to add it to a 1.7 ml tube containing 100 µl of extraction buffer. The mixture was briefly vortexed and incubated at 95°C for 10 min. Then 100 ml of the dilution solution was added to the tube and briefly vortexed to neutralise inhibitory substances present in the extract. The mixture was centrifuged for 2 min at 3,220 x g and the supernatant was transferred to a new 0.6 ml tube and stored at -20°C until used for PCR.
The ribosomal DNA (rDNA) was amplified using the REDExtract-N-Amp™ PCR ready mix (Sigma Aldrich) using the ITS-1 (5‟-TCCGTAGGTGAACCTGCGG-3‟; White et al., 1990) and NL4 (5‟-GGTCCGTGTTTCAAGACGG-3‟; O‟Donnell, 1992) primers following the manufacturer‟s recommendation. Each 20 µl amplification reaction contained 10 µl of the REDExtract-N-AmpTM PCR ready mix, 4 µl of the DNA extract, 1 µl each of each primer (5 µM) and 4 µl of sterile nanopure water (SNW). Negative controls using SNW instead of DNA template were included in every PCR reaction. Samples were briefly vortexed and centrifuged for 3,220 x g for 5 s before placing into the PCR machine (Biorad
Laboratories, California, USA). The thermal cycle was performed with initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation for 1 min at 94°C, annealing for 30 s at 55°C and extension for 1 min at 72°C, followed by a final extension at 72°C for 7 min.
After amplification, 5 ul of every PCR product were separated by electrophoresis (10 V /cm for 45 min) in a 1% agarose gel (Bioline, London, UK) immersed in 1x TAE buffer (40 mM Tris acetate, 2 mM Na2EDTA, pH 8.5). The 1 Kb Plus DNA Ladder™ (0.1 ng/µl) (Invitrogen, California) molecular weight marker was run in the first or last lane of each gel. The agarose gels were transferred to plastic containers containing ethidium bromide (0.5 µg/ml) and allowed to stain in a shaker for 15 min, then destained in water for 10 min. Stained gels were photographed under UV light using the Versa DocTM Imaging System Model 3000 (BIO-RAD Laboratories Inc.).
Once the ribosomal DNA was amplified, each ~1200 bp PCR product was digested separately with one or more restriction enzymes. The choice of restriction enzymes was determined in an iterative and sequential process (Figure 2.2). Restriction digestions using HaeIII, SacII and NciI were done following the manufacturer‟s recommendations in a
final volume of 20 µl containing 2 U of enzyme, 3 µl of 10x NE Buffer 4 (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT), 12.8 µl of SNW and 5 µl of the PCR product. The HaeIII and SacII reactions were incubated at 37°C for 16 h using the PCR machine and inactivated by heating to 80°C and 65°C, respectively, for 20 min. The NciI digest was incubated at 37°C for 14 h. For TaqI digestion, the reaction contained 2 units of enzyme, 3 µl of 10x NE Buffer 4, 0.3 µl of bovine serum albumin (BSA), 16.5 µl of SNW and 5 µl of the PCR product. Reactions were incubated at 65°C for 2 h and inactivated at 80°C for 30 min. DNA fragments were separated by electrophoresis in 1.5% agarose gels using 1X TAE buffer under constant voltage of 100 V for 1 h. The 1 Kb Plus DNA Ladder™ (0.1 ng/ul) molecular weight marker, was run in the first or last lane of each gel. The gel was stained and visualised as previously described. Species identity was assigned to each sample by comparing the DNA fragment patterns with those previously described (Alves et al., 2005; Sammonds et al., 2009; Baskarathevan et al.,
2011).
The initial digestionwith TaqI gave a pattern that allowed the isolate to be placed into one of three groups (Alves et al., 2005). Isolates with four bands of 364, 292, 189 and 53-92 bp were placed in Group A and were either N. australe or N. luteum. Those with three bands of 427-428, 291-292 and 53-117 bp were placed in Group B (N. parvum or N. ribis)
and those with four bands of 426-432, 291-292, 173-189 and 51-63 bp were placed in Group C (N. parvum, D. seriata, Do. sarmentorum or Do. Iberica; Figure 2.2). The isolates in Group A were differentiated with the SacII enzyme which resulted in two banding patterns, with N. luteum (Group D) having two bands of 1071 and 102 bp and N. australe
Baskarathevan et al., 2011). The Group B and C isolates were further digested using the
HaeIII enzyme to differentiate N. parvum (Group G with five bands of 258, 254, 203, 157 and 58-83 bp) and B. dothidea (Group H with three bands of 716, 157 and 58-83 bp) from
N. ribis,D. seriata or D. mutila (Group F). The isolates belonging to Group F were further digested using the NciI enzyme to differentiate D. mutila (Group K with two bands of 1079 and 93 bp),D. seriata (Group J with three bands of 334, 747 and 93 bp)and N. ribis which was not cut by the enzyme (Group I with one band of 1173 bp). Those isolates that were not differentiated using ARDRA analysis (Group L) were subjected to DNA sequencing.
Figure 2.2 Diagram of the iterative restriction digestion analyses used for identifying the botryosphaeriaceous species isolated from grapevine nursery plants and propagation materials. Group D N. luteum Group E N. australe Group G N. parvum Group F N. ribis D. seriata, D. mutila Others NciI digest Group H B. dothidea All isolates TaqI digest Group A SacII digestion Group B and C
HaeIII digest
Group I N. ribis Group J D. seriata Group K D. mutila Group L Unidentified spp.
2.2.5.2 Sequencing of ribosomal DNA
To confirm the identity of the isolates of each species identified by ARDRA, four isolates of N. luteum, four of N. parvum, two of N. ribis, and one each of N. australe, D. mutila,D. seriata and B. dothidea were randomly selected for DNA sequencing of the internally transcribed spacer region (ITS) of the rDNA using the ITS1 primer as described in Section 2.2.5.1. The selected PCR products for DNA sequencing were sequenced at the Lincoln University Sequencing Facility using an ABI PRISM® 310 Genetic Analyzer (Applied Biosystems, Foster City, California). The resulting sequences and chromatographs were analysed using the DNAMAN 5.2 (Lynnon Biosoft©) and Chromas Lite 2.1© (Technelysium PTY Ltd) softwares. For the eight botryosphaeriaceous species found in the nurseries, three to six published sequences of the ribosomal DNA, including the ITS region with at least 500 bp, were obtained from the NCBI database (www.ncbi.nlm.nih.gov) and aligned with the sequences of the above mentioned isolates using MEGA Version 4 (Tamura et al., 2007). One rDNA sequence that included the ITS region of Guignardia philoprina
(Berk. & M.A. Curtis) was further obtained from the NCBI database and included as an outgroup. This species was used as the outgroup for a similar phylogenetic study by Slippers et al. (2004a) because it was closely related to the botryosphaeriaceous group. The aligned sequences were tested for phylogeny and a neighbour joining tree was generated. The robustness of the trees obtained was evaluated by 1000 bootstrap replicates (Crous et al., 2006).
2.2.5.3 Amplification and sequencing of -tubulin and elongation factor -1 genes
The novel species and the two isolates whose rDNA gene sequences were not 100% homologous with any of the accessions in Genbank were further identified by amplifying and sequencing the -tubulin gene using primers: Bt2a (5‟-
GGTAACCAAATCGGTGCTGCTTTC-3‟) and Bt2b
(5‟-ACCCTCAGTGTAGTGACCCCTTGGC-3‟; Glass & Donaldson, 1995) and the
elongation factor -1 gene primers: EF1-728F (5‟-CATCGAGAAGTTCGAGAAGG-3‟) and EF1–986R (5‟-TACTTGAAGGAACCCTTACC-3‟; Carbone et al., 1999).
The -tubulin gene was amplified using the REDExtract-N-AmpTM PCR ready mix following the manufacturer‟s recommendation. PCR was conducted using the conditions described in Section 2.2.5.1.
For the amplification of elongation factor -1 gene, each 25 µl PCR reaction contained 1.25 U of Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany), 1x PCR buffer (10 mM Tris-HCL, 1.5 mM MgCl2, 50 mM KCl) (Roche Diagnostics, Basel, Switzerland), 200 μM each of dGTP, dCTP, dATP, dTTP, 1.0 mM MgCl2, 20 pmoles of each primer, and 20-25 ng genomic DNA. Thermal cycling conditions were: denaturation at 94°C for 2 min, then 40 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s and 90 s primer extension at 72°C with a final extension at 72°C for 5 min (Slippers et al., 2004a).
The resulting PCR products were analysed by 1% agarose gel electrophoresis before being sequenced. Although many other studies have used forward and reverse sequence of the nominated regions as the main method of identification, this study needed only confirmation of identity already determined by morphological characteristics, ARDRA techniques and sequence analyses of the ITS region. Therefore the sequencing of - tubulin and elongation factor genes was only done for one strand of the amplicons using the Bt2a and EF1-728F -1, respectively, with previously described methods. The returned sequences were analysed following the same methods as described in Section 2.4.2. A species was considered a match if it had 100% homology with a continuous section of at least 370 bp of the -tubulin gene and 220 bp of the elongation factor -1 gene of the reference species.