sdu faculty of forestry journal special edition 2009 - Orman Fakültesi

SDÜ ORMAN FAKÜLTESİ DERGİSİ
Ronquist and Huelsenbeck, 2003). Additional ITS sequences derived from
Genbank were included in a broader ITS phylogeny to look at the relationship
between L. pinastri isolates from Scotland and from other parts of the globe.
2.4. Amplified Fragment Length Polymorphism and Corresponding Analysis
AFLP (Amplified Fragment Length Polymorphism) markers were obtained using
the protocol of Vos et al. (1995). Eight primer combinations were chosen which
gave 549 markers across all L. pinastri isolates (Table 2). Bands were scored as
present or absent, a distance matrix was calculated using the Jaccard coefficient,
and this was used to conduct a principal coordinate analysis in PAST.
Table 2: Numbers of AFLP markers found in each putative Lophodermium species
Primer Combination Total Clade Ia Clade Ib Clade II
AAC/CAA 74 27 23 24
AAC/CG 86 31 33 33
AAC/CT 98 24 39 48
AAC/CC 91 26 33 46
AAC/CAGA 53 26 7 28
AAC/CTA 58 18 10 34
AAC/CCG 60 20 13 32
AAC/CAG 29 12 5 12
2.5. Culture Morphology
Isolates collected as described above from Glen Affric, Loch Maree and Abernethy
(Table 1) were identified to clade on the basis of their ITS sequence. Isolates were
then chosen at random within clades and populations. Inoculum measuring 5mm
diameter was cut from cultures and transferred onto a 2% malt agar Petri dish.
Radial growth was measured once a week. The experiment followed a randomised
block design with four blocks and included two replicates of each isolate. Results
were analysed using R 2.8.0 (R Development Core Team, 2008)
3. RESULTS
3.1. Phylogenetic and Genetic Marker Analysis
Phylogentic analysis showed a total of five monophyletic clades with strong bootstrap
or prior probability support within the Lophodermium complex on Pinus sylvestris.
Isolates derived from shed needles in Scottish pinewoods, and previously classified as
L. pinastri, fell into three of these clades named Ia, Ib and II. These clades are
consistent when using both Parsimony and Baysian inference of phylogeny on
combined data from the ITS and Actin loci (Fig. 1 and 2). Isolates from these three
clades also form three distinct groupings in the Principal Coordinate analysis based on
genetic distances obtained by scoring AFLP markers.
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