A total of 180 trees were selected from four different forest stands in the Otago region of New Zealand in 2006. In each stand, an equal number of pruned and unpruned trees of the same cohort were selected for sampling. Stand 1 was planted in 1997 (40 trees sampled), stand 2 in 1998 (40 trees sampled), stand 3 in 2000 (50 trees sampled), and stand 4 in 2002 (50 trees sampled). Stands 1 and 2 had been pruned 4–5 months prior to the first assessment in February 2006, while Stand 3 had been pruned 18 months prior to the first assessment. Stand 4 had two pruning lifts, 25 and 11 months prior to the first assessment. Two pruned and six unpruned trees were accidently removed in a thinning operation midway through the trial, and these trees were therefore not included in the analysis. At the end of the study in 2008, all trees were classified as either fluted or unfluted based on the presence of depressions in the stem in 2008 that it was believed might later develop into flute cankers, given that trees were generally too young in the study for fully developed flute cankers (as illustrated by Bulman 2007) to be present.
Samples were collected from trees using an increment borer that removed 5 mm diameter cores. The increment corer was surface sterilised in bleach and rinsed with commercially bottled water between every tree to avoid cross contamination. To sample pruned trees, the increment corer was inserted directly above a branch stub while unpruned trees were sampled as close to the whorl as possible. The same trees were sampled in 2006, 2007 and 2008.
Detection of Neonectria fuckeliana infection
The presence of N. fuckeliana in the samples was determined using visual identification of microbial cultures and also using DNA-based methods. To culture N. fuckeliana a 5 mm sample was cut from each core, surface sterilised in 70% ethanol, followed by rinsing in 10% bleach and three rinses in sterile distilled water, all for 30 seconds per step. The pieces of wood were then placed on 2% malt extract agar (MEA) and incubated at 20°C in the dark. Mycelial growth that emerged from the wood sample was subcultured onto fresh MEA and N. fuckeliana was identified visually, based on the morphological characteristics of the Acremonium anamorph. Microbiological culturing was only conducted on samples collected in 2007 and 2008.
The presence of N. fuckeliana was assessed for all samples using the PCR method of Langrell (2005). Ten small disks were randomly cut from the length of the increment core and then ground in liquid nitrogen using a mortar and pestle. A FastDNA Kit (with extraction buffers CLS-VR and PPS) and a FastPrep instrument (Qbiogene Inc, CA) were used to extract DNA from a volume of 200–500 μl of ground tissue following the manufacturer's protocol. Homogenization was carried out using lysing matrix A at speed 5 for 20 seconds, repeated 3 times, with a 2 minute incubation on ice between 20 second bursts. Following DNA extraction, the concentration of DNA in every sample was measured using a FLUOstar Galaxy fluorometer (BMG Lab Technologies) and then diluted to a concentration of 1 ng/μl with sterile water for PCR analysis.
The DNA of N. fuckeliana was detected using a nested-PCR protocol, which used the universal ITS primers ITS-1F (5'-CTT GGT CAT TTA GAG GAA GTA A-3') (Gardes and Bruns 1993) and ITS-4 (5'-TCC TCC GCT TAT TGA TAT GC-3') (White et al. 1990) to amplify all fungal DNA within the sample. The PCR products were then subjected to a second round of PCR using the N. fuckeliana specific PCR primers Cct1 (5'-ACC CCA AAC CCT TAT TTC TG-3') and Cct2 (5'-ACG GCG TGG CCG CGC CGC TT-3') developed by Langrell (2005). The first round of PCR was carried out in a 15 μl reaction volume, containing 2.5 ng of template DNA, 0.05 pmol/μl of PCR primers ITS-1F and ITS-4, 0.45 U Taq DNA polymerase (Roche), 1 x reaction buffer (Roche), 1.5 mM MgCl2, 0.2 mM each of dATP, dGTP, dCTP, and dTTP (Roche). The PCR conditions were 95°C for 6 min, followed by 35 cycles of 95°C for 30 s, 60°C for 40 s, 72°C for 40 s, and then one cycle of 72°C for 5 min and a 4°C hold. The PCR reaction contents of the second round reaction were the same as the first round, but the N. fuckeliana PCR primers were used and 1:50 dilution of the first-round PCR products were used as the DNA template for the second round PCR reaction. The second round PCR profile was 94°C for 3 min, followed by 30 cycles of 94°C for 1 min, 62°C for 1 min, 72°C for 1 min, then one cycle of 72°C for 10 min and a 4°C hold. Prior to use in this study, a pilot study was conducted using DNA from fungi that are commonly isolated from Pinus radiata to ensure that the test was specific to N. fuckeliana. Additionally, the amplicon from N. fuckeliana was sequenced to validate the assumption that a band of 360 base pairs was diagnostic for the presence of N. fuckeliana.
Products obtained from the PCR procedure were subjected to electrophoresis on 1% agarose gels, stained with ethidium bromide and visualised and photographed under UV light. If a band of 360 base pairs was present following the second round PCR reaction, it was assumed that N. fuckeliana DNA was present in the sample and the sample was thus scored as N. fuckeliana positive. The PCR test was repeated on all samples that gave an initial positive result to confirm the initial positive result.
A logistic regression analysis was performed to test for a difference in the presence or absence of N. fuckeliana between pruned and unpruned trees. In this analysis, a tree was considered to be infected with N. fuckeliana if the fungus was either isolated by culturing or detected by the PCR test in any of the three years. The following logistic regression model was fitted using the SAS (Version 9.2) procedure GENMOD:
where y was a binary variable coded as 1 for infected trees and 0 for uninfected trees. An analysis of deviance associated with this model, with the variance scaled to allow for overdispersion, was used to determine significance of pruning in relation to the presence of N. fuckeliana.
To test whether stems with early evidence of fluting were more likely to be infected, a further term was added to the model:
To assess change in infection rates over time, and to determine whether this varied between pruned and unpruned trees, a Generalised Estimation Equation (GEE) model was fitted using PROC GENMOD. For this analysis a binary variable, y, representing presence of N. fuckeliana detected by the PCR method in each assessment year was the dependent variable. The model was of the following form:
This model was used to test for any change in infection level within each tree over the course of the study and used an unstructured covariance structure between assessment years.