Mixermill

Individual adult trees were sampled from the FDP at DVCA, LHNP, and PFR [14 (link),19 (link)], which are integrated within the CTFS–ForestGEO global network of forest plots [56 (link)]. Parashorea tomentella sampled from SFR by Kettle et al. [4 (link)] followed a stratified sampling approach over a much larger spatial scale, and therefore a 50 ha subsection of this dataset was used (full details are provided in S2 File).
Published microsatellite genotype and coordinate datasets are available for P. tomentella from SFR [4 (link)], S. parvifolia from LHNP [19 (link)] and S. leprosula from PFR [14 (link)]. Using identical methods, we analyzed patterns of FSGS for the same species from the DVCA plot. Consistent with the comparison datasets, all individuals with a DBH > 30 cm were sampled, and coordinates recorded using a handheld GPS (Garmin GPSmap 60CSx). Cambium samples were taken using a 2 cm diameter leather punch and hammer, following the procedure of Colpaert et al. [61 ]. Samples were desiccated in silica gel and then stored at -4°C prior to DNA extraction. DNA was extracted from roughly 0.025g of lyphosized sample using Qiagen DNeasy™ 96-well-plate extraction system, after first milling samples to a fine powder using a Qiagen Mixer-Mill™. Details of sampling and DNA extraction from LHNP, PFR and SFR are described in the original papers [4 (link),14 (link),19 (link)].

Mosquito pools from in-house bred Culex pipiens were used to determine suitability of the JCV primer sets in mosquito surveillance. Serial dilution of JCV 61V2235 were spiked into pools of 50 Cx. Pipiens in the range of 6 log₁₀ PFU/mL to 4 log₁₀ PFU/mL. Pools were process as previously described [33 (link)]. Briefly, pools of 50 adult Cx. Pipiens were homogenized with 1 mL BA-1 media in a Mixer Mill (Qiagen), clarified by centrifugation, RNA was extracted using the MagMax Viral Pathogen Nucleic Acid kit (Thermo Fisher) and eluted in 100µL. Ten microliters of eluate from each pool and a negative pool were tested in duplicate with each JCV primer set with the conditions described above.

Plant roots were flash-frozen in liquid nitrogen then pulverized using a mixer mill (QIAGEN, Germany). Total RNA was extracted using RNeasy Plant Mini Kit (QIAGEN). Quantitative real-time reverse-transcription PCR (qRT-PCR) was performed using One Step SYBR PrimeScript RT-PCR Kit ІІ (Takara, Japan) and 7300 Real Time PCR System (Applied Biosystems, USA). Three biological replicates were run for each photoperiod. An ubiquitin gene (Accession number: AF240445) was used as a reference gene in hybrid aspen T89. The Ubiqutin primers for qRT-PCR were the following: UBIQUTIN-F (5′-TGAACCAAATGATACCATTGATAG-3′) and UBIQUTIN-R (5′-GTAGTCGCGAGCTGTCTTG-3′). The gene expression analysis primers for PtKUP1, PttHAK-like1, PttHAK-like2, PttCNGC1-like1, and PttCNGC1-like2 are listed in Table 1. Significant differences between each gene expression level were confirmed by one-way ANOVA.

Permission to collect samples was received from all land owners. Leaves with typical symptoms of target spot were surface sterilized with 10% chlorine for 1 min and a section of tissue at the edge of a lesion was excised and placed onto RA-amended water agar media (rifampicin 25 ppm, ampicillin 100 ppm, 20 g agar and 1 L water). Hyphal-tips were transferred to RA-amended V8 agar media (15 g agar, 3 g calcium carbonate + 160 mL V8 juice + 840 mL water) and maintained at -4 °C.
For genomic DNA extraction for Whole Genome Sequencing (WGS), mycelium was grown 2 weeks at room temperature in 250 ml flasks containing 10 ml RA-V8 liquid broth (above, minus agar). The resulting mycelium was transferred to 2 ml tubes containing 2–3 glass beads, freeze dried, and powdered with a Mixer-Mill (Qiagen). Genomic DNA was extracted using a standard phenol-chloroform protocol. DNA extraction for targeted-sequencing was accomplished in a 96 well plate as described by Lamour and Finley [38 (link)].