Supplemental Table 1. Site location and sample sizes of the collection and isolation in Pinuscontortaof bacteria from Dendroctonusponderosae, Dendroctonusponderosae galleries, and unattacked tree phloem during the summers of 2008 and 2009.

Site Location / DendroctonusHost / Sample Type / Sample Size
Mackenzie, British Columbia / P. contorta / D. ponderosae
Gallery
Phloem / 26
26
7
McBride, British Columbia / P. contorta / D. ponderosae
Gallery / 300
150
Grande Prairie, Peace River, &
Slave Lake, Alberta / P. contorta / D. ponderosae
Gallery
Phloem / 52
15
8
Whitecourt, Alberta / P. contorta X P. banksiana hybrid / D. ponderosae
Gallery
Phloem / 41
13
24
Grand Prairie, Alberta / P. contorta X P. banksiana hybrid / D. ponderosae
Gallery / 300
150
Athabasca, Alberta / P. banksiana / D. ponderosae
Gallery
Phloem / 150
8
18

Beetles were washed in sterile phosphate buffered saline (PBS) at pH 7.4, transferred to 1 ml fresh PBS, crushed, and 100 µl of solution was cultured on 5% Tryptic Soy Agar (TSA) with a Whatman No. 1 9 cm filter paper containing 1 mllodgepole pine turpentine affixed to the lid of a 10 cm plastic Petri dish. Bacteria were incubated at 25°C, 30% relative humidity (RH) for 2 weeks. Another 100 µl of this solution was inoculated into 3 ml Bushnell Haas broth, a minimal medium, with 50 µl lodgepole pine turpentine (LPP) to screen for putatively terpene-tolerant bacteria (BH broth + LPT solution). Cultures were shaken for 2 weeks at room temperature, then 100 µl of undiluted culture was plated as before. Bacteria were incubated at 25°C, 30% RH for 4 days. Unique morphotypes were isolated on 20% TSA.

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Supplemental Table 2.Bacteria isolated from Dendroctonus ponderosae adults and assayed for potential ability to metabolize terpenes.

Isolate (Accession number) / Closest named match
(Acc. No.) / Sequence Length (base pairs) / S_ab Score
JN194137 / Serratia marcescens (AB061685) / 1414 / 0.975
JN194138 / Brevundimonas vesicularis (AJ227780) / 1320 / 0.985
JN194126 / Serratia marcescens (AB061685) / 1394 / 0.984
JN194139 / Pseudomonas brenneri (AF268968) / 1392 / 0.975
JN194140 / Pseudomonas mandelii (AF058286) / 952 / 0.978
JN194133 / Pantoea agglomerans (AJ233423) / 1289 / 0.921
JN194135 / Rahnella aquatilis (AJ233426) / 1398 / 0.917
JN194127 / Pseudomonas migulae (AF074383) / 1386 / 0.973

Target sequences were amplified by performing a polymerase chain reaction (PCR) using the primers 27f (5’-GAGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). Sequencing of PCR products was performed by the University of Wisconsin Biotechnology Center. Sequence data were aligned using Sequencher (version 4.4 build 1415, Gene Codes Corporation, Ann Arbor, MI). Sequences of bacteria used in bioassays were deposited in GenBank. The closest match, S_ab score and accession number of the closest match were obtained using the Ribosomal Database Project ( by searching for type strain, source isolates, ≥1200 base pairs in size and good quality.

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Supplemental Table 3.Details of materials and procedures used in bioassays. Listed in order of presentation in text.

Material / Vendor / Details
Tryptic Soy Broth / MP Biomedicals, Solon, OH, USA
Lodgepole pine turpentine / Synergy Semiochemicals Corp, Burnaby, BC, Canada
Filter paper / Whatman
TSA / Agar; Difco, Sparks, MD, USA
PTFE thread tape seal / AA Thread Seal Tape Co., Wauconda, IL, USA / 1.3 cm wide
Clear screw-cap glass vials / Fisher Scientific, Pittsburg, PA, USA / 25 X 83mm 6 dram
Monoterpenes / Aldrich, St, Louis, MO, USA / α-pinene [98%; blend: (-):(+)-α-pinene: 44:54]; (-)-β-pinene (>88%), 3-carene (90%)
Resolv hexanes / Fisher Scientific, Pittsburg, PA, USA
Autosampler vials / National Scientific
isobutylbenzene / Aldrich, St. Louis, MO, USA / 98%
Gas chromatography / Shimadzu 17-A GC-FID / Temperature cycle: 10 minutes at 60˚C, then 5˚C min-1 to 160˚C. Injectors and detectors at 220˚C.
Chiral column / Agilent Technologies / Cyclodex-B; 30 m, 0.25 mm i.d., 0.25 µm film thickness
Kruskal-Wallis Test / Systat Software, Point Richmond, CA, USA / SYSTAT 11
Media for abiotic acid assays / 0.3 g/L yeast extract, 6.0 g/L Na2HPO4, 3.0 g/L KH2PO4, 0.5 g/L NaCl, 1.0 g NH4Cl, 1.0g KNO3, 1M MgSO4
Vacuum centrifuge / Savant / Speedvac
High-pressure liquid chromatography / Hewlett Packard; 1090 with diode array detector (DAD); ODS 5 µm 4.6 x 25 mm column; Beckman Ultrasphere / Mobile phase consisted of MeOH:1.7% acetic acid (91:9) run isocratically at 1.0 ml/min with column heater at 30C. We injected 20 µl of each solution and compared retention times and peak areas at 240 nm to abietic acid standard. We collected UV data from 190-400 nm and confirmed identity in each sample with resulting spectra. All peak areas fell within linear range (R2 = 1.0, P < 0.001) of standard.
Abietic acid / Acros Organics, Waltham, MA, USA / 85%
ANOVA / SAS Institute, Cary NC, USA / SAS version 9.1, 2003; PROC GLM
Petri dishes / Fisher Scientific, Pittsburg, PA, USA
Yeast Malt Extract Agar / 4 g/L yeast extract, 10 g/l malt extract broth, 4 g/l dextrose, 20 g/l agar; Spores were dislodged from hyphae using a bacterial spreader.

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