High-Throughput Sequencing to Investigate Phytopathogenic Fungal Propagules Caught in Baited Insect Traps

Studying the means of dispersal of plant pathogens is crucial to better understand the dynamic interactions involved in plant infections. On one hand, entomologists rely mostly on both traditional molecular methods and morphological characteristics, to identify pests. On the other hand, high-throughput sequencing (HTS) is becoming the go-to avenue for scientists studying phytopathogens. These organisms sometimes infect plants, together with insects. Considering the growing number of exotic insect introductions in Canada, forest pest-management efforts would benefit from the development of a high-throughput strategy to investigate the phytopathogenic fungal and oomycete species interacting with wood-boring insects. We recycled formerly discarded preservative fluids from the Canadian Food Inspection Agency annual survey using insect traps and analysed more than one hundred samples originating from across Canada. Using the Ion Torrent Personal Genome Machine (PGM) HTS technology and fusion primers, we performed metabarcoding to screen unwanted fungi and oomycetes species, including Phytophthora spp. Community profiling was conducted on the four different wood-boring, insect-attracting semiochemicals; although the preservative (contained ethanol) also attracted other insects. Phytopathogenic fungi (e.g., Leptographium spp. and Meria laricis in the pine sawyer semiochemical) and oomycetes (mainly Peronospora spp. and Pythium aff. hypogynum in the General Longhorn semiochemical), solely associated with one of the four types of semiochemicals, were detected. This project demonstrated that the insect traps’ semiochemical microbiome represents a new and powerful matrix for screening phytopathogens. Compared to traditional diagnostic techniques, the fluids allowed for a faster and higher throughput assessment of the biodiversity contained within. Additionally, minimal modifications to this approach would allow it to be used in other phytopathology fields.

C6 and C8 were verified 99% pure by gas chromatography-mass spectrometry (GC-MS) by the Canadian Forest Service, and loaded into polyethylene pouches by Contech Inc (Delta, BC, Canada).
Each pouch contained 1.4 g of either C6 or C8. The release rates (at 20°C) were 20 mg/d for C6, and 25 mg/d for C8. Two C6 and two C8 pouches (semiochemicals) were both placed on a trap to obtain cumulative release rates of 40 to 50 mg/d. Second semiochemical set: UHR_E_AP.
The second semiochemical set (UHR_E_AP) consisted of UHR EtoH and UHR alpha-pinene. The two chemicals were loaded into separate release devices and placed on an insect trap. The UHR ethanol (95% purity, 121.5 g loaded/pouch) and UHR alpha-pinene [95% (-) enantiomer, 172 g/pouch] lures (Contech Inc., Delta, BC, Canada) had release rates (at 20°C) of 275 mg/d and 2 g/d, respectively. The UHR ethanol and UHR alpha-pinene chemicals were exactly the same throughout this project.

Supplementary Material S2: Fungal OTU, prior to species subtraction.
The most abundant fungi for all semiochemical treatments at the phylum, genus and species (top 10 per semiochemical) taxonomic levels are listed in Supplementary Table S4.
To visualize sampling depth, examples of the rarefaction curves obtained for the spore traps (negative control) and their respective original insect dataset, and subtracted insect dataset are shown in Supplementary Figure S5. The spore trap data demonstrated the highest sequencing depth as saturation was obtained for all of the samples tested with a sequence number per species ranging between approximately 5,000 and 25,000 (sequences). The lowest species number obtained by all samples tested was just below 150. In contrast, only part of the samples from both the original insect and the subtracted data reached saturation in their respective rarefaction curves.