Susceptibility of Canadian Flora to EU2 Lineage of Phytophthora ramorum and Pathogen Sporulation Potential Simon Francis Shamoun, Grace Sumampong, Robert Kowbel, and Katherine Bernier, Canadian Forest Service, Pacific Forestry Centre, Victoria, BC, Canada; [email protected]; Marianne Elliott, Washington State University, Puyallup Research and Extension Centre, Puyallup, WA, USA; Danny Rioux and Martine Blais, Canadian Forest Service, Laurentian Forestry Centre, Sainte-Foy, Québec, Canada; Alexandra Schlenzig, Science and Advice for Scottish Agriculture, Plant Biosecurity and Inspection, Edinburgh, UK Abstract Results and Discussion Phytophthora ramorum is an oomycete pathogen and a causal agent of a disease There was a large variation in aggressiveness and sporulation potential among the evaluated hosts. Among the non-conifer species, the EU2 isolate commonly referred to as Sudden Oak Death (SOD). The pathogen causes foliar blight and EU2-specific EU1-specific produced the largest lesions on Pacific dogwood (Cornus nuttallii), and Camellia japonica, in the west; red oak (Quercus rubra), yellow birch (Betula shoot dieback of nursery plants, including Rhododendron and Viburnum. It is also EU1: TA A C A G G T G T G G C - - A A AT C C AT T T T T G alleghaniensis), and white ash (Fraxinus americana) in the east. For conifer hosts, we found that the EU2 isolate was most aggressive on balsam fir responsible for the widespread mortality of tan oak and coast live oak in coastal (Abies balsamea) in the east and grand fir (Abies grandis), western hemlock (Tsuga heterophylla), and western larch (Larix occidentalis) in the west. California and southwestern Oregon, USA, as well as Japanese larch in the UK. A total of EU2: TA A C T G G T G T G G C - - A A AT T C AT T T T T G As for sporulation potential, red alder (Alnus rubra) and bigleaf maple (Acer macrophyllum) in the west produced significantly more sporangia than 33 host species commonly found in eastern (8) and western (25) Canadian landscapes bay laurel. Sugar maple (Acer saccharum) (Fig. 7) in the east was a potential spore producer but not significantly different from bay laurel. For the and forest sites were selected for this study. Detached leaves/needles were inoculated NA1: TA A C A G G T G T G G C - - A A AT T C AT T T T T G sporangia per unit lesion area, Himalayan blackberry (Rubus discolor), raspberry (Rubus idaeus), and Garry oak (Quercus garryana) were with P. ramorum EU2 lineage mycelia which was isolated from stream bait near an NA2: TA A C A G G T G T G G C - - A A AT T C AT T T T T G significantly higher than California bay laurel (Umbellularia californica), although both Himalayan blackberry and raspberry were asymptomatic. infected larch plantation in Scotland, U.K. There was a large variation in aggressiveness These results confirm the potential threat of EU2 lineage to Canadian flora. and sporulation potential among the evaluated hosts. Among the non-conifer species, Figure 1. Differences in Cox1 gene sequences (bolded & underlined) provided a diagnostic the EU2 isolate produced the largest lesions on Pacific dogwood, (Cornus nuttallii), Foliar hosts were classified by relative leaf area into five groups and these groups were analyzed Camellia japonica in the west; red oak, yellow birch, and white ash in the east. For test to discriminate between all four lineages of Phytophthora ramorum, conifer hosts, we found that the EU2 isolate was most aggressive on balsam fir in the including EU1 and EU2 lineages. (Source: King, K.M. et al. 2015. Plant Pathology 64 :1168-1175). separately: east and grand fir, western hemlock, and western larch in the west. As for sporulation Group A, conifer needles: Western hemlock needles were asymptomatic but produced a small amount of sporangia. The potential, red alder (Alnus rubra) and bigleaf maple (Acer macrophyllum) in the west conifer host that produced the most sporangia/mm2 lesion area was white spruce, which produced significantly more than produced significantly more sporangia than bay laurel. Sugar maple (Acer saccharum) in bay laurel in trial 1 but not in trial 2. Lesion area was biggest on grand fir, balsam fir, and western larch. the east was a potential spore producer but not significantly different from bay laurel. These results confirm the potential threat of EU2 lineage of P. ramorum to Canadian Group B, small broadleaf: This group included manzanita and wintergreen, both evergreen broadleaf plants. Lesion area was flora. similar for both hosts, which tended to cover approximately 50% of the leaf area on the average (data not shown). Both hosts produced fewer sporangia than bay laurel. Western red cedar was in this size class but was asymptomatic. Sporangia Introduction production was highly variable on western red cedar and ranged from 0-157 sporangia/mm2. Phytophthora ramorum is an aggressive invasive alien pathogen that has caused extensive mortality of oak and tanoak trees in western USA and Japanese larch trees in the UK. P. Group C, medium broadleaf: Blueberry and Oregon grape had smaller lesion area than paper birch and currant. The only host ramorum is also present on Rhododendron, Camellia, and Viburnum and other susceptible that produced more sporangia per mm2 lesion area was Oregon grape, but it was not significantly more than bay laurel. landscape plants in the nursery industry, which is thought to have been the pathway for its spread into new geographic regions including forests and natural ecosystems. Group D, large broadleaf: The largest lesions were formed on yellow birch. The average total sporangia per lesion produced by There are four distinct clonal lineages of P. ramorum, one originally discovered in Europe, but yellow birch was 21 and bay laurel 186. also found in western North America (EU1), a new lineage detected in Europe (EU2), and two lineages only present in North America (NA1 and NA2) (Figure 1). The host range of P. Group E, extra large broadleaf: lesion area on these three hosts tended to be small, but significantly more sporangia were ramorum is very broad (more than 120 host plants). Many of the host species are currently produced on bigleaf maple when compared to California bay laurel. present in forested and urban areas in the west coast of the US and Canada (Shamoun et al. 2018). Figure 2. Aerial photo of Phytophthora ramorum symptoms on Japanese To assess the risk posed by an invasive alien pathogen such as Phytophthora ramorum, it is Lesion area, broadleaf often a good strategy to evaluate its capacity to infect plants prevalent in the area of interest. Larch (Crown dieback) in Southwest England. (Courtesy: Mick Biddle, Forestry This approach has been used successfully where potential hosts were identified by artificial Commission England, Tree Health Team) Lesion area, conifer Range lesion area Mean lesion area Mean leaf area infections before being found naturally infected by P. ramorum. Establishment of P. ramorum Range lesion area Mean lesion area Mean leaf area in Canadian nurseries and landscapes could result in large economic losses and limitations to 9000 80 trade in ornamental plants and threats to biodiversity and sustainability of forest ecosystems. 8000 To date, we have investigated the susceptibility and sporulation potential of selected eastern Figure 5. Distribution map of Phytophthora ramorum lineages EU1 and EU2 2 70 and western Canadian host plants to three lineages (NA1, NA2 and EU1) (Elliott et al. 2011; in the UK. (Courtesy of Dr. Joan Webber, Principal Pathologist, Head of 7000 Jinek et al. 2008; Jinek et al. 2011). However, there is an urgent need to determine the Forest Tree Health, Forest Research, UK). mm 60 2 6000 susceptibility, sporulation potential and risk assessment of the newly emerged lineage EU2 in 50 the UK (Van Poucke et al. 2012) and its potential threat to Canadian flora and impact on mm 5000 Table 1. Foliar host species collected from the Western Canadian region 40 nursery and forestry. For instance, Kalmia latifolia was first identified as highly susceptible to 4000 P. ramorum under laboratory conditions (Tooley et al. 2004) and was thereafter found in the 30 Area, UK (DEFRA 2008, Plant Health Portal) Biological Name Common Name 3000 20 [https://planthealthportal.defra.gov.uk/assets/factsheets/pramparks.pdf]. We published Arbutus menziesii Arbutus 2000 similar results when a larch (Larix laricina) was found susceptible for the first time after needles, 3 of Area 10 artificial inoculations in 2008 (Jinek et al. 2008) before another larch (L. kaempferi) was Quercus garryana Garry Oak 1000 0 reported heavily infected in plantations in the UK in 2009 (Webber and Brasier 2010; Webber Acer macrophyllum Bigleaf Maple 0 et al. 2010) (Figs. 2, 3 & 4). In the U.K., the current distribution of the EU2 lineage is limited to southwest Scotland and Alnus rubra Red Alder Salal Grape Poplar Northern Ireland (NI).The EU2 lineage is almost exclusively the only lineage of P. ramorum in Sumac Grand fir Grand Populus trichocarpa Poplar Currant Balsam fir Red oak Red Camellia Douglas fir Douglas NI. The EU2 lineage has not been found in England and Wales. Only EU1 lineage is present Madrone Red alder Red Garry oak Garry Blueberry Bay laurel White ash White Sitka spruce Sitka Raspberry Manzanita White spruce White there (Figure 5). The objectives of the present study are to determine the susceptibility of Cornus nuttallii Pacific Dogwood Paper birch Western larch Yellow birch Sugar maple Sugar Wintergreen Lodgepole pine Lodgepole Bigleaf maple Bigleaf selected Canadian flora to EU2 lineage and pathogen sporulation potential, and its threat to Oregon grape Rhododendron Betula papyrifera Paper Birch hemlock Western Canadian flora and impact on nursery industry and forest ecosystems.