First Report of Phytophthora Pluvialis in Douglas Fir Plantations in Belgium

Following a report of Phytophthora pluvialis in the United Kingdom in 2021 (Pérez-Sierra et al., 2022), surveillance for this forest pathogen started in 2023 in southern Belgium. Douglas fir (Pseudotsuga menziesii) was surveyed as it is an economically important conifer species and a major host for P. pluvialis. Fifteen Douglas fir plantations close to streams were visited between May and October. Two monitoring methods were used: checking trees for symptoms of the disease (shoot dieback, resinous cankers, needle loss) and baiting in streams using a qPCR detection method developed by McDougal et al. (2021). Baiting was done using Western hemlock (Tsuga heterophylla) twigs placed in cheesecloth bags attached to the riverbank which were left floating on the water surface in the river for two weeks. In spring, none of the surveyed trees displayed symptoms and no plant samples were collected. However, Phytophthora pluvialis was detected by qPCR in the baits placed in streams close to two of the fifteen forest sites (sites A and B). These two sites were located c. 3.5 kilometres apart in the province of Luxembourg. Further baiting tests in streams were undertaken at site A in September 2023 and a Phytophthora with the morphological characteristics of P. pluvialis (Reeser et al., 2015) (Figure 1) was isolated on selective Phytophthora medium (PARP-carrot agar). Incubations were done at 17°C in the dark for two-three weeks as recommended by Dick et al. (2014). The colonies were transferred to carrot agar medium (CA). Sequencing of the ITS region with the ITS5/ITS4 PCR primers confirmed that it was P. pluvialis (GenBank Accession No. OR752037). In October 2023, 38 trees selected at random were sampled at site B. Needles were collected by shaking the branches over a net connected to a container (Figure 2). Two trees tested positive using qPCR. There was no needle collection at site A as the trees were mature and the branches inaccessible. Douglas fir twigs were inoculated by placing them in demineralised water together with ten mycelium plugs from 10-day-old cultures grown on CA. After 10 days at 17°C in the dark, needles with typical olive-green lesions (Gomez-Gallego et al., 2017) were observed (Figure 3). Ten twigs were also inoculated by placing a mycelium plug of the pathogen in a wound made in the bark. After one month at 20°C under an approximately 9 hour day / 15 hour night photoperiod, typical necrosis was visible in the inner bark (Figure 4). Isolations confirmed the presence of P. pluvialis in the needles and bark. There were no symptoms in the negative controls used in the two tests. To our knowledge, this is the first report of P. pluvialis in continental Europe. Its presence in the natural environment in two watercourses in June as well as September suggests that it may be established in southern Belgium. The research that yielded these results, was funded in part by the Belgian Federal Public Service Health, Food Chain Safety and Environment through the contract RT 23/03 EMPHYPEST. We wish to thank the Walloon Observatory of Forest Health for its support, Dr Gomez-Gallego (INRAE, France) and Dr LeBoldus (Oregon State University, U.S.A) for providing us with P. pluvialis isolates, and Dr Pérez-Sierra (IVIA, Spain) for her advice on how to carry out baiting and needle collection from trees.