First report of ‘Candidatus Phytoplasma ulmi’ on elm in Belgium

n April 2017 the project ‘Belgian Plant Sentinel Network’ started as part of the Euphresco-IPSN2 network. The botanical gardens and arboreta of the network contain very diverse plant collections with both exotic and native taxa which can be used as sentinels, by recording their sensitivity to either native or to (newly) introduced pests and diseases. One of the specifically chosen case studies focused on monitoring phytoplasma diseases in trees, especially on Ulmus spp. To monitor these Ulmus spp., a total of 117 root samples were taken from collection trees in 5 participating botanical gardens and arboreta in Flanders, Belgium. Total genomic DNA was extracted by a CTAB protocol (Doyle & Doyle, 1990). The 16S rRNA gene was partially amplified using the phytoplasma universal primer pairs P1/P7 (Deng & Hiruki, 1991) followed by nested PCR with primer pairs R16F2/R2 (Lee et al. 1993), as diagnostic test, and P1/Tint (Smart et al. 1996) on positive samples for sequencing reasons. Additionally, specific group V nPCR targeting the secY (primer sets FD9f2L/FD9r followed by FD9f3L/FD9r2L) gene, according Arnaud et al. (2007) was done to yield a nested PCR product of 1174 bp, and finally, a short fragment from the tuf gene was generated following the Q-Bank methodology for Q-phytoplasma (www.Q-bank.eu). In one location in Vlaams Brabant, one sample, roots from an Ulmus minor subsp. canescens tree (approx. 8 years old) tested positive for the presence of a phytoplasma. The positive sample was taken in March 2018 (early spring) and only consisted of roots. An amplified fragment of approx. 1600 bp (P1/Tint) was gel purified (SmartPure, Eurogentec), and directly sequenced (Macrogen, Amsterdam, the Netherlands). The obtained partial sequence of the 16S ribosomal RNA gene (1516 bp) was submitted to the NCBI GenBank® (Accession MH583020.2). This sequence revealed the closest homology (99%) with ‘Candidatus Phytoplasma ulmi’ strains from the USA (AF189214.1), and Czech Republic (EU184021). In addition, the secY and tuf fragment were directly bi-directionally sequenced and the sequences of the resulting contigs, 1027bp (MH727566) and 367 bp (MK491655), respectively, were also submitted to the GenBank®. Phylogenetic analysis was undertaken (MEGA version 7.0.26) on the 16S, secY, and tuf fragments, indicating the phytoplasma to be a member of 'Ca. Phytoplasma ulmi', 16SrV subgroup A (Figs. 1A, B and C), and showing the genetic relationship with other ‘Ca. P. ulmi’ isolates previously reported in Europe. Virtual RFLP on the 16S fragment using iPhyClassifier (Zhao et al. 2009) confirmed this result. All other Ulmus trees, from the 5 botanical gardens and arboreta, as well as related Zelkova trees (Z. carpinifolia and Z. serrata), and a Celtis aetnensis tree tested negative. In addition, the phytoplasma survey on elm has been expanded the complete affected botanical garden (28 Ulmus or related trees) and to lane trees from the area surrounding the botanical garden. In addition, to confirm the finding, a second sampling of the same tree in May 2018 confirmed the presence of ‘Ca. P. ulmi’ on new root and leaf samples. From July onwards, yellowing and partial branch decay could be observed on the affected tree (Fig. 2). All additional samples from the botanical garden and surrounding area tested negative. To our knowledge, this is the first report of ‘Ca. Phytoplasma ulmi’ on Ulmus in Belgium. Considering the other recent findings in Europe, we suspect that ‘Ca. P. ulmi’ is likely to be distributed throughout the region, but with few previous reports, rather than being a new emerging risk.