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

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  • Kris De Jonghe
  • Anne-Marie Deeren
  • Thomas Goedefroit
  • Anne Ronse
In 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 diverse plant collections with both exotic and native taxa that can be used as sentinels by recording their sensitivity to either native or to (newly) introduced pests and diseases. One of the case studies focused on monitoring phytoplasma diseases in trees, especially on Ulmus spp. To monitor these species, a total of 117 root samples were taken from collection trees in five botanical gardens and arboreta in Flanders, Belgium. Total genomic DNA was extracted by a CTAB protocol (Doyle and Doyle 1990). The 16S rRNA gene was partially amplified using the phytoplasma universal primer pairs P1/P7 (Deng and Hiruki 1991) followed by nested PCR with primer pairs R16F2/R2 (Lee et al. 1995) 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 to Arnaud et al. (2007), was done to yield a nested PCR product of 1,174 bp, and finally, a short fragment from the tuf gene was generated following the Q-Bank methodology for Q-phytoplasma ( In one location in Vlaams Brabant, one sample, roots from an Ulmus minor subsp. canescens tree (∼8 years old) tested positive for the presence of a phytoplasma. The sample was taken in March 2018 (early spring) and only consisted of roots. An amplified fragment of ∼1,600 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 (1,516 bp) was submitted to GenBank (accession MH583020.2). This sequence revealed the closest homology (99%) with ‘Candidatus Phytoplasma ulmi’ strains from the U.S.A. (AF189214.1) and Czech Republic (EU184021). In addition, the secY and tuf fragment were directly bidirectionally sequenced and the sequences of the resulting contigs, 1,027 bp (MH727566) and 367 bp (MK491655), respectively, were also submitted to 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. P. ulmi’, 16SrV subgroup A, 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 five 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 expanded to the complete affected botanical garden (28 Ulmus or related trees) and to lane trees from the surrounding area. 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 onward, yellowing and partial branch decay could be observed on the affected tree. All additional samples from the botanical garden and surrounding area tested negative. To our knowledge, this is the first report of ‘Ca. P. 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.
Original languageEnglish
JournalPlant Disease
Issue number7
Pages (from-to)1763-1763
Number of pages1
Publication statusPublished - 2019
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