First Report of ‘ <i>Candidatus</i> Phytoplasma asteris’ Affecting Woody Hosts ( <i>Fraxinus excelsior</i> , <i>Acer campestre</i> , <i>Acer platanoides</i> , <i>Ulmus minor</i> , and <i>Sambucus nigra</i> ) of an Urban Forest in Hungary

Symptoms of growth abnormalities including shoot proliferation, leaf rosetting and witches' broom symptoms, reduction in leaf size, downward rolling of leaf margins, distortion and chlorotic foliage were observed in several tree species in a Hungarian park forest in Martonvásár in the summer of 2022. In subsequent years, we observed the progression of the disease. This was manifested by the death of shoots and twigs, as well as an overall reduction in growth. The affected woody species were: <i>Fraxinus excelsior</i>, <i>Acer campestre</i>, <i>Acer platanoides</i>, <i>Ulmus minor</i> and <i>Sambucus nigra</i>. The observed symptoms were reminiscent of those observed in phytoplasma infections of other woody plants. The present study aimed to investigate whether the disease in these species was also caused by a phytoplasma. Shoot samples were taken from ten symptomatic, and three asymptomatic plants of each species. Total nucleic acids were extracted from the phloem tissue using the CTAB method (Ahrens and Seemüller 1992). To specifically amplify phytoplasma DNA, the universal primer pairs fP1/rP7 (Smart et al., 1996), followed by R16F2n/R16R2 (Gundersen and Lee, 1996)or fU5/rU3 (Lorenz et al., 1995) were used in a nested PCR. Amplicons of the expected sizes were obtained from 90% of the symptomatic samples (fP1/rP7 1783 bp, R16F2n/R16R2: 1.2 kb, fU5/rU3: 876 bp), but none from the asymptomatic ones. The R16F2n/R16R2-amplified products obtained from the five species were cloned (at least three from each PCR reaction), sequenced and deposited in GenBank under the following accession numbers PX491300 (<i>F. exelsior</i>), PX491301 (<i>S. nigra</i>), PX491302 (<i>A. campestre</i>), PX491303 (<i>U. minor</i>) and PX491304 (<i>A. platanoides</i>). The sequences of the five isolates were almost identical, showing a 99.92%-100% identity with each other. A BLAST search of GenBank yielded 99.92% sequence similarity in the case of the <i>S. nigra</i> isolate and 100% in the case of the other 4 isolates with the onion yellows phytoplasma strain OY-M (GenBank AP006628) from the '<i>Candidatus</i> Phytoplasma asteris' 16SrI-B subgroup. To confirm the pathogen's identity in subsequent experiments, the elongation factor Tu (<i>tuf</i>) and the translocase protein (<i>secY</i>) genes were amplified using the universal primer pairs fTuf1/rTuf1 (Schneider and Gibb, 1997), and the aster yellows-specific AYsecY_F-46/AYsecY_R1450 primer pairs (Viczián et al. 2023), respectively. Amplicons of the expected sizes (1085 bp for fTuf1/rTuf1, and 1520 bp for AYsecY_F-46/AYsecY_R1450) were produced from 84% of the symptomatic plants across all five species, but not from the asymptomatic ones. The PCR fragments were cloned and sequenced, and sequences obtained for the <i>tuf</i> and <i>secY</i> genes were deposited in GenBank under the accession numbers PV648950-PV648954, and PV854856-PV854860, respectively. The sequences obtained from the five plant species were almost identical, sharing 99.81% identity with the Aster yellows phytoplasma strains IRap (AJ271316.1) and ORN (MN526022.1) in the case of the elongation factor TU, and 100% identity with the strains De Villa (CP035949.1) and M8 (CP128414.1) in the case of the SecY translocase protein. In <i>i</i>PhyClassifier analysis, the virtual RFLP patterns derived from the query 16S rDNA F2nR2 fragments were identical (similarity coeffcient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession: AP006628). These results clearly indicate that the five tree species under study were affected by the aster yellows phytoplasma, which is assigned to the 16SrI-B subgroup of the 16SrI group and is caused by the bacterial agent '<i>Candidatus</i> Phytoplasma asteris'. Although this phytoplasma is known to infect a wide range of hosts (Lee et al., 2004), aster yellows has only been reported on <i>F. exelsior</i> in Poland (Kamińska and Berniak, 2009) of the five plant species studied here. To our knowledge, this is the first report of aster yellows on <i>A. campestre</i>, <i>A. platanoides</i>, <i>U. minor</i> and <i>S. nigra</i> worldwide, and on <i>F. exelsior</i> in Hungary.