First report of a ‘Candidatus Phytoplasma asteris’ strain affecting lingonberry (Vaccinium vitis-idaea) and causing lingonberry stunt phytoplasma disease
A.-S. Brochu and A. Methot contributed equally to this work.
Vaccinium vitis-idaea is a red berry-producing woody shrub, native to North America, Europe, and Scandinavia, known commonly as lingonberry (Hendrickson, 1997). During the spring/summer seasons of 2019 and 2020, producers from Quebec, Canada, have noticed symptoms of yellowing leaves and stunted growth, resembling symptoms caused by phytoplasma.
To confirm whether phytoplasmas were indeed associated with these reported symptoms, fifteen lingonberry plants from different producers from the region of Chaudière-Appalaches were analysed (Table 1). Of these, nine had symptoms of stunting, little leaves and yellowing, while the rest showed no visible symptoms when collected (Figure 1). For each plant, total DNA was extracted from leaves and stem tissue using a CTAB-based method. The DNA was used as a template for a cpn60UT PCR with primers H279p/H280p (Dumonceaux et al., 2014), and the positive samples were subjected to direct PCR with phytoplasma 16S primers R16F2n/R16R2 as previously described (Perez-Lopez et al., 2019). Amplicons of the expected size (∼600 bp for cpn60UT) were obtained for 9/15 samples, while those for R16F2n/R2 sequences were obtained for 8/9 samples positives using the cpn60UT assay (Table 1). The cpn60UT and R16F2n/R2 amplicons obtained from two symptomatic samples (named LbSP14 and LbSP16 for lingonberry stunt phytoplasma) were purified and sequenced directly with the amplification primers (Centre Hospitalier de l'Université Laval de Quebec, Canada). The R16F2n/R2 sequence from LbSP14 was cloned into the pGEMT-Easy Vector (Promega, USA), and two clones were sequenced. Sequences were deposited in GenBank with Accession Nos OL739234 and OL739235 for LbSP14 and LbSP16 cpn60UT, respectively, and OL773369 and OL773370 for LbSP14 and LbSP16 R16F2n/R2 sequences. Accession Nos. OL773367 and OL773368 were used to register the sequences obtained from clone 1 (LbSP14c1) and clone 2 (LbSP14c2), respectively. A group/subgroup classification was performed on the LbSP cpn60UT sequences using CpnClassiPhyR (Muirhead et al., 2019) and R16F2n/R2 sequences with iPhyClassifier (Zhao et al., 2009). Phylogenetic relationships were determined using cpn60UT and R16F2n/R2 sequences of the LbSP phytoplasma and those from GenBank with the MEGA program version X (Kumar et al., 2018) and the neighbour-joining method with 1000 bootstraps. The CpnClassiPhyR classification suggested that the LbSP phytoplasma strain is a ‘Candidatus Phytoplasma asteris’-related strain and a member of the cpn60UT I-I(E/AI)AI subgroup (Figure 2a), with a similarity coefficient of 1.00 compared with blueberry stunt phytoplasma (BbSP; MH279496). The phylogenetic tree showed that LbSP strains branches with cpn60UT I, closely related to BbSP isolates from Quebec, Nova Scotia and New Brunswick (Figure 3). The 16S classification linked to the cpn60UT identified is 16SrI- (E/AI)AI (Perez-Lopez et al., 2019). We also found that LbSP displays 16S rRNA-encoding gene sequence heterogeneity with two distinct rrn operons, rrnE (OL773367) and rrnAI (OL773368), as previously reported for BbSP (Figure 2b and Figure 4).
| Cultivar | Symptoms | Sample | Tissue | Date of sample preparation | Date of DNA extraction | [DNA] ng/μL | A260/A280 | A260/A230 | Cpn60 PCR | 16S PCR |
|---|---|---|---|---|---|---|---|---|---|---|
| Sussi | Little leaves and yellowing | 1 | Leaf | 9-9-21 | 12-10-21 | 409.1 | 1.95 | 1.04 | Neg | Neg |
| 2 | Stem | 9-9-21 | 15-11-21 | 892.4 | 2.10 | 1.78 | Pos | Pos | ||
| Magenta | Little leaves and yellowing | 3 | Leaf | 9-9-21 | 12-10-21 | 189.1 | 1.85 | 0.89 | Neg | Neg |
| 4 | Stem | 9-9-21 | 15-11-21 | 981.2 | 2.09 | 1.78 | Pos | Pos | ||
| Koralle | Little leaves and yellowing | 5 | Leaf | 9-9-21 | 12-10-21 | 433.7 | 1.98 | 1.11 | Neg | Neg |
| 6 | Stem | 9-9-21 | 15-11-21 | 732.7 | 2.11 | 1.75 | Pos | Pos | ||
| Sussi | Asymptomatic | 7 | Leaf | 9-9-21 | 12-10-21 | 186.5 | 1.89 | 0.99 | Neg | Neg |
| 8 | Stem | 9-9-21 | 15-11-21 | 560 | 2.07 | 1.45 | Pos | Pos | ||
| Magenta | Asymptomatic | 9 | Leaf | 9-9-21 | 12-10-21 | 142.7 | 1.75 | 0.74 | Neg | Neg |
| 10 | Stem | 9-9-21 | 15-11-21 | 211.5 | 1.97 | 1.16 | Neg | Neg | ||
| Koralle | Asymptomatic | 11 | Leaf | 9-9-21 | 12-10-21 | 170.2 | 1.91 | 0.93 | Neg | Neg |
| 12 | Stem | 9-9-21 | 15-11-21 | 673.6 | 2.06 | 1.65 | Pos | Pos | ||
| Koralle | Little leaves and stunted plants | 13 | Leaf | 9-9-21 | 20-10-21 | 546.9 | 1.98 | 1.21 | Neg | Neg |
| 14 | Leaf | 9-9-21 | 14-9-21 | 1512.5 | 2.01 | 1.8 | Pos | Pos | ||
| 15 | Stem | 9-9-21 | 15-11-21 | 2909.5 | 2.12 | 1.72 | Neg | Neg | ||
| Magenta | Little leaves and yellowing | 16 | Leaf | 9-9-21 | 15-11-21 | 228.3 | 1.51 | 0.69 | Pos | Pos |
| 17 | Leaf | 9-9-21 | 14-9-21 | 184 | 1.47 | 0.63 | Neg | Neg | ||
| 18 | Stem | 9-9-21 | 15-11-21 | 3824.8 | 1.98 | 0.94 | Neg | Neg | ||
| Magenta | Little leaves and yellowing | 19 | Leaf | 9-9-21 | 12-10-21 | 324.4 | 0.91 | 0.27 | Neg | Neg |
| 20 | Stem | 9-9-21 | 15-11-21 | 1047.8 | 1.94 | 0.94 | Neg | Neg | ||
| Koralle | Little leaves and stunted plants | 21 | Leaf | 9-9-21 | 12-10-21 | 142.4 | 1.13 | 0.30 | Neg | Neg |
| 22 | Stem | 9-9-21 | 15-11-21 | 3418.8 | 1.94 | 0.96 | Neg | Neg | ||
| Koralle | Little leaves and stunted plants | 23 | Leaf | 9-9-21 | 12-10-21 | 195.7 | 1.17 | 0.39 | Neg | Neg |
| 24 | Stem | 9-9-21 | 15-11-21 | 631.2 | 1.92 | 1.06 | Neg | Neg | ||
| Koralle | Little leaves and stunted plants | 25 | Leaf | 9-9-21 | 20-10-21 | 307.6 | 0.92 | 0.25 | Neg | Neg |
| 26 | Leaf | 9-9-21 | 14-9-21 | 364 | 1.73 | 0.94 | Neg | Neg | ||
| 27 | Stem | 9-9-21 | 15-11-21 | 1670.7 | 1.83 | 0.54 | Neg | Neg | ||
| Erntesegen | Asymptomatic | 28 | Leaf | 9-9-21 | 20-10-21 | 101.7 | 1.13 | 0.36 | Neg | Neg |
| 29 | Leaf | 9-9-21 | 15-11-21 | 389 | 1.68 | 0.94 | Neg | Neg | ||
| 30 | Stem | 9-9-21 | 15-11-21 | 1366.8 | 2.03 | 1.3 | Pos | Neg | ||
| Koralle | Asymptomatic | 31 | Leaf | 9-9-21 | 20-10-21 | 139.4 | 0.88 | 0.27 | Neg | Neg |
| 32 | Leaf | 9-9-21 | 15-11-21 | 196.1 | 1.55 | 0.89 | Neg | Neg | ||
| 33 | Stem | 9-9-21 | 15-11-21 | 1697 | 2.00 | 1.19 | Neg | Neg | ||
| Koralle | Asymptomatic | 34 | Leaf | 9-9-21 | 12-10-21 | 226.8 | 1.76 | 0.87 | Neg | Neg |
| 35 | Stem | 9-9-21 | 15-11-21 | 704.7 | 2.08 | 1.46 | Pos | Pos |
To our knowledge, this is the first report of lingonberry as a possible phytoplasma host. Other Vaccinium species, for example blueberry (Vaccinium corymbosum), are known to be affected by phytoplasmas in the same 16SrI- (E/AI)AI subgroup in Quebec (Perez-Lopez et al., 2019), and by 16SrI-E phytoplasmas in Canada and the USA (Lee et al., 2004; Arocha-Rosete et al. 2019). The fact that blueberries and lingonberries can be affected by phytoplasma strains in the same phytoplasma subgroup may suggest a common insect vector with variable feeding habits. Our findings are the basis for the understanding of the colonisation of a new plant host in the family Ericaceae by phytoplasmas.
ACKNOWLEDGMENTS
We would like to thank the MAPAQ and Les Airelles des Frères, specially to F. Philippe Roy for the support and collaboration, and to Prof. Jean Collin who through the class PLG-3305 supported the completion of the research.
