T. Olivier1*, E. Demonty1, G. Peusens2, T. Beliën2 and S. Steyer1
1 Walloon Agricultural Research Centre, 4 Rue du Liroux 5030 Gembloux, Belgium
2 Proefcentrum Fruitteelt vzw, Department Zoology, 1 Fruittuinweg 3800 Sint-Truiden, Belgium
Received: 19 Sep 2012; Published: 09 Dec 2012
Pear decline phytoplasma (PD) is a quarantine pest (‘harmful organism’) for the European Union that has been assigned to the Candidatus taxon, ‘Candidatus Phytoplasma pyri’. In October 2010, 137 samples consisting of leaves or roots from symptom-bearing pear trees showing early yellow or red discoloration (Fig. 1) were collected in pear production orchards in northeast Belgium.
Total DNA was extracted from 0.5 g of leaf midribs using a simplified CTAB extraction method (ANSES, 2010). Total DNA was used as a template for phytoplasma testing in a direct PCR assay using the universal primer pair fU5/rU3 (Lorenz et al., 1995) that targets the phytoplasma 16S rRNA gene. Amplified 16S rDNA fragments of expected size (approximately 880 bp) were directly sequenced and each obtained sequence was compared to those of reference phytoplasmas in GenBank by BLAST. In order to further study the genetic diversity of the phytoplasma isolates, some positive samples were additionally amplified and directly sequenced using the fHflB3_1/rHflB3 primer pair (Seemüller et al., 2011) that targets the putative transmembrane metalloprotease gene (HflB) for phytoplasmas.
Out of the 137 samples, approximately 15% gave positive results for ‘Ca. Phytoplasma pyri’ through PCR and sequencing. BLAST analysis of the consensus fU5/rU3 sequence (HF547271) revealed a 100% of sequence identity with that of ‘Ca. Phytoplasma pyri’ isolates (16SrX group, subgroup C). Four HflB amplicons of expected size (approximately 530 bp) from samples of four different municipalities were directly sequenced. The obtained sequences were 100% identical in the available regions, and the consensus HflB sequence (HE984352) showed 98.3% sequence identity with that of ‘Ca. Phytoplasma pyri’ partial HflB gene, strain PD1 (GenBank Accession No. FM201271). To our knowledge this is the first report of the molecular identification of ‘Ca. Phytoplasma pyri’ in Belgian pear orchards.
This work was partly supported by a grant of the Federal Public Service: Health, Food Chain Safety and Environment (RF-6206 research project).
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Olivier T, Demonty E, Peusens G, Beliën T, Steyer S, 2012. First report of molecular identification of 'Candidatus Phytoplasma pyri' in pear trees in Belgium. New Disease Reports 26, 29. [http://dx.doi.org/10.5197/j.2044-0588.2012.026.029]
©2012 The Authors