While performing a routine field survey on 2-year-old canes of Rubus fruticosus (cv. Evergreen Thornless) in the region of Plovdiv (central southern Bulgaria), severe stunting of single or grouped plants (3 to 4 in a row) was found in late August of 2009. It was noteworthy that the leaves of these plants were curved upwards and stayed green until the end of the season. The bushy aspect of the diseased plants led to the assumption of a phytoplasma origin; therefore, specific PCR and sequence based identification methods were applied on leaves, petioles, and stems from three infected Rubus plants grown in different rows of the field (midsummer, nine samples in total) and the same number of asymptomatic samples. Partial amplification of the 16S ribosomal RNA gene with generic phytoplasma primer pairs P1/P7 and fu5/ru3 (3), followed by a nested PCR specific for all members of the Phytoplasma stolbur subgroup by means of the stol11 primers (1), and an RFLP analysis of the tuf gene (elongation factor Tu) fragment produced with PCR primers tufAY/r tufAY (3), were used for the identification and characterization of the pathogen. All target amplicons were also sequenced by Macrogen (Seoul, South Korea) following gel purification (Nucleospoin Plant II, Macher-Nagel). Identical sequences were obtained from each of the P1/P7-derived amplicons (100% homology between samples) and a consensus 1,142 bp sequence was delineated and submitted to NCBI GenBank with accession no. JF293091. It had the highest similarity (99 to 100%) to sequences of ‘Bois noir’ phytoplasma (e.g. HQ589193; Candidatus Phytoplasma solani, position 29 to 1,171). The fu5/ru3 amplicons produced sequences that showed 99.5% homology to the Ca. Phytoplasma solani strains of a southern Russian and Romanian phytoplasma survey on different hosts (potato, tomato, Convolvulus) (GenBank Accession No. HM449999 to HM4450002). The stolbur specific primers also produced an amplicon in all samples and again the consensus sequence was identified (100% homology between the samples) and deposited in GenBank (JN561701). RFLP analysis of the tuf gene with the enzymes HindIII, HinfI, HpaII, and TaqI (Fermentas) produced the same profile types for the different samples and clearly allocated the phytoplasma in the tuf type-b (VKII), according to (2). This type is commonly reported as associated with bindweed (Convolvulus arvensis). Additionally, the sequenced tufAY fragment also confirmed a 100% correspondence with the submitted Tu elongation factor fragments of Ca. Phytoplasma solani strains in GenBank. No phytoplasma was detected in symptomless blackberry plants that were sampled from the same plot. In the molecular identification tests, a stolbur phytoplasma control (potato isolate), a Rubus stunt (EY subgroup, 16SrV) and an apple proliferation phytoplasma (AP subgroup, 16SrX) were used as controls. Based on the symptoms and the laboratory results, we concluded that the Rubus plants were infected by Ca. Phytoplasma solani, a species belonging to the stolbur subgroup (16SrXII-A). To our knowledge, this is the first report of Ca. Phytoplasma solani on Rubus fruticosus in Bulgaria. The disease is not likely to be an isolated case in the future because of the pathogen's spread on other hosts and the expected increase in blackberry fields.
References: (1) X. Daire et al. Eur. J. Plant Pathol. 103:507, 1997. (2) M. Langer and M. Maixner. Vitis 43:191, 2004. (3) K.-H. Lorenz et al. Phytopathology 85:771, 1995.