The epidemiology and ecology of the Bois Noir disease in Eger: Tuf gene based molecular characterisation of 'Candidatus Phytoplasma solani'

Topic & objectives: Bois Noir disease Bois Noir (BN) disease is a grapevine yellows (GY) disease that can cause severe symptoms on grapevines resulting in significant reduction in yield and fruit quality. BN is transmitted by insect vectors from alternative host plants (namely bindweed and stinging nettle) to grapevine and cannot be transmitted from grapevine to grapevine. Therefore, the disease cycle is very complex and BN management is difficult. Since the number of BN cases has increased in recent years causing severe economic losses to viticulture, and because of the limited knowledge on driving mechanisms of this disease, the epidemiology of BN still needs to be investigated. Such knowledge may help the development of environmental-safe pest management programmes for more profitable grape production. Methods: The 'Candidatus Phytoplasma solani' infection status of 15 experimental plants (situated on Kőlyuktető in Eger wine region, in Hungary)was confirmed using PCR/RFLP with P1/P7 universal primers, followed by R16F2n/R16R2 primers, that target the 16S rDNA phytoplasma. Then, for genotyping, Tuf gene was amplified by nested PCR using StoltufF0/R0 and fTufAY/rTufStol primers. All the nested PCR products were analyzed by electrophoresis in 1.2 % agarose gel, and visualized under UV. The PCR products were excised from the agarose gel and the fragments of each sample were purified by gel extraction kit. The purified samples were verified on 1.2 % agarose gel and finally 7 out of 15 150 ng fTufAY/rTufStol PCR products were sent for sequencing. For assembling and sequence editing Staden Package Version 3.3 was used. Nucleotide sequences were aligned with CLUSTAL W. Sequences were compared to Tuf genotypes Tuf-type a, Tuf-type b1, Tuf-type b2, Tuf-type b3 and Tuf-type c. Results: Tuf-type b1 strain was found in all of the 15 examined BN infected grapevines. These results are in accordance with the fact that Tuf-type b1 is prevalent in Eastern Europe, while the Tuf-type b2 is prevalent in Western Europe. Furthermore, my results are consistent with the fact that in Hungary, only Tuf-type b strain was observed and, so far, the Tuf-type a phytoplasma has not been detected, neither on stinging nettle nor on grapevine. Thus, my hypothesis that Tuf-type b1 is the dominant strain in the experimental vineyard, and perhaps in Eger region was confirmed. Main conclusions: Bois Noir disease is an emergent problem in Hungary. Presumably the Tuf-type b1 strain is dominant in Eger region. According to this, bindweed, Convolvolus arvensis, is the main 'Ca. P. solani' reservoir and measures should be focused against bindweed.