Phytoplasmas associated with grapevine yellows : an overview

Phytoplasmas are obligate intracellular bacterial parasites restricted to the phloem sieve elements of the infected plants and are transmitted by phloem-sucking insects belonging to the families Cicadellidae, Cixidae, Psyllidae, Delphacidae and Derbidae. They are associated with diseases of several hundred plant species, including some economically important crops. Grapevine yellows (GY) is a worldwide disease complex caused by genetically different phytoplasmas. GY-affected Vitis vinifera shows leaf enrollment accompanied by yellowing or reddening, rubbering of the canes and desiccated clusters. Epidemiology of the different GY diseases, undistinguishable based on symptoms observation, strictly depends on the involved phytoplasma because of the insect-vector specificity and their behavior. GY diseases are attributed to infections by at least seven distinct phytoplasmas. In Europe, the GY diseases Flavescence dor\ue9e (FD) and Palatinate grapevine yellows (PGY, present only in Germany), are associated with phytoplasmas classified in the ribosomal group 16SrV, while Bois noir (BN) is attributed to phytoplasmas classified in stolbur group (ribosomal subgroup 16SrXII-A). In Australia, Australian grapevine yellows is attributed to \u2018Ca. Phytoplasma australiense\u2019 (ribosomal subgroup 16SrXII-B), and to \u2018Ca. Phytoplasma australasia\u2019 (ribosomal group 16SrII). Grapevine yellows in Virginia is associated with \u2018Ca. Pytoplasma asteris\u2019-related strains (ribosomal group 16SrI) and X-disease group (ribosomal group 16SrIII) phytoplasmas. In order to distinguish each GY from the others, an important research topic focuses on developing molecular tools for specific identification of GY-associated phytoplasmas. In Europe, the employment of such methods for the certain exclusion of FD and BN phytoplasmas from grapevine certified propagating material is becoming urgent. PCR-based techniques allowed development of useful tools for the identification of phytoplasmas associated with GY diseases. Standard protocols include nested PCR amplification of phytoplasma 16S rDNA using universal or group specific primers and RFLP analyses in order to determine the taxonomic (ribosomal group/subgroup) affiliation. Further molecular characterization, performed by sequence analyses on genes less conserved than 16S rDNA, found additional markers useful for developing suitable analytical tests for faster and specific detection of FD and BN phytoplasmas. Up to now, innovative molecular approaches developed to this aim are: (i) Real Time PCR and reverse transcription \u2013 Real Time PCR for the detection of phytoplasmas causing FD and BN; (ii) nanobiotransducer for detecting FD phytoplasmas; (iii) multiplex nested PCR for simultaneous identification of FD and BN phytoplasmas; (iv) Ligase Detection Reaction (LDR) DNA microarray to detect and distinguish FD and BN phytoplasmas. Furthermore, multiple gene sequence analyses (Multi Locus Sequence Typing, MLST) on ribosomal (rplV-rpsC) and extra ribosomal (secY, map, uvrB, degV, hlyC, vmp, and tuf) genes highlighted an unexpected genetic heterogeneity among both FD and BN phytoplasma populations, identifying different FD- and BN-phytoplasma strains that can be associated with specific ecological niches (plant hosts, insect vectors, geographic origin). MLST analyses improved the chance to associated phytoplasma-specific molecular markers with biological features, opening new perspectives for the studies of FD and BN epidemiology