Fitoplasmosi dell'erba medica in Argentina: caratterizzazione del fitoplasma associato all' "Escoba de bruja" e identificazione di un potenziale vettore

The work focused on the phytoplasma disease known as "escoba de bruja", which infects alfalfa crops in Argentina. The research was conducted in part in Argentina, at the Instituto de Patología Vegetal (IPAVE-CIAP-INTA) of Córdoba and was completed in Italy, in the laboratories of the Istituto di Virologia Vegetale (IVV-CNR) and the Dipartimento di Scienze Agrarie Forestali e Alimentari (DISAFA), section of Entomologia Agraria, University of Turin. Phytoplasmas are transmitted by phloem-feeding insects, belonging to the order Hemiptera, families Cixiidae (planthoppers), Cicadellidae (leafhoppers) and Psyllidae (psyllids). The identification of causative agents and carrier insects' species are the prerequisite to design rational phytoplasma control strategies. This project aims at clarifying the epidemiology of the ¿escoba de bruja¿, by characterising the pathogen and seeking putative vector insects. Besides, we wanted to lay the foundations for a future collaboration between the research groups in Argentina and Italy. During October 2012, several fields in the Mendoza and San Juan's provinces (in Argentina) have been inspected. The symptomatic alfalfa plants have been sampled, and associated insects have been collected, by using sweeping nets. Afterwards, molecular analyses have been carried out in the laboratory: DNA was extracted from both plants and insects; PCR amplification (direct and nested) was performed using primers pairs which amplify a phytoplasma-specific ribosomal DNA region (P1/P7 and R16F2/R2); RFLP analysis were done on the PCR products; amplicons were cloned and sequenced in order to carry out a sequence analysis. Concerning the insects, some specimens of Cicadellidae have been stored under ethanol to achieve a species identification based on the observation of morphological characters. For 18 out of the 33 samples of alfalfa examined with molecular analysis, it has been possible to associate the observed symptoms to the phytoplasmas of the group 16Sr VII-C, ArAWB (Argentinian alfalfa withces'-broom). For the remaining 15, originated by the the same sampling, no phytoplasma has been identified. Analyses of insects have shown that some specimens of the Membracide Ceresa nigripectus were infected by ArAWB. It is therefore possible that this species is involved in phytoplasma transmission in alfalfa crops. Transmission tests shall be done to confirm this hypothesis. It is worthy to note that so far no Membracidae species has been reported as phytoplasma vector. Moreover, the mitochondrial gene coding for the subunit cytochrome oxidase I (COI) of C. nigripectus has been amplified and sequenced in order to input the sequence in the Life Data Systems Barcode. This database collects all the known insect mtCOI sequences and use them as a species-specific identification tool. The highest number of vector COI sequences in the Barcode of Life the easiest will be the vector identification for future epidemiological studies. In conclusion, it has been proven that: group 16Sr VII-C, ArAWB (Argentinean alfalfa witches'-broom) phytoplasmas are associated with the ¿escoba bruja¿ disease; a variable incidence of this phytoplasmosis occurs in the different inspected areas; the membracid treehopper Ceresa nigripectus, a phloem-feeding insect species feeding on alfalfa in Argentina, hosts the phytoplasma associated with the ArAWB, and therefore is a putative vector. However, further aspects of the ArAWB epidemiology in Argenti