INVESTIGATIONS ON THE PSYLLID (HEMIPTERA: PSYLLIDAE)VECTORS OF ‘Candidatus Phytoplasma mali’ IN TRENTINO

Phytoplasmas are cell wall-less, phloem-limited and so far uncultured bacteria which are associated with plant diseases of large economic impact. They are transmitted in a persis-tent propagative manner by phloem feeding insects like leafhoppers, planthoppers and psyl-lids. ‘Candidatus Phytoplasma mali’ is the etiological agent of apple proliferation (AP), a phytoplasma disease which may cause severe losses in many central-European apple grow-ing regions including Trentino. AP can affect the vigour of the apple trees and fruits of in-fected trees can not be commercialised because of their small size and poor taste. Two psyl-lid species (Cacopsylla picta and C. melanoneura) and a leafhopper (Fieberiella florii) have been demonstrated to be able to transmit ‘Ca. P. mali’. Whereas F. florii can be ex-cluded as important vector of the disease in Trentino, contradictory data have been reported for the role of the two psyllid species in the natural disease spread. This research had there-fore two specific aims: the study of the acquisition and transmission efficiencies of the two psyllid vectors and an analysis of the biology and genetics of populations of C. melanoneura applying bioassays and molecular tools. The acquisition and transmission efficiency of the different developmental instars of the two psyllids were studied in three consecutive years in experiments under controlled condi-tions, in which individuals were fed on micropropagated infected plants and then moved onto healthy test plants. The minimum acquisition access periods were established for the overwintered adults. After each trial, the insects were analysed by real-time PCR in order to estimate the phytoplasma level within the individuals. The experiments demonstrated that, in spite of a good acquisition efficiency found in all the developmental instars of the two species, only the nymphal stages and the new generation adults of C. picta were able to transmit the disease to healthy test plants. These results can be explained by a higher per-centage of high-titre individuals found for C. picta but not for C. melanoneura. In contrast, overwintered adults of both species acquired the phytoplasma already after short acquisition access periods of one to four days, but no significant further multiplication after the acqui-sition was observed. Thus, these results revealed a relationship between the phytoplasma level in the individuals and their transmission efficiency and explain why C. picta is in Trentino a more efficient vector of AP than C. melanoneura. Contradictory hypotheses have been proposed for the role of the oligophagous C. melanoneura in the epidemiology of AP. Therefore, the natural infection rate was studied by qualitative and quantitative PCR in overwintered adults collected from different plants (apple, hawthorn and conifers) in different areas (Trentino, Veneto, Aosta Valley and France). The analyses revealed different ratios of infected individuals among the different areas and even within the different areas of Trentino. Populations collected in northeastern Italy, where C. melanoneura was demonstrated to be an efficient vector, showed similar in-fection levels in samples collected from apple or hawthorn, while in Trentino only some populations captured on apple exhibited high natural infection rates. The presence of AP-infected individuals in samples collected on conifers demonstrates that the pathogen is re-tained within the insect body during winter. On the other hand, a positive correlation be-tween the infection rate and the incidence of the disease in apple orchards was found indi-cating an acquisition of the phytoplasma within the same season. The effect of the infection of ‘Ca. P. mali’ in C. melanoneura was studied in bioassays conducted under controlled conditions. The experiments demonstrated a detrimental effect of ‘Ca. P. mali’ on the fitness of this psyllid species, affecting both the number of eggs laid and the hatching rate, whereas the survival of the overwintered adults and the development of the nymphal stages seemed not to be damaged. Detrimental effects of the pathogen on its vector may indicate a recent co-evolution between the phytoplasma and C. melanoneura as a vector. As for C. melanoneura two different host plants (hawthorn and apple) are known, host-switching experiments were carried out to investigate the relationships between the psyllid and the two plant species. This study revealed that the two populations are dependent on their native host plant for egg laying and also for the development of the juvenile instars. A molecular approach was therefore applied to assess the genetic bases of these differences. The genetic variability of the populations was studied using microsatellite markers devel-oped for C. melanoneura and DNA sequences from the mitochondrial cytochrome oxidase subunit I. Data obtained from microsatellite analyses indicate a low, but statistically signifi-cant difference between the ‘apple’ and the ‘hawthorn’ populations. Mitochondrial DNA diversity was too low to differentiate the two populations. Furthermore, a genetic boundary was found separating the Aosta Valley populations from the others. Behavioural and ge-netic results indicate a differentiation among C. melanoneura populations linked to the host plants. In conclusion, C. picta was confirmed as most efficient vector of AP whereas the con-tradictory data reported previously for C. melanoneura could be attributed to the existence of different populations with different transmission efficiencies and to a lower multiplica-tion efficiency of the phytoplasma within the individuals of the Trentino population.