UNIVERSITA' DEGLI STUDI DI PADOVA ___________________________________________________________________ SCUOLA DI DOTTORATO DI RICERCA IN SCIENZE DELLE PRODUZIONI VEGETALI INDIRIZZO PROTEZIONE DELLE COLTURE - CICLO XX Dipartimento di Agronomia Ambientale e Produzioni Vegetali INVESTIGATIONS ON THE PSYLLID (HEMIPTERA: PSYLLIDAE) VECTORS OF ‘ Candidatus Phytoplasma mali’ IN TRENTINO Direttore della Scuola : Ch.mo Prof. Andrea Battisti Supervisore : Ch.mo Prof. Vincenzo Girolami Dottorando : Federico Pedrazzoli DATA CONSEGNA TESI 2 febbraio 2009 To M. Elisabetta, an example of honesty, diligence and humanity Declaration I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of the university or other institute of higher learning, except where due ac- knowledgment has been made in the text. A copy of the thesis will be available at http://paduaresearch.cab.unipd.it/ Dichiarazione Con la presente affermo che questa tesi è frutto del mio lavoro e che, per quanto io ne sia a conoscenza, non contiene materiale precedentemente pubblicato o scritto da un'altra perso- na né materiale che è stato utilizzato per l’ottenimento di qualunque altro titolo o diploma dell'università o altro istituto di apprendimento, a eccezione del caso in cui ciò venga rico- nosciuto nel testo. Una copia della tesi sarà disponibile presso http://paduaresearch.cab.unipd.it/ Index Index .......................................................................................................................................7 Summary.................................................................................................................................9 Riassunto ..............................................................................................................................13 Introduction..........................................................................................................................17 I. Phytoplasmas ...............................................................................................................17 a) Morphology and ultrastructure.................................................................................18 b) Molecular characterisation of phytoplasmas............................................................19 c) Symptoms of phytoplasma-infected plants...............................................................20 d) Transmission and spread of phytoplasmas...............................................................20 e) Geographic and ecological distribution of phytoplasmas.........................................21 f) Host specificity of phytoplasmas..............................................................................22 g) Ecological niches and evolution of new phytoplasma strains ..................................23 h) Disease control .........................................................................................................24 II. Phytoplasma transmission by insect vectors................................................................25 a) Taxonomic groups of phytoplasma vectors..............................................................26 b) Phytoplasma-insect vector interactions ....................................................................27 c) Phytoplasma-insect vector specificity......................................................................30 d) Transovarial transmission.........................................................................................32 e) Effects of phytoplasma on the vector .......................................................................32 f) Factors mediating the transmission efficiency .........................................................34 g) Phytoplasma vector dispersal ...................................................................................34 III. ‘Candidatus Phytoplasma mali’...................................................................................37 a) Host plants................................................................................................................38 b) Symptoms.................................................................................................................39 c) Diagnosis ..................................................................................................................43 d) Transmission.............................................................................................................47 e) The situation in Trentino ..........................................................................................48 IV. Transmission of ‘ Ca. Phytoplasma mali’ by psyllid vectors.......................................49 a) The research on psyllid vectors in Trentino .............................................................50 b) Other experiences .....................................................................................................52 7 c) Biology of psyllids ...................................................................................................54 d) Cacopsylla picta Förster (1848) ...............................................................................60 e) Cacopsylla melanoneura Förster (1848) ..................................................................64 f) Biology of Cacopsylla picta and C. melanoneura in Trentino.................................69 V. Aims of the research ....................................................................................................71 VI. References....................................................................................................................72 Chapter 1 Acquisition capacities of the overwintering adults of the psyllid vectors of 'Candidatus Phytoplasma mali' ...................................................................................101 Chapter 2 Acquisition and transmission of ' Candidatus Phytoplasma mali' by its psyllid vectors in Trentino .......................................................................................................103 Chapter 3 Detection of ' Candidatus Phytoplasma mali' in different populations of Cacopsylla melanoneura Förster (Hemiptera: Psyllidae) ..........................................133 Chapter 4 A preliminary study of the effects of ' Candidatus Phytoplasma mali' on the psyllid Cacopsylla melanoneura (Hemiptera: Psyllidae) ..........................................149 Chapter 5 Characterization of microsatellite loci in Cacopsylla melanoneura Förster (Homoptera: Psyllidae) ................................................................................................157 Chapter 6 Differences in populations of Cacopsylla melanoneura (Hemiptera, Psyllidae): insights from ecological and molecular studies .......................................161 Conclusions ........................................................................................................................189 Aknowledgements...............................................................................................................191 8 Summary 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,