Detection and molecular characterization of viruses infecting Actinidia spp.

Kiwifruit (genus Actinidia) is an important horticultural crop grown in the temperate regions. The four world’s largest producers are China, Italy, New Zealand and Chile. More than 50 species are recognized in the genus but the principal species in cultivation are A. deliciosa and A. chinensis. In Italy, as well as in many other countries, the kiwifruit crop has been considered to be relatively disease free and then no certification system for this species has been developed to regulate importation of propagation plant material in the European Union. During the last years a number of fungal and bacterial diseases have been recorded such as Botrytis cinerea and Pseudomonas syringae pv. actinidiae. Since 2003, several viruses and virus-like diseases have been identified and more recent studies demonstrated that Actinidia spp can be infected by a wide range of viral agents. In collaboration with the University of Auckland we have been detected thirteen different viral species on kiwifruit plants. During the three years of my PhD I worked on the characterization of Cucumber mosaic virus (CMV) and Pelargonium zonate spot virus (PZSV). The determination of causal agents has been based on host range, symptom expression in the test plant species and morphological properties of the virus particles using transmission electron microscopy (TEM) and using specific oligonucleotide primers in reverse transcription-polymerase chain reaction (RT-PCR). Both viruses induced several symptoms on kiwifruit plants. Moreover with new technologies such as high-throughput sequencing we detected additional viruses, a new member of the family Closteroviridae and a new member of the family Totiviridae. Taking together all results of my studies it is clear that, in order to minimize the risk of serious viral disease in kiwifruit, it is vital to use virus-free propagation material in order to prevent the spread of these viruses.

Produzione di virus sintetici per lo studio dei meccanismi di interazione coinvolti nell'induzione di resistenza

Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV) are members of Benyvirus genus. BSBMV has been reported only in the United States while BNYVV has a worldwide distribution. Both viruses are vectored by Polymyxa betae, possess similar host ranges, particles number and morphology. Both viruses are not serologically related but have similar genomic organizations. Field isolates consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs 1 and 2 are essential for infection and replication while RNAs 3 and 4 play important roles on plant and vector interactions, respectively. Nucleotide and amino acid analyses revealed BSBMV and BNYVV are different enough to be classified in two different species. Additionally in BNYVV/BSBMV mixed infections, a competition was previous described in sugar beet, where BNYVV infection reduces BSBMV accumulation in both susceptible and resistant cultivars. Considering all this observations we hypothesized that BNYVV and BSBMV crossed study, exploiting their similarities and divergences, can improve investigation of molecular interactions between sugar beets and Benyviruses. The main achievement of our research is the production of a cDNA biologically active clones collection of BNYVV and BSBMV RNAs, from which synthetic copies of both Benyviruses can be transcribed. Moreover, through recombination experiments we demonstrated, for the first time, the BNYVV RNA 1 and 2 capability to trans-replicate and encapsidate BSBMV RNA 3 and 4, either the BSBMV RNA 1 and 2 capability to replicate BNYVV RNA2 in planta. We also demonstrated that BSBMV RNA3 support long-distance movement of BNYVV RNA 1 and 2 in B. macrocarpa and that 85 foreign sequence as p29HA, GFP and RFP, are successfully expressed, in C. quinoa, by BSBMV RNA3 based replicon (RepIII) also produced by our research. These results confirm the close correlation among the two viruses. Interestingly, the symptoms induced by BSBMV RNA-3 on C. quinoa leaves are more similar to necrotic local lesions caused by BNYVV RNA-5 p26 than to strongly chlorotic local lesions or yellow spot induced by BNYVV RNA- 3 encoded p25. As previous reported BSBMV p29 share 23% of amino acid sequence identity with BNYVV p25 but identity increase to 43% when compared with sequence of BNYVV RNA-5 p26. Based on our results the essential sequence (Core region) for the longdistance movement of BSBMV and BNYVV in B. macrocarpa, is not only carried by RNA3s species but other regions, perhaps located on the RNA 1 and 2, could play a fundamental role in this matter. Finally a chimeric RNA, composed by the 5’ region of RNA4 and 3’ region of RNA3 of BSBMV, has been produced after 21 serial mechanically inoculation of wild type BSBMV on C. quinoa plants. Chimera seems unable to express any protein, but it is replicated and transcript in planta. It could represent an important tool to study the interactions between Benyvirus and plant host. In conclusion different tools, comprising a method to study synthetic viruses under natural conditions of inoculum through P. Betae, have been produced and new knowledge are been acquired that will allow to perform future investigation of the molecular interactions between sugar beets and Benyviruses.