First Report of 'Candidatus Phytoplasma fragariae' Infecting Hazelnut in Italy.

Abstract

Italy is the second largest producer of hazelnuts in the world after Turkey. The Viterbo province (Latium region) is one out of the four main productive districts, accounting for approximately 30% of the total Italian production. Several pathogens can severely affect hazelnut trees (Corylus avellana), mainly fungi and bacteria that are commonly spread in all the growing areas. However, other pathogens such as phytoplasmas, are known to infect this crop worldwide. Among these, 'Candidatus Phytoplasma fragariae' (16SrXII-E) has been recently associated with the decline and death of hazelnut trees in UK where it was identified for the first time on this plant species (Hodgetts et al., 2015), and in Slovenia (Mehle et al., 2018; Mehle et al., 2019). In July 2020, a survey was carried out in two orchards located in the Viterbo province, where symptoms resembling those induced by phytoplasmas and consisting in yellowing and rolling of leaves and tree decline (Supplementary figure 1) were recorded by growers. To ascertain the possible presence of phytoplasma infections, leaf samples were collected from five symptomatic plants: one plant from orchard 1, cv. Tonda Gentile Romana and four plants from orchard 2, cv. Tonda di Giffoni. Samples from four non-symptomatic plants (two per each orchard) were also collected and used as negative controls in the detection assays. DNA was extracted from 0.6 g of leaf ribs homogenised in CTAB buffer according to Marzachì et al. (1999). DNA extracts were amplified by direct and nested PCR employing the universal primer pairs P1/P7 and R16F2n/R2 according to the EPPO Standard PM7/133(1) - Appendix 2 (EPPO, 2018). Nested amplicons of the expected size (1,250 bp) were obtained from the five symptomatic samples whereas no bands were observed for the symptomless ones. The R16F2n/R2 fragments were submitted to both Sanger sequencing and RFLP analysis for the phytoplasma identification. Nucleotide BLAST analysis (NCBI) of the obtained sequences showed an identity percentage ranging from 99.65% and 99.91% with numerous strains of 'Ca. Phytoplasma fragariae' (16SrXII-E). The RFLP analysis was performed with the MseI restriction enzyme, and the obtained profiles were compared in the Codon Code Aligner software to those virtually generated from reference sequences of phytoplasmas belonging to the 16SrXII subgroups identified so far (Kazeem et al., 2021). All the five samples showed a common RFLP profile which was ascribable to that virtually generated from the 16SrXII-E reference sequence DQ086423 (Valiunas et al., 2006) (Supplementary figure 2). To our knowledge, this is the first finding of 'Ca. P. fragariae' on hazelnut in Italy since the previous records on woody plant hosts regarded Cornus sanguinea and Sambucus nigra (Filippin et al. 2008). Further surveys are ongoing to assess the spread of 'Ca. P. fragariae' in the Italian hazelnut orchards and to identify the putative insect vectors. The nucleotide sequences obtained in this study were deposited in GenBank NCBI database (accession numbers: MW767127, MW767128, MW767129, MW767130, MW767131).