Detection and preliminary identification of phytoplasmas in hazelnut in Chile

Introduction Decline and yellows are diseases reported since longtime in several European hazelnut (Corylus avellana L.) growing areas and were associated since 20 years to 16SrX group phytoplasma presence (1). More recently in stunted, but also in asymptomatic plants a 16SrIII-B phytoplasma was identified in Oregon (USA) (2). Hazelnut infection by phytoplasmas appears therefore a puzzling issue. During a survey in the Campo Experimental Maquehue, Universidad de La Frontera, Temuco, hazelnut plants cv. Barcelona 12 year-old showing leaf and catkin malformation, and yellowing in some branches were sampled to verify phytoplasma presence. Materials and Methods Leaf and small branch samples from symptomatic and asymptomatic hazelnut plants were collected at the beginning of the Spring (December) 2016 and, after a chloroform-based DNA extraction of phloematic tissues, they were subjected to nested PCR on the 16S ribosomal gene (3) followed by RFLP analyses and direct amplicon sequencing for phytoplasma identification and phylogenetic clustering, respectively. Results and Dıscussıon Only the symptomatic samples resulted positive to phytoplasma presence and RFLP analyses on 500 bp amplicons (M1/M2 primers) indicate the presence of 16SrI, 16SrIII, and 16SrV phytoplasmas. In particular, in samples showing yellow leaves 16SrI and 16SV phytoplasmas were identified in single or mixed infection; moreover RFLP analyses on amplicons obtained with the 16SrI group specific primers show the presence in the 16SrI phytoplasmas of a Tru1I profile that was different from all those reported so far. In the small branches with yellow in the leaflets surrounding the young nuts 16SrIII phytoplasmas were preliminary identified on 500 bp amplicons. The sequencing confirmed the phytoplasma clustering and the polymorphism determined by RFLP analyses. The yellowing observed in Chilean hazelnut was believed to be due to the presence of nutritional disorders related to the quite new environment for the crop. The finding of phytoplasmas is prompting to verify now their relationship with the symptomatology observed. The detection of several phytoplasmas in Chilean hazelnuts confirms the species susceptibility to these bacteria (1, 2) and their possible influence on the plant metabolism. Vegetative propagation together with still unknown insect vector(s) could be the main responsible for presence of several diverse phytoplasmas in the relatively restricted area where the hazelnut is starting to be grown in Chile.