Transmission of 16SrIII-J phytoplasma by Paratanus exitiosus leafhopper.

Phytoplasmas found in Chilean grapevines showing yellows symptoms were identified as belonging to the ribosomal subgroups 16SrI-B and 16SrI-C (‘Candidatus Phytoplasma asteris’), 16SrIII-J (X-disease group), 16SrV-A (‘Ca. P. ulmi’), 16SrVII-A (‘Ca. P. fraxini’), 16SrXII-A (stolbur or “bois noir”) (Gajardo et al., 2009; González et al., 2010). The presence of these pathogens in the plants depends on both propagation of infected plants and spreading by different insect species which feed on grapevine and also on the weeds growing near and/or in vineyards. In infected vineyards, several insects belonging to the family Cicadellidae positives to phytoplasmas were found. The most common was Paratanus exitiosus (Beamer) in which phytoplasmas of 16SrI-B, 16SrIII-J, 16SrVII-A, and 16SrXII-A subgroups were detected. In the present work, we verified the phytoplasma transmission ability of the leafhopper P. exitiosus. Materials and Methods During 2011 (since September till December) and 2012 (since January till May) leafhopper survey was carried out in Chilean vineyards infected by phytoplasmas, two located in Metropolitana Region (1 and 2) and one in Valparaiso Region (3), to know biological characteristics of P. exitiosus and its ability to transmit phytoplasmas. The insects were captured by sweeping with an entomological net. During the sampling period adults of P. exitiosus captured have been released into entomological cages to let them feed on three plants of periwinkle (Catharanthus roseus (L.) G. Don) grown from seed and previously tested to ascertain the absence of phytoplasmas. A total of 81 plants were used. Periwinkle plants were tested starting five months after transmission trials, while dead insects were then preserved in 70% ethanol. Insects and periwinkles were tested in order to identify the phytoplasma presence. Total nucleic acids (TNAs) were extracted with chloroform/phenol methods, dissolved in Tris-EDTA pH 8.0 buffer, and maintained at 4°C; 20 ng/µl of nucleic acid were used for amplification. After direct PCR with primer pair P1/P7, nested PCR with R16F2n/R2 primers (Gundersen and Lee, 1996) was performed. PCR and nested PCR reactions were carried out following published protocol (Schaff et al., 1992). P1/P7 amplicons were purified using Concert Rapid PCR Purification System and DNA fragments were cloned. Putative recombinant clones were analyzed by colony PCR. Selected fragments from cloned DNAs were sequenced in both directions using the BIG DYE sequencing terminator kit. The sequences were then aligned with BLAST engine for local alignment (version Blast N 2.2.12). Identification was done using in silico restriction fragment length polymorphism (RFLP) analyses on sequences amplified with primer pair R16F2n/R2 with BstU1 and HhaI restriction enzymes (Wei et al., 2007). Results and Discussion In the vineyard 1 the P. exitiosus capture rate remained constant during all months, with a decrease in October 2011 and April 2012. In the vineyard 2 the highest number of individuals was obtained in December 2011 and January 2012. In the vineyard 3 P. exitiosus was less abundant in September 2011, remained constant during the other months and increased only during February 2012. These results indicate that P. exitiosus tends to be more abundant during summer time. Three out of 81 periwinkles used for transmission trials were positive to phytoplasmas. Two (VC28C and VC31C) correspond to the transmission trials carried out with insects captured in the vineyard 1 in two different months (November and December 2011 respectively), the third (VC33A) was infected from insects captured in the vineyard 3 during December 2011. Cloned P1/P7 fragments were sequenced and there was no sequence difference between the cloned fragments from the three periwinkles (1,819 bp). The similarity percentages of VC28C, VC31C, and VC33A phytoplasmas, showed a close correlation (99.6, 99.7, 99.6 % respectively) with the strain Ch10 (AF147706...