Transmission trials using in vitro plants: a new protocol to confirm phytoplasma insect vector species

In Chile the phytoplasmas associated to grapevine yellows belong to the ribosomal subgroups 16SrI-B and 16SrI-C (‘Candidatus Phytoplasma asteris’-related), 16SrIII-J (‘Ca. P. pruni’-related), 16SrV-A (‘Ca. P. ulmi’), 16SrVII-A (‘Ca. P. fraxini’), and 16SrXII-A (‘Ca. P. solani’ or “stolbur”). The prevalent are phytoplasmas 16SrIII-J that are widely distributed mainly in South America. The "flavescence dorée" phytoplasmas and its vector Scaphoideus titanus, have not been found in Chile. Previous epidemiological studies indicate that weed, shrub, and non-grapevine woody plants from different botanical families present in the vineyards and in their surroundings (Convolvulus arvensis, Galega officinalis, Polygonum aviculare, Malva sp., Brassica rapa, Rubus ulmifolius, and Rosa sp.) are reservoirs of 16SrIII-J phytoplasma, and the leafhoppers Paratanus exitiosus and Bergallia valdiviana are vectors of the same pathogen. Other leafhopper species, Bergallia sp., Amplicephalus ornatus, Amplicephalus curtulus, Amplicephalus pallidus, and Exitianus obscurinervis, captured in a vineyard planted with Pinot noir, were identified as new potential vectors of the 16SrIII-J phytoplasma. To carry out the transmission trials (TT), adult individuals were captured monthly from October 2017 to June 2021, using an entomological sweeping net. The insects were identified at the species level based on morphological characteristics. Periwinkle and grapevine cultivar Cabernet Sauvignon micropropagated phytoplasmas-free plantlets, were used in the TT. Between 4 and 6 insects of the same species were introduced to each in vitro plant tube. For each TT, the insects have been allowed to feed for a maximum of 7 days and were collected as they dye or after 7 days and stored in 70% ethanol. At the end of the TT, the plants were treated with fungicides (Captan and Tebuconazole), transferred in a solid sterilized substrate composed of peat and perlite in a 2:1 ratio, and kept in a conditioned incubator at 25ºC under 16 h/day light. To date, 235 in vitro TT have been carried out, using 123 and 112 grapevine and periwinkle plants, respectively. Eight plants without contact with insects have been used as control. Plants and insects used in the TT have been analyzed by nested-PCR with P1/P7 followed by R16F2n/R2 primers. The amplification products (1,250 bp) were sequenced, and the identification of phytoplasmas was carried out by in silico RFLP using the enzymes HhaI, BstUI and RsaI. All the plants were analyzed 3, 10 and 18 months after the start of TT. All the insect species were able to transmit the 16SrIII-J phytoplasma to grapevine and/or periwinkle plants: A. ornatus (1 grapevine); A. pallidus (4 grapevine, 1 periwinkle); Bergallia sp. (3 grapevine, 2 periwinkle); E. obscurinervis (1 grapevine, 3 periwinkle); A. curtulus (5 grapevine, 2 periwinkle). The plants used as control were negative for phytoplasma presence. The plants positive for phytoplasmas, start to show symptoms two months after the beginning of TT. Plants that were kept in contact with insects and that were negative for phytoplasmas, are still under observation and will be tested again in the next months. The use of in vitro plants for TT has been successful in identifying new insect vector species. This is due very likely to the reduced stress the insects suffer during the experiments of transmission.