New genetically distinct phytoplasmas and insect carriers associated with pine tree disease revealed by a survey in Curonian Spit, Lithuania

Authors: IVANAUSKAS, ALGIRDAS - Nature Research Centre; VALIUNAS, DEIVIDAS - Nature Research Centre; RIMSAITE, JOLANTA - Nature Research Centre; DANILOVAS, JURIJUS - Vilnius University; SÖDERMAN, GUY - Finnish Environment Institute; ŠNEIDERIS, DONATAS - Nature Research Centre; GENOVAITE ŽIŽYTE-EID, MARIJA - Nature Research Centre; Wei, Wei

Submitted to: Canadian Journal of Forest Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/9/2021
Publication Date: 8/11/2022
Citation: Ivanauskas, A., Valiunas, D., Rimsaite, J., Danilovas, J., Söderman, G., Šneideris, D., Genovaite Žižyte-Eid, M., Wei, W. 2022. New genetically distinct phytoplasmas and insect carriers associated with pine tree disease revealed by a survey in Curonian Spit, Lithuania. Canadian Journal of Forest Research. 52(2): 201-208. https://doi.org/10.1139/cjfr-2021-0152.
DOI: https://doi.org/10.1139/cjfr-2021-0152

Interpretive Summary: In Curonian spit, Lithuania, pine trees play a key role in the stabilization of sand dunes and afforestation. In addition, their unique “dancing or drunken” appearance also attracts many tourists and photography enthusiasts. Since 2008, the diseased pine trees exhibiting excessive branching and shortened needles have been observed. Phytoplasmas, small phloem-restricted, and insect transmissible intracellular bacteria have been identified as the etiological agents that cause the disease. However, the insects that transmit pine phytoplasmas have not reported yet. In this study, ARS scientists, in collaboration with Lithuanian scientists, conducted a field survey in the Spit. Insects were collected from symptomatic pine trees and adjacent areas. Through insect identification, no known phytoplasma insect vectors were found. Furthermore, a literature search did not find any recognized phytoplasma insect vectors (leafhoppers, planthoppers, and psyllids) feeding on pine trees. These facts suggest that other phloem feeding insects may transmit phytoplasmas associated with pine diseases. In our insect collection, a total of 1018 insects were identified as phloem-feeding insects, of which aphids accounted for the majority. By employing PCR amplification, phytoplasmas were detected in scots pine aphids (Cinara pini), waxy grey pine needle aphids (Cinara pineti), and some unknown aphids. The results indicated that aphids were the insect carriers/hosts of pine phytoplasmas. Interestingly, in silico RFLP analysis based on 16S rRNA gene sequences revealed genetically distinct phytoplasma strains that represent a new subgroup lineage. The findings will benefit researchers and students who are interested in phytoplasma diversity, insect host and epidemiology of phytoplasma diseases.

Technical Abstract: Pines are widely cultivated due to their ornamental and commercial values, as well as their important roles in dune stabilization and afforestation. Pines have been severely affected in many countries, by infections of phytoplasmas, which are known as small, phloem-restricted, and insect-transmissible bacteria. However, the insect vectors that transmit pine phytoplasmas remain elusive. This study investigated the potential insect hosts of pine phytoplasmas in Curonian spit, Lithuania, from 2016 to 2019. Surprisingly, 1018 phloem-feeding insects (order Hemiptera) were identified in our collection, of which 98.60% were aphids (Aphididae), and no known phytoplasma vectors such as leafhoppers, planthoppers and psyllids were found. Moreover, a literature search revealed that recognized phytoplasma vectors rarely feed on pine trees. This suggests that pine phytoplasmas may be transmitted by other phloem-feeding insects. In this study, employing nested PCR with phytoplasma specific primers, the phytoplasmas were detected in scots pine aphids (Cinara pini), waxy grey pine needle aphids (Cinara pineti), and species-unknown aphids. Sequence analysis of aphid-harbored phytoplasma strains revealed that they were closely related to the reference strain of 'Candidatus Phytoplasma pini' (16SrXXI-A). Subsequent virtual RFLP analysis showed that phytoplasmas detected in aphids were mainly 16SrXXI-A variants, which were also main strains identified in diseased pine trees. In addition to main 16SrXXI-A variant strains, three new genetically distinct subgroups were also identified from previously overlooked Lithuanian pine phytoplasma strains (16SrXXI-C and 16SrXXI-D), and new aphid-harbored phytoplasmas (16SrXXI-E). Further transmission trial study on these pine aphids will provide insights into the epidemiology, and pathosystem of pine phytoplasma diseases, as well as the disease management.