First Report of the Spittlebug Lepyronia quadrangularis and the Leafhopper Latalus sp. as Vectors of the Elm Yellows Associated Phytoplasma, Candidatus Phytoplasma ulmi in North America.

Abstract

Elm yellows (EY) is a lethal disease of American (Ulmus americana L.) and other elm species (1). On the Pennsylvania State University campus, EY, together with Dutch elm disease, has killed 82 of about 400 mature elms since 2007, the year of first EY detection. Candidatus Phytoplasma ulmi, associated with EY, has been reported to be transmitted by the whitebanded elm leafhopper Scaphoideus luteolus Van Duzee, the meadow spittlebug Philaenus spumarius L., and the leafhopper Allygus atomarius Fabricius (1) in North America, but correlation of these insects with EY in the eastern United States has not been reported. Three Cicadellidae collections using sweep nets and aspirators were performed from July to September 2012 on branches of an EY infected red elm (U. rubra Muh; 40°48.408'N, 77°52.208'W) and on vegetation within a 0.5 km radius. The red elm is in close proximity to trees, shrubs, and a managed meadow and has repeatedly tested positive for EY since 2007. During each collection, about 200 cicadellids were captured in BioQuip No-See-Um catch bags with cups, and the bags were hung around the red elm branches, forcing the insects to feed on the infected tree for 24 h. Insects were transferred to BugDorm rearing tents containing wild grasses, elm seedlings, cowpeas, celery, carrots, and basil, all grown from seed, and were kept for 3 weeks in a controlled environment chamber at 28°C and 70% humidity with a 16-h photoperiod. Insects easily recognized in the same species or individual insects of uncertain identity were then isolated for about 1 week in cages each containing one 6-month-old healthy American elm seedling (grown from seed in growth chamber). Up to 10 morphospecies were found in each collection, with 1 to 20 individuals per morphospecies. The total number of unique morphospecies used in the three transmission trials and later identified as different species was 8. Dead insects collected daily were stored in 80% ethanol and later identified to genus or species level. About 70% insect mortality was recorded, but about 60 individuals from each collection survived the change of diet and environment. After 3 months, individual elm seedlings were tested by RT-PCR (3) for the presence of phytoplasmas using universal primers fU5/rU3 (2). PCR products were visualized on 1.5% agarose gel, and if DNA was amplified, it was cloned and sequenced. Three of 30 seedlings tested positive for phytoplasmas and sequencing of the cloned products (24 clones were sequenced per transformation, per each of the three positive seedlings) confirmed that only Ca. P. ulmi was present in the 3 infected seedlings but not in the remaining 27 or in 46 unexposed control seedlings. The 3 seedlings were each exposed to a single insect and the same insects that were used in the transmission trial were identified. One spittlebug (Cercopidae) Lepyronia quadrangularis Say, one P. spumarius, and one leafhopper in the genus Latalus (Cicadellidae: Deltocephalinae) were identified as vectors. The phytoplasma-positive seedlings showed stunting and yellowing, and died shortly after testing. Other insects captured and identified in the survey were A. atomarius, Neophilaenus lineatus L., Metcalfa pruinosa Say, Amblysellus curtisii Fitch and individuals in the genera Draeculacephala, Elymana, Empoasca, Mesamia, Stroggylocephalus, and Ceratagallia. S. luteolus was not captured during this sampling but was captured on yellow sticky traps and in light traps in previous years at other locations on the campus. This is the first report suggesting that L. quadrangularis and Latalus sp. can serve as natural vectors of EY.