Transmission of the Mycopathogen, Hirsutella spp., to Nymphs and Adults of the Glassy-Winged Sharpshooter, Homalodisca Vitripennis (=Coagulata), in the Greenhouse

The mycopathogen Hirsutella spp. produces regular epizootics in Florida populations of the glassy-winged sharpshooter (GWSS), Homalo disca vitripennis, a polyphagous, xylem-feeding cicadellid native to the southeastern U.S. and northeastern Mexico (Boucias et al. 2007). The GWSS transmits a lethal phytopathogenic bacte rium, Xylella fastidiosa, and thereby threatens the production of economically important plants (Redak et al. 2004). Recent introduction of GWSS into southern California, French Polynesia, Tahiti and Hawaii has stimulated interest in identifying potential biocontrol agents against invasive pop ulations (Boucias et al. 2007; Hunnicutt et al. 2008). During a field survey in Quincy, FL, a new species of Hirsutella, H. homalodiscae nom. prov., was detected in -50% of mycosed sharpshooters (Boucias et al. 2007). Virtually nothing is known about the transmission of Hirsutella within GWSS populations. Our field observations have shown that Hirsutella-infected sharpshooters at tach via rhizoids to the plant bark in the summer and often remain attached throughout the winter. Placed in a moist environment, overwintered ca davers produce conidiospores suggesting they serve as microhabitats protecting Hirsutella and facilitating transmission to the new generation of GWSS. This study examined potential transmis sion routes of Hirsutella under greenhouse condi tions. Experimental treatments included topical spore application and choice-exposure to sporu lating cadavers on host plants. For insect rearing, field-collected, healthy adults without hyphal bodies (HBs) in their hemolymph were identified by non-destructive antennal bleeding (Breaux 2005) and maintained on caged, potted plants (soybean, Glycine max, cotton, Gossypium hirsutum, and cowpea, Vigna unguiculata) in a greenhouse (temperature 26 30°C, 14:10 h light:dark photoperiod). Leaves with egg masses were removed and hatching neo nates were transferred to caged lemon basil (Oci mum basilicum). Soil was watered to saturation once daily. In each bioassay replicate, individual cotton or basil plants housing groups of 10-20 adults or nymphs, respectively, were covered with clear, gauze-covered acryl cylinders (15 cm diam eter x 45 cm high). Mortality and infection were recorded daily by removing dead individuals from the soil surface and examining their hemolymph for HB propagation. Cadavers were maintained on water agar to record mycosis. After 3 weeks, each plant was examined for mycosed cadavers, and surviving GWSS were subjected to hemolymph examination. Statistical analyses were conducted using the Proc Genmod procedure and Ls means statement of the Statistical Analy sis System (SAS) for Windows to compare mortal ity or infection responses by logistic regression (Neter et al. 1990; SAS 2004). For topical application of Hirsutella spores, healthy adults were treated in three replicate as says by touching their ventral surface to either sporulating in vitro colonies of strain 3A (main tained at the UF Entomology/Insect Pathology lab, Gainesville, FL) (total n = 46 adults), to GWSS cadavers displaying spores of Hirsutella (n = 33), or to a nutrient agar plate (control, n = 35). Mortality in control, in vitro, and cadaver treat ments was similar with 56 ± 11%, 74 ± 12%, and 60 ± 8%, respectively (x2= 2.48, P = 0.1155). Con tac with agar, in vitro colonies or cadavers pro duced 0%, 13 ± 6% and 42 ± 5% infection, respec tively, and only the latter treatment produced my cosis (28 ± 6%). Infection transmitted from cadav ers was significantly higher than that transmitted from in vitro colonies (x2 = 8.02, P = 0.0046). To examine whether co-existence of healthy and mycosed GWSS on a plant would result in disease tr nsmission, either overwintered cadav ers collected in Jan (stored at -80°C) or new ca davers collected in Jul/Aug were pinned to plants (10 per plant) and groups of healthy nymphs or adults were maintained on each plant for up to 3 weeks. Controls were conducted on plants with out cadavers. The majority of pinned cadavers displayed sporulating Hirsutella mycelium within one week. New cadavers developed an un usually thick, white mycelium overgrowing the entire insect (Fig. 1A). Disease transmission was observed to varying degrees (Table 1). Dead ex posed nymphs and adults (Fig. IB and C), at tached to the plant and displaying Hirsutella-in duced mycosis, were seen as early as 7 and 12 d after exposure, respectively. Hemolymph-borne HBs were detected as early as 8 d after exposure. New cadavers were more efficient in disease transmission when compared with overwintered cadavers (Table 1). When nymphs were exposed to new cadavers, no survivors were found and all dead insects were overgrown with thick mycelium