Morphological and molecular characterization of a Hirsutella species infecting the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), in Florida

The Asian citrus psyllid, Diaphorina citri Kuwayama, is an invasive pest that vectors citrus greening disease, which recently was detected in Florida. Mycosed adult D. citri were collected at four sites in central Florida between September 2005 and February 2006. Observation of the cadavers using scanning electron microscopy revealed that the pathogen had branched synnemata supporting monophiladic conidiogenous cells. A high-fidelity polymerase chain reaction (PCR) assay was used to amplify the 18S rRNA, 28S rRNA and beta-tubulin genes of the pathogen for phylogenetic analysis. The morphological and genetic data indicated that the pathogen was a novel isolate related to Hirsutella citriformis Speare. PCR assays using isolate-specific primers designed from the unique putative intron region of the beta-tubulin sequence distinguished the psyllid pathogen from five related Hirsutella species. The pathogen was maintained in vivo by exposing healthy D. citri to the synnemata borne on field-collected cadavers. Infected psyllids had an abundance of septate hyphal bodies in their hemolymph and exhibited behavioral symptoms of disease. In vitro cultures of the pathogen were slow-growing and produced synnemata similar to those found on mycosed D. citri. In laboratory bioassays, high levels of mortality were observed in D. citri that were exposed to the conidia-bearing synnemata produced in vivo and in vitro.