Non-culture Dependent Survey of the Microbiota of the Glassy-winged Sharpshooter Using 454 Pyrosequencing

The glassy-winged sharpshooter (GWSS; Homalodisca vitripennis) is an invasive pest that has spread across the southern and western United States. It is highly polyphagous, feeding on at least 100 species in 31 families (Hoddle et al., 2003; Turner and Pollard, 1959), and a voracious feeder, having been known to consume up to 100 times its weight in xylem fluid daily. This insect is a vector of the phytopathogen Xylella fastidiosa (Xf), which is the causative agent of Pierce’s disease (PD) in grapevines. In order to evaluate the microbial flora associated with GWSS hemolymph, alimentary canal excretions and whole insect bodies were subjected to 16S pyrosequencing using the bTEFAP methodology and the resulting sequences (370520 bp) were compared to a curated high quality 16S database derived from NCBI’s GenBank. Species from the genera Wolbachia, Delftia (formerly Pseudomonas), Pectobacterium, Moraxella, Serratia, Bacillus and many others were detected and a comprehensive picture of the microbiome associated with GWSS was established. Some of the bacteria identified in this report are initial discoveries and having a breadth of knowledge as to the microbial flora of this insect pest can serve as a reservoir of information for developing biological control strategies. One method for biological control can be the genetic engineering of a particular bacterium to deliver certain molecules known to affect the life stage development of an insect. Another method, could be isolating a bacterium that competes with Xf, and re-delivering it to wild populations in excess of their natural bacterial load. Within this study, we have identified the types of bacteria which may be ubiquitous among GWSS providing us with targets to begin to investigate these future directions. LAYPERSON SUMMARY The glassy-winged sharpshooter is an insect pest that spreads the bacterium Xylella fastidiosa, the causal agent of Pierce’s disease in the grapevine. Both wine and table grape production is affected by this disease and it has become a major financial burden on the industry. Bacterial DNA can be used to screen for such pathogens in insect populations and having knowledge of the bacterial pathogens of the insect can be used to develop a biological control strategy. Hemolymph, alimentary canal excretions and whole insect tissue were subjected to DNA sequencing and the resulting sequences were matched to groups of bacteria using NCBI’s GenBank database. This study shows that bacteria such as Wolbachia, Delftia, Pectobacterium, Moraxella, Serratia, and Bacillus spp may be useful targets to develop biological control strategies. INTRODUCTION The glassy-winged sharpshooter (GWSS) is a highly mobile pest and transmits the xylem-limited bacterium Xylella fastidiosa (Xf). This bacterium can cause disease in many economically important plants including the grapevine, peach, and citrus and has become a major limiting factor in their mass production. The bacterium can also cause disease in ornamentals such as the oak, elm and sycamore. Once a plant has become infected with Xf, it becomes a reservoir for bacterium and can be easily spread from plant to plant by the near-continuous feeding of GWSS. Although pesticides are available for the control of the insect, resistance and harm to non-target insects is an issue of concern. Naturally occurring forms of control have been successful and should be further pursued as a complimentary strategy for insect and disease control. Many insect taxa have obligate endosymbionts that supplement nutrition in exchange for vertical or horizontal transfer among individuals (Moran et al 2005, Buchner 1965). This mutualism has allowed insects to occupy or thrive in otherwise hostile niches. The GWSS, a xylem feeder, is known to host several bacterial species including Baumannia cicadellinicola and Sulcia muelleri (Wu et al 2006). The B. cicadellinicola genome is devoted to the biosynthesis of vitamins and cofactors but lacks most amino acid biosynthetic pathways, whereas S. muelleri apparently produces most of the amino acids needed for the host. DGGE has been used to find other symbiotic bacteria including Wolbachia, Bacillus, Pseudomonas, Pedobacter, Methylobacterium, and Curtobacterium flaccumfaciens which the authors suggest could be used as forms of symbiotic control (Lacava et al 2007). Curly et al (2007) identified bacteria closely related to Stenotrophomonas and Acinetobacter in hemolymph samples. OBJECTIVES 1. Identify major groups of bacteria in the hemolymph, alimentary canal and whole insect. 2. Identify species of bacteria for possible transgenesis and biological control.