ANALYSIS OF THE BACTERIAL COMMUNITY INHABITING GLASSY-WINGED SHARPSHOOTER FOREGUT BY CULTURE-DEPENDENT TECHNIQUES AND DGGE Project Leaders:

The glassy-winged sharpshooter (GWSS) is an important vector of Xylella fastidiosa (Xf), the bacterial pathogen that causes several economically important plant diseases, including citrus variegated chlorosis (CVC), oleander leaf scorch (OLS) and Pierce’s disease (PD) of grapevines. In recent years there has been an increasing interest in the potential use of biological control agents to halt the spread of Xf. One such strategy is the exploration of symbiotic microorganisms to reduce the spread of the pathogen (symbiotic control). In a symbiotic control strategy a bacterium symbiont that occupies the same niche as the pathogen must be identified. The study of the bacterial community of GWSS foreguts by isolation and DGGE revealed the presence of several potential symbiotic candidates such as Bacillus sp., Pseudomonas sp., Methylobacterium sp. and Curtobacterium flaccumfaciens (C. flaccumfaciens). Members of genus Methylobacterium and C. flaccumfaciens are frequently isolated as endophytes from citrus plants with CVC symptoms and without disease symptoms. Recently, an interaction between Methylobacterium, C. flaccumfaciens and Xf was strongly indicated, reinforcing that these bacteria could interact inside the host plant and vector insect. In the future, the genus Methylobacterium and C. flaccumfaciens could be an interesting candidate in a strategy of symbiotic control to Xf. INTRODUCTION The glassy-winged sharpshooter (GWSS) is one of the main vectors of Xylella fastidiosa (Xf). It is a xylophagous insect that has a wide array of host plants, including many ornamental and crop plants (Purcell and Hopkins 1996, Purcell and Saunders 1999). One new potential management strategy for Pierce’s disease (PD) of grapevine is the use of symbiotic control. Symbiotic control exploits the interactions among a pathogen-transmitting organism, its bacterial symbionts, and the pathogenic organism itself (Beard 2002). For symbiotic control to be effective in limiting the spread of PD, a culturable symbiont that inhabits the pre-cibarium and cibarium of GWSS is required, since these areas are colonized by the pathogen, Xf. A previous biochemical analysis of the GWSS foregut microdiversity encountered three bacterial species that meet these requirements: Chryseomonas spp, Ralstonia spp, and Alcaligenes spp (Bextine 2004). The Alcaligenes species were of particular interest because they were frequently isolated from wild GWSS (Kuzina 2004). Although Alcaligenes spp. can colonize GWSS, this specie does not colonize grapevines well (Bextine 2005). According to Bextine et al (2005), the amount of colonization by Alcaligenes xylosoxidans subsp. denitrificans decreased in the following order: orange (Citrus sinensis ‘‘sweet orange’’) > chrysanthemum (Chrysanthemum grandiflora cv. ‘‘White Diamond’’) > periwinkle (Vinca rosea) > crepe myrtle (Lagerstroemia indica) > grapevine (Vitis vinifera cv. Chardonnay). Therefore, steps in a symbiotic control strategy should be directed in finding a bacterial symbiont that colonizes well the pre-cibarium and cibarium of GWSS as well as the target host plant: grapevines. The CelectrophoreticCT separation of PCR products of variable regions of genes encoding 16S rDNA (by use of primers homologous to conserved regions of the gene) in a polyacrylamide matrix over a denaturing gradient (DGGE) is a technique recently introduced in microbial ecology (Muyzer et al. C1993CT). The denaturing gradient can be achieved either chemically with urea and formamide in DGGE. This technique is reported to be interchangeable, giving comparable fingerprints of microbial communities. Recently, Reeson et al. (2003) demonstrated the efficiency of DGGE to study the bacterial communities associated to insects (wasp Vespula germanica). OBJECTIVES 1. The aim of this work was to characterize the bacterial community inhabiting GWSS foreguts by using isolation in culture medium and DGGE techniques. RESULTS The study of bacterial community by isolation and DGGE (Figures 1, 2, and 3) revealed the presence of several genera of bacteria such as Bacillus sp., Cryocola sp., Microbacterium sp., Micrococcus sp., Pedobacte sp.r, Pseudomonas sp., Methylobacterium extorquens, C. flaccumfaciens, Baumannia cicadellinicola, and Wolbachia spp. Members of genus