ENDOPHYTIC BACTERIA ASSOCIATED WITH SHARPSHOOTERS, INSECT VECTORS OF XYLELLA FASTIDIOSA SUBSP. PAUCA Principal Investigator:

Xylella fastidiosa (Xf) subsp. Pauca causes citrus variegated chlorosis (CVC) disease in Brazil, resulting in significant production losses in the citrus industry. Xf is mainly transmitted by three species of sharpshooters (Hemiptera: Cicadellidae) in Brazil; Dilobopterus costalimai (Young), Acrogonia citrina Marucci & Cavichioli and Oncometopia facialis (Signoret). We identified bacterial communities associated with the heads of surface-sterilized insect vectors of Xf that were collected from CVC affected citrus groves in Brazil. Bacteria were isolated and analyzed by amplified ribosomal DNA restriction analysis (ARDRA) and sequencing, revealing the presence, among the most abundant genera, of the well-known citrus endophytes Methylobacterium spp. and Curtobacterium spp. Specific PCR systems for the detection of these genera indicated high frequencies of presence of these bacteria in sharpshooters. The remaining bacterial community was compared in distinct vector species and at different period of the year by denaturing gradient gel electrophoresis (DGGE), showing its responsiveness to the climate change over the year. These results represent a new basis for the knowledge about the interaction symbiotic-pathogenic bacteria inside insect vectors and provides a basis for further work on the biocontrol of phytopathogens like Xf. LAYPERSON SUMMARY We report the bacterial communities associated with the heads of surface-sterilized insect vectors of Xylella fastidiosa (Xf) that were collected from citrus variegated chlorosis (CVC) affected citrus groves in Brazil. Many aspects can influence the transmission of a pathogen by an insect vector such as the low concentration of Xf cells in the citrus plants and the low number of colonized vessels in affected plants. The interaction between different bacteria inside the insect foregut can also influence the transmission, as once inside the foregut, bacterial interaction, such as competition for nutrients, space and other complex interactions could occur. C. flaccunfaciens is a potential candidate for biological control of CVC because an antagonism between C. flaccumfaciens and Xf was strongly indicated in vitro and in vivo including inhibition of growth of Xf and reduced severity of the disease symptoms in the presence of this phytopathogen. The ability demonstrated by C. flaccumfaciens to colonize plant tissues in the presence of Xf and the reduction of disease symptoms caused by Xf are prerequisites for the use of this endophytic bacterium as a biocontrol agent. Since members of the genus Curtobacterium were consistently detected in the insect vectors of Xf as demonstrated in the present study, they fulfill another requirement of candidates for biological control of Xf. INTRODUCTION Brazil is the largest producer of citrus fruit in the world, also supplying most of the international market for concentrated orange juice. By 2005, the percentage had increased to 43%, and citrus variegated chlorosis (CVC) was present in all citrus growing regions of Brazil (Bové & Ayers 2008). The disease is caused by the xylem-limited gram-negative bacterial pathogen, Xylella fastidiosa (Xf) subsp. Pauca (Hartung et al. 1994). Endophytic microorganisms live within plants without causing apparent harm to the host. We have studied the possible use of endophytes as vehicles to control both phytopathogens and insects (Azevedo et al. 2000). Endophytes colonize ecological niches similar to that of phytopathogens (Gai et al 2009a), which gives them access as possible candidates as biocontrol agents. In citrus, several endophytic bacteria were isolated and Methylobacterium spp. and Curtobacterium flaccumfaciens were further determined as the main endophytic species interacting with Xf (Araújo et al. 2001). These species vary in population density when CVC-affected and asymptomatic plants are compared. Later, Lacava et al. (2004) reported that the growth of Xf was inhibited by endophytic C. flaccumfaciens and stimulated by Methylobacterium sp. A similar effect was demonstrated by the reduced severity of the Xf colonization in plants priory colonized by C. flaccumfaciens (Lacava et al. 2007a). In Brazilian citrus groves, Dilobopterus costalimai, Oncometopia facialis and Acrogonia citrina are the most common sharpshooters found. The transmission efficiency of bacteria is a measure of the ability to successfully acquire bacteria from an affected plant and transmit to healthy ones. The efficiency of Xf transmission leafhopper species ranges from 0 to 100%, however the lower values are found when