CULTURE AND SEROLOGICAL DETECTION OF THE XYLEM-LIMITED BACTERIUM CAUSING CITRUS VARIEGATED CHLOROSIS AND ITS IDENTIFICATION AS A STRAIN OF XYLELLA-FASTIDIOSA
A xylem-limited bacterium resembling Xylella fastidiosa has been shown previously by electron microscopy to be associated with citrus variegated chlorosis (CVC), a new disease of sweet orange trees in Brazil. A bacterium was consistently cultured from plant tissues from CVC twigs of sweet orange trees but not from tissues of healthy trees on several cell-free media known to support the growth of Xylella fastidiosa. Bacterial colonies typical of X. fastidiosa became visible on PW, CS20, and PD2 agar media after 5 and 7-10 days of incubation, respectively. The cells of the CVC bacterium were rod-shaped, 1.4-3 mum in length, and 0.2-0.4 mum in diameter, with rippled walls. An antiserum against an isolate (8.1.b) of the bacterium gave strong positive reactions to double-antibody-sandwich (DAS), enzyme-linked immunosorbent assay (ELISA) with other cultured isolates from CVC citrus, as well as with several type strains of X. fastidiosa. This result indicates that the CVC bacterium is a strain of X. fastidiosa. ELISA was also highly positive with all leaves tested from CVC-affected shoots. Leaves from symptomless trees reacted negatively. Sweet orange seedlings inoculated with a pure culture of the CVC bacterium supported multiplication of the bacterium, which became systemic within 6 months after inoculation and could be reisolated from the inoculated seedlings. Symptoms characteristic of CVC developed 9 months post inoculation.