Primary Culturing of the Western X Mycoplasmalike Organism from Colladonus montanus Leafhopper Vectors

Abstract

Anterior 1-type lobes of salivary glands of Colladonus montanus leafhopper vectors infected with Western X mycoplasmalike (WXM) organisms were cultured in a variety of media using an organ culture method. The suitability of various media was judged by the preservation of the ultrastructure of the spherical electron transparent bodies of WXM. The best medium found was that used for maintaining leafhopper cells lines supplemented with 0.2 M sucrose. Infected lobes cultured 1 week in this medium exhibited some infectivity.Direct culturing of WXM was tried in 3 variations of the standard leafhopper tissue culture medium. The variations were made by the addition of 0.2 M or 0.9 M sucrose, or by forming a 'conditioned' medium by adding medium and cells from the actively growing monolayers of the C. montanus cell line. The primary cultures were started using crushed WXM-infected anterior 1-type lobes of the salivary glands of C. montanus.One week after beginning the cultures, little infectivity was detected. After 2 weeks, the assay results (expressed as the percentage of inoculated test insects that transmitted the WXM agent) were 22.5, 62.5, and 32.5 for the media containing 0.2 M, 0.9 M sucrose, and 'conditioned' medium, respectively. Spherical dense bodies were the main morphological form in the 'plasmolysing' (0.9 M sucrose) medium 1 week after the start. After 2 weeks the large polymorphic, as well as the small spherical, electron transparent bodies appeared. It was presumed that some multiplication of WXM had occurred.