A New Technique to Select Mild Strains of Cucumber mosaic virus

An efficient technique to select a good attenuated virus to control Cucumber mosaic virus (CMV) disease was developed. Preliminary screenings were conducted to assess the virulence of virus recovered from dark-green islands and yellow tissues of mosaic leaves of Nicotiana rustica after co-inoculation with an attenuated mutant P2bR46C of CMV and its original severe isolate Pepo. All single-lesion isolates (SLIs) obtained from dark-green islands had the attenuated P2bR46C phenotype, but the SLIs from yellow tissue had either the virulent Pepo or the P2bR46C phenotype. When Pepo-infected N. rustica and tomato plants were grown at 15 or 36°C for 30 days, 17 of 288 SLIs obtained from the treated leaves elicited mosaic and dark-green spots without malformation. Dark-green tissue from each plant infected with 1 of these 17 SLIs then was used to inoculate one plant of N. rustica. All 17 plants had either very mild mosaic or no visible symptoms. One of these potential mild strains, 36R37, had an amino acid substitution on the 2b gene encoding the 2b protein. Isolate 36R37 also was highly cross-protective, and its symptom attenuation was stable for three serial host passages. After cold or heat treatment, the dark-green tissue proved to be a good source for isolating mild strains of the virus.