A New Technique to Select Mild Strains of Cucumber mosaic virus

Abstract

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.

Studies on molecular mechanisms of viral attenuation and cross protection

“…Thus, an efficient technique to select a good attenuated virus to control CMV disease had to be developed. In our study, mild CMV isolates appeared to be more efficiently selected from dark-green leaf tissue, suggesting that isolations from dark-green tissues after mutagenesis may be advantageous for several plant viruses (Kobori et al 2005).…”

Immunocapture and simple-direct-tube-reverse transcriptase polymerase chain reaction (RT-PCR) for detection of Rice yellow mottle virus

“…This is the first report on the use SDT-and IC-RT-PCR for detection and assay of RYMV. This method has been used for detection of other plant viruses (Kobori et al, 2005;Suehiro et al, 2005;Temaja et al, 2012). The results indicate that SDT-RT-PCR and IC-RT-PCR detected RYMV in infected leaf samples similar to the virus extraction RNA-RT PCR technique.…”

“…This protocol involves grinding of plant tissues in phosphate buffer saline with 0.5% Tween-20 (PBST 1X) and placing the extract in a micro-centrifuge tube for two minutes and allowing adsorption of virus particles to the tube wall. Simple direct tube method was successfully used for detection of Turnip mosaic virus, Cucumber mosaic virus and Cucumber green mottle mosaic virus in infected plants (Kobori et al, 2005;Suehiro et al, 2005). The purpose of this work was to optimize and evaluate the efficiency of IC-and SDT-RT-PCR methods for RYMV detection in infected plant materials.…”

Attenuated plant viruses: preventing virus diseases and understanding the molecular mechanism

“…The point mutation in the 2b of CR95 is responsible for binding to siRNA (Goto et al 2007). Another attenuated strain of CMV, 36R37, also had a different amino acid substitution in 2b (Kobori et al 2005) (Fig. 1b).…”