Infectious clones of cotton leafroll dwarf virus strains used for genotype evaluation in cotton

Cotton blue disease (CBD) and atypical‐CBD are the most important viral diseases of cotton plants in the southern region of South America. Common and atypical strains of cotton leafroll dwarf virus (CLRDV and CLRDV‐at, respectively) are thought to be causative agents of CBD and atypical‐CBD, respectively. Inoculation of test plants via aphid vectors is difficult, as is determining strains via molecular diagnosis; accordingly, it is difficult for breeders to evaluate the effects of blue disease‐associated virus infections in cotton lineages. In the present study, we attempted to circumvent these difficulties by producing six full‐length cDNA infectious clones from CLRDV and CLRDV‐at strains using the Gibson Assembly protocol. For inoculation of the infectious clones, a vacuum chamber‐mediated agroinfiltration protocol was adapted and applied. Using this protocol, 90%–100% of cotton plants became infected with the clones, which was not possible via syringe‐based agroinfiltration. A genotyping protocol based on RT‐qPCR targeting a specific region of the virus P0 protein was also developed, allowing rapid differentiation of CLRDV and CLRDV‐at. Applying this protocol to 68 field samples revealed that CLRDV‐at was dominant (50%) over CLRDV (5.8%) in single virus infections. These preliminary results imply that CLRDV‐at might occupy the ecological niche of CLRDV in the cotton fields of Brazil.