Sequencing, improved detection, and a novel form of Kalanchoë top-spotting virus

Virions of Kalanchoë top-spotting virus (KTSV) were purified from infected leaf tissue of Kalanchoë blossfeldiana using a procedure that prevented loss of virus in the initial extraction step. The double-stranded DNA viral genome was cloned and sequenced. The KTSV genome was 7,591 bp in size and contained three open reading frames capable of encoding proteins of 21, 14, and 223 kDa, respectively. The size and organization of the KTSV genome were similar to those of other mealybug-transmitted badnaviruses. Several oligonucleotide primer pairs, based on the KTSV genomic sequence, were used to efficiently detect the virus in plants, thereby removing a major constraint to reliable screening of kalanchoë propagating stock and breeding lines for KTSV infection. Two KTSV sequences, one symptom-inducing and the other not, were identified and differentiated by polymerase chain reaction (PCR) amplification and digestion of the resulting amplicon with restriction endonucleases. Preliminary results from graft-transmission tests and PCR indexing suggest that the nonsymptomatic form of KTSV may represent an integrated viral element. The occurrence of such integrated pararetroviral elements poses practical problems for routine PCR indexing of breeding and propagating stock, and also raises the possibility of symptomatic episomal infections arising from these viral integrants.