New Disease Reports

Leaf samples from 13 diseased and four healthy hibiscus plants were collected. Total DNA was isolated and subjected to PCR using degenerate begomovirus primers (CP-F: 5′-GCATCTGCAGGCCCACATYGTCTTYCCNGT-3′ and CP-R: 5′-AATACTGCAGGGCTTYCTRTACATRGG-3′). All samples of symptomatic leaves yielded a ~1.2 kb PCR product indicative of a begomovirus. Full-length genomes were amplified by the rolling circle amplification method (~2.7 kb) from two samples, and cloned and sequenced (GenBank Accession Nos. JN880418 and JN807763). Nucleotide sequence analysis revealed 99% identity between the two isolates, and 90-93% identity and close phylogenetic relationships with isolates of Cotton leaf curl Multan virus (CLCuMV) (Fig. 2). Hence, two new variants of CLCuMV were identified. Association with a betasatellite was tested by PCR with specific primers (Beta 01: 5′-AGCCTTAGCTACGCCGGAGC-3′ and Beta 02: 5′-GCTGCGTAGCGTAGAGGTTT-3′). Again, all 13 samples of symptomatic leaves produced the expected product of ~1.3 kb. One amplicon was cloned and sequenced (KT447040). The sequence had 98% nucleotide identity and close phylogenetic relationships with Cotton leaf curl Multan betasatellite (CLCuMB) isolates (Fig. 3), and was concluded to be an isolate of CLCuMB. The disease incidence was confirmed during a survey of the same garden in September 2015. Twenty three of the 42 hibiscus plants investigated by dot blot analysis using a CLCuMV-specific probe (derived from clone JN880418) were found to be infected (Fig. 4).