During a survey in February 2011, severe symptoms of upward leaf curling, vein enation on lower side of the leaves, and shortening of internodes were observed on 20 out of 117 Amaranthus hypochondriacus plants (17% disease incidence) examined at breeding plots of CSIR-NBRI, Lucknow. These symptoms are typical of begomovirus infection. PCR with begomovirus-specific primers (3) produced the expected ~1.1-kb product from DNA extracts of 20 symptomatic plants but not from a non-symptomatic plant, suggesting the association of a begomovirus. The full-length begomoviral genome from a representative sample was amplified by rolling circle amplification using Ø-29 DNA polymerase and digested by BamHI, which resulted in a ~2.7 kb product when electrophoresed in 1.0% agarose gel. The product obtained was cloned, sequenced, and sequence data of 2,753 nucleotides was deposited in GenBank (Accession No. JF682242). BLASTn analysis revealed 97 to 98% nucleotide identity and forms a distinct clade with Ageratum enation virus (AEV) isolates. This shows the virus in A. hypochondriacus to be an isolate of AEV. The separate PCRs were also performed with betasatellite and alphasatellite specific primers (1,2) that resulted in ~1.3-kb amplicons from all samples, suggesting their association. The amplification products were cloned and sequenced. An analysis of betasatellite (JX512904) revealed highest 98% nucleotide identity and close phylogenetic relationship with Ageratum leaf curl betasatellite (ALCB, JQ710745). The alphasatellite (JX512905) showed highest 95% identity and close relationship with Hibiscus leaf curl alphasatellite (HLCA, FN794199). This shows the betasatellite and alphasatellite in A. hypochondriacus to be isolates of ALCB and HLCA, respectively. The partial direct repeat clones of the begomovirus (pCAM-AEV), betasatellite (pCAM-ALCB), alphasatellite (pCAM-HLCA) were generated and mobilized into Agrobacterium tumefaciens strain GV3101 and infiltrated in A. hypochondriacus seedlings. The plants inoculated with pCAM-AEV, pCAM-ALCB, and pCAM-HLCA; pCAM-AEV and pCAM-ALCB developed severe leaf curl and enation symptoms on 5/5 plant at 35 days post inoculation, which were similar to those of naturally infected plants, satisfying Koch's postulates. On the other hand, plants inoculated with pCAM-AEV alone or in combination with pCAM-HLCA developed mild symptoms. Plants inoculated with pCAM-ALCB and pCAM-HLCA did not develop symptoms. The results here show that leaf curl and enation disease of A. hypochondriacus in India is caused by AEV and ALCB and that an alphasatellite may be associated with symptomatic plants.
References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2) S. E. Bull et al. Mol. Biotechnol. 23:83, 2003. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.