Begomoviruses (family Geminiviridae) have a circular, single-stranded DNA genome, encapsidated within a twin-icosahedral particle, transmitted by the whitefly Bemisia tabaci and infect dicot plants (Fauquet and Stanley 2005). Most begomoviruses have a bipartite genome consisting of two DNA components (DNA-A and DNA-B), but an increasing number of monopartite begomoviruses have only a single genomic component homologous to DNA-A. Monopartite begomoviruses are usually associated with DNA-β satellites that are required for symptom induction (Briddon et al. 2003). Some nanovirus-like DNA components known as alpha-satellites (DNA 1) have also been reported with many begomovirus disease complexes (Briddon et al. 2004). Ageratum enation virus (AEV) is a monopartite begomovirus, mainly reported to infect some weed plants. It is important to investigate the emergence of new begomoviruses, especially in weeds that may act as their reservoirs during the non-cropping season (Packialakshmi et al. 2010).

In a previous study, leaf curl disease of Zinnia elegans was found to be associated with Ageratum enation virus (AEV) and a nanovirus-like DNA 1 component in Palampur region of Himachal Pradesh, India, which has a subtemperate climate (Kumar et al. 2010). Crassocephalum crepidioides (Benth.) S. Moore and Ageratum conyzoides L., common weeds of this region, also had vein yellowing symptoms, indicative of a begomoviral infection (Fig. 1a, b). Leaf samples of these symptomatic plants were collected to investigate whether these weeds act as reservoirs of AEV, responsible for zinnia leaf curl disease. Three samples of each weed were collected, and total DNA was extracted with an AuPrep DNA Extraction Kit (Life Technologies, Delhi, India).

Fig. 1
figure 1

Vein yellowing symptoms on a Crassocephalum crepidioides and b Ageratum conyzoides. c Phylogenetic analysis of all available complete sequences of Ageratum enation virus (AEV) reported from various host plants. The tree shows that the isolates group together according to geographical origin. Names of specific host plants and accession numbers of sequences are also shown. Vertical distances are arbitrary, horizontal branches are proportional to the genetic distances, and the numbers at the nodes are the bootstrap values (1000 replicates). IN India, PK Pakistan, NE Nepal

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Samples were subjected to rolling circle amplification (RCA) using a TempliPhi DNA Amplification Kit (GE Healthcare, Piscataway, NJ, USA) and a protocol described previously (Kumar et al. 2008). RCA products (1 μL samples) were digested with a number of popular restriction enzymes to identify an enzyme having a single site in the genomic components. Restriction digestion with SalI, HindIII and BamHI produced ca. 2.8-kb DNA fragments, whereas KpnI and SacI produced ca. 1.3-kb DNA fragments (data not shown). Complete components of ca. 2.8 and 1.3 kb from both weeds were cloned into SalI and SacI sites of pBluescriptII KS+ vector (Stratagene, La Jolla, CA, USA). The clones were sequenced using a primer walking strategy and an automated DNA sequencer (ABI Prism 3130XL genetic analyzer) and ABI prism Big Dye Terminator v3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). PCR was carried out using universal primers for DNA-B (Rojas et al. 1993) and DNA-β (Cui et al. 2004) under the described conditions, but no amplification was obtained from any of the samples (data not shown).

In a BLAST search for homology, AEV and a nanovirus-like DNA 1 were identified to be associated with C. crepidioides and A. conyzoides. AEV and DNA 1 sequences from C. crepidioides were submitted to GenBank database under accession numbers FN794201 and FN794202, respectively. Accessions FN794198 and FN794199 were obtained for AEV and DNA 1 sequences from ageratum. The present sequences of AEV had 99% nucleotide identity with the zinnia isolate (FN543099) reported previously from this region, but DNA 1 sequences had only 90% identity with DNA 1 associated with the zinnia isolate. The AEV sequences from C. crepidioides and A. conyzoides had 99% identity with one another and <94% identity with all other reported AEV isolates. The present DNA 1 sequences showed the maximum identity (93%) with a DNA 1 sequence reported from Pakistan, associated with Sonchus arvensis (AM930248).

Phylogenetic analysis for all AEV complete sequences available in GenBank was done by neighbor-joining method using the ClustalW program available online (http://clustalw.ddbj.nig.ac.jp/top-e.html). The phylogenetic trees were viewed and printed with the help of TreeView software (Page 1996), selecting 1,000 bootstrap replicates. In the tree, all Palampur isolates formed a separate clade, while all Pakistan and other Indian isolates grouped separately showing their geographical relatedness (Fig. 1c). An isolate of Euphorbia leaf curl virus-[India:2007] (EU194914) was used as an outgroup. The sequences were also checked for potential recombination events using recombination detection program (RDP) 3 beta 26 and default settings (Martin and Rybicki 2000), but no significant event was detected for the present isolates (data not shown).

Crassocephalum crepidioides occurs widely throughout tropical Africa and has been introduced and naturalized throughout tropical and subtropical Asia. The plant has several medicinal uses, and its leaves are also used as vegetable (Denton 2004). So far, there is only one report of a begomovirus infecting this plant in China (Dong et al. 2008). In this study, we identified and characterized AEV and a nanovirus-like satellite DNA 1 associated with C. crepidioides and A. conyzoides in a subtemperate region in India. To the best of our knowledge, this report is the first report of a begomovirus associated with C. crepidioides in India. It is also the first report of AEV associated with this weed worldwide and A. conyzoides in India.