New Disease Reports (2015) 32, 6. [http://dx.doi.org/10.5197/j.2044-0588.2015.032.006]
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First report of Ageratum enation virus and Ageratum leaf curl betasatellite infecting Calendula officinalis in India

Meraj Jaidi, S. Kumar, A. Srivastava and S.K. Raj*

*skraj2@rediffmail.com

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Received: 03 Jul 2015; Published: 10 Aug 2015

Keywords: agroinfectivity, begomovirus, betasatellite, calendula, yellow vein net disease

Calendula officinalis (Asteraceae), commonly known as pot marigold, is grown as an annual ornamental plant in tropical countries including India. In January 2014, yellow vein net disease was observed on a number of C. officinalis plants (Fig. 1A) growing in a garden at Lucknow, India. The severely infected C. officinalis plants exhibited yellow vein net symptoms on leaves and leaf deformation (Fig. 1B) as compared to healthy plants (Fig. 1C). The severely infected plants remained stunted and bore smaller sized and fewer flowers. Based on the typical yellow net symptoms described earlier on C. officinalis plants caused by a begomovirus (Khan et al., 2005), begomovirus infection was suspected.

Total DNA isolated from fifteen infected samples and one healthy leaf sample was subjected to PCR amplification with begomovirus degenerate (Rojas et al., 1993) and betasatellite specific primers (Briddon et al., 2002). Following PCR amplification, products of the anticipated sizes of respectively ~1.2 and ~1.3 kb were successfully generated from all fifteen infected samples with no amplicon being generated from a healthy plant. These results suggest the association of a begomovirus and a betasatellite with the yellow net disease of C. officinalis. For molecular identification of the begomovirus, full length DNA genome was amplified from three independent DNA preparations of representative samples by the rolling circle amplification method using the RCA kit (GE healthcare, Buckinghamshire, UK) followed by restriction enzyme analysis. The resulting ~2.7 kb amplicons from three isolates (NBRI 1-3) were digested with BamHI cloned, sequenced and the sequences were deposited in GenBank with Accession Nos. KM066975 (NBRI-1), KM262822 (NBRI-2) and KM262823 (NBRI-3). These three NBRI begomovirus isolates shared 99% sequence identity together and similar identities (98-99%) with various strains of Ageratum enation virus (AEV; JX436472, FN543099, FN794198, FN543099, JF682242 and JF728864). These isolates also shared a close phylogenetic relationship with well recognised strains of AEV (FN543099, HM149260 and AJ437618) as defined by the ICTV Geminiviridae study group (Brown et al., 2015) (Fig. 2). Based on these findings we propose that the begomovirus isolated from C. officinalis is an isolate of AEV. The three ~1.3 kb amplicons obtained by PCR using betasatellite specific primers from the same samples were also cloned and sequenced (KR922821, KR922822 and KR922823). Sequence analysis of associated betasatellites showed 99% sequence identity together and 92-96% identity and close phylogenetic relationships with Ageratum leaf curl betasatellite (ALCuB) (Fig. 3), and is nominated ALCuB. 

To determine the infectivity of these new AEV and ALCuB isolates, infectious clones of both were generated using the pCAMBIA1300 vector backbone and agroinoculated in ten seedlings of C. officinalis. Combined agroinoculation of C. officinalis with AEV and ALCuB induced yellow vein net symptoms at 35 days post inoculation (Fig. 1D), which were similar to those observed in C. officinalis plants in the field, suggesting fulfilment of Koch’s postulates. Reports of natural occurrence of Cucumber mosaic virus (Naqvi & Samed, 1985), an unidentified begomovirus (Khan et al., 2005) from India and Bidens mottle virus from Taiwan (Huang & Jan, 2011) on C. officinalis are known. However, this is the first report of AEV and ALCuB infecting C. officinalis in India.

Figure1+
Figure 1: Naturally infected Calendula officinalis plant showing yellow vein net disease in field (A); a close view of leaf of infected plant showing severe yellow vein net symptoms (B) as compared to a healthy plant (C); and yellow vein net symptoms induced on C. officinalis seedling 35 days post agro-inoculation (D).
Figure 1: Naturally infected Calendula officinalis plant showing yellow vein net disease in field (A); a close view of leaf of infected plant showing severe yellow vein net symptoms (B) as compared to a healthy plant (C); and yellow vein net symptoms induced on C. officinalis seedling 35 days post agro-inoculation (D).
Figure2+
Figure 2: Phylogenetic analysis of begomovirus isolates under study: NBRI-1, NBRI-2 and NBRI-3 showing their close relationships with strains of AEV. N-J trees were constructed by MEGA v6.0 tool (http://www.megasoftware.net/) and the percentage of replicate trees in which the associated taxa clustered together in bootstrap test (1000 replicates) are shown next to the branches. Names of viruses indicated by abbreviations may be found by reference to the GenBank accessions.
Figure 2: Phylogenetic analysis of begomovirus isolates under study: NBRI-1, NBRI-2 and NBRI-3 showing their close relationships with strains of AEV. N-J trees were constructed by MEGA v6.0 tool (http://www.megasoftware.net/) and the percentage of replicate trees in which the associated taxa clustered together in bootstrap test (1000 replicates) are shown next to the branches. Names of viruses indicated by abbreviations may be found by reference to the GenBank accessions.
Figure3+
Figure 3: Phylogenetic relationships of betasatellite molecule under study (KR922821, KR922822 and KR922823) with various isolates of ALCuB. Methodology as in Fig. 2.
Figure 3: Phylogenetic relationships of betasatellite molecule under study (KR922821, KR922822 and KR922823) with various isolates of ALCuB. Methodology as in Fig. 2.

References

  1. Briddon RW, Bull SE, Mansoor S, Amin I, Markham PG, 2002. Universal primers for the PCR-mediated amplification of DNA β. Molecular Biotechnology 20, 315-318. [http://dx.doi.org/10.1385/MB:20:3:315]
  2. Brown JK, Murilo Zerbini F, Navas-Castillo J, Moriones E, Ramos-Sobrinho R, Silva JCF, Fiallo-Olivé E, Briddon RW, Hernández-Zepeda C, Idris A, Malathi VG, Martin DP, Rivera-Bustamante R, Ueda S, Varsani A, 2015. Revision of Begomovirus taxonomy based on pairwise sequence comparisons. Archives of Virology 160, 1593-1619. [http://dx.doi.org/10.1007/s00705-015-2398-y]
  3. Huang CH, Jan FJ, 2011. First Report of Bidens mottle virus infecting Calendula in Taiwan. Plant Disease 95, 362. [http://dx.doi.org/10.1094/PDIS-10-10-0753]
  4. Khan AA, Naqvi QA, Khan MS, Singh R, Raj SK, 2005. First report of a begomovirus infecting Calendula in India. Plant Pathology 54, 569. [http://dx.doi.org/10.1111/j.1365-3059.2005.01220.x]
  5. Naqvi QA, Samad A, 1985. Purification and properties of Calendula yellow net virusIndian Journal of Virology 1, 143-146.
  6. Rojas MR, Gilbertson RL, Russell DR, Maxwell DP, 1993. Use of degenerate primers in the polymerase chain reaction to detect whitefly transmitted geminiviruses. Plant Disease 77, 340-347. [http://dx.doi.org/10.1094/PD-77-0340]

To cite this report: Jaidi M, Kumar S, Srivastava A, Raj SK, 2015. First report of Ageratum enation virus and Ageratum leaf curl betasatellite infecting Calendula officinalis in India. New Disease Reports 32, 6. [http://dx.doi.org/10.5197/j.2044-0588.2015.032.006]

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