Australasian Plant Pathology

, Volume 47, Issue 2, pp 215–225 | Cite as

Development and validation of PCR assays for detection of alfalfa dwarf disease-associated viruses in Australian lucerne pastures

  • S. Samarfard
  • N. E. Bejerman
  • M. Sharman
  • V. Trucco
  • F. Giolitti
  • R. G. Dietzgen
Original Paper


In 2010, a severe disease of lucerne (Medicago sativa L.) named alfalfa dwarf disease (ADD) was reported in Argentina. High throughput sequencing of diseased plants indicated the presence of five viruses, the (−) ssRNA virus, alfalfa dwarf virus (ADV), the (+) RNA viruses, alfalfa mosaic virus (AMV), bean leafroll virus (BLRV) and alfalfa enamovirus 1 (AEV-1) and the ssDNA virus, alfalfa leaf curl virus (ALCV). In this study, we determined which ADD-associated viruses are present in Australian lucerne that showed diverse virus-like symptoms. A duplex RT-PCR was developed for simultaneous detection of ADV and AMV using a cloned non-infectious ADV RNA fragment as positive control. Similarly, the presence of BLRV and AEV-1 was determined by duplex RT-PCR, and ALCV by PCR. Only AMV and BLRV that are endemic to Australia were detected. None of the novel exotic viruses ADV, AEV-1 and ALCV were detected in lucerne samples collected between 2015 and 2017. However, AMV and BLRV were detected in 78% and 70% of tested samples, respectively. Based on analysis of coat protein (CP) nucleotide sequences, Australian BLRV isolates are closely related to each other and to the Argentine Manfredi isolate. Phylogenetic analyses based on CP gene nucleotide sequences confirmed separation of AMV isolates into two subgroups. The majority of AMV isolates, including all those from Argentina and Australia, clustered in subgroup I with isolates from various hosts and geographic origins. All Australian AMV isolates were closely related to AMV isolated from ADD-affected lucerne in Argentina.


Diagnostics Biosecurity Next generation sequencing Rhabdovirus Geminivirus Genetic diversity 



We thank James De Barro and Ben Farmer (Alpha Group Consulting) and Mohammad Aftab (Agriculture Victoria) for their generous assistance in collecting lucerne field samples. This research was jointly supported by the Queensland Department of Agriculture and Fisheries and the University of Queensland (UQ) through the Queensland Alliance for Agriculture and Food Innovation, and by AgriFutures Australia project PRJ-009751. Samira Samarfard was supported by a living allowance scholarship from AgriFutures Australia and a tuition fee scholarship from UQ Graduate School.

Supplementary material

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ESM 1 (DOCX 26 kb)
13313_2017_533_MOESM2_ESM.docx (15 kb)
ESM 2 (DOCX 15 kb)


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Copyright information

© Australasian Plant Pathology Society Inc. 2017

Authors and Affiliations

  • S. Samarfard
    • 1
  • N. E. Bejerman
    • 2
  • M. Sharman
    • 3
  • V. Trucco
    • 2
  • F. Giolitti
    • 2
  • R. G. Dietzgen
    • 1
  1. 1.Queensland Alliance for Agriculture and Food InnovationThe University of QueenslandSt. LuciaAustralia
  2. 2.Instituto de Patología Vegetal (IPAVE), Centro de Investigaciones AgropecuariasInstituto Nacional de Tecnología AgropecuariaCórdobaArgentina
  3. 3.Department of Agriculture and FisheriesAgriScience Queensland, Ecosciences PrecinctDutton ParkAustralia

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