Virus Research 184 (2014) 87–92

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Short communication

Hardenbergia mosaic virus: Crossing the barrier between native and introduced plant species M.A. Kehoe a,b,∗ , B.A. Coutts a,b , B.J. Buirchell a,b , R.A.C. Jones a,b a b

School of Plant Biology and Institute of Agriculture, Faculty of Science, University of Western Australia, Crawley, WA 6009, Australia Crop Protection and Lupin Breeding Branches, Department of Agriculture and Food Western Australia, Bentley Delivery Centre, Perth, WA 6983, Australia

a r t i c l e

i n f o

Article history: Received 20 January 2014 Received in revised form 17 February 2014 Accepted 18 February 2014 Available online 1 March 2014 Keywords: Virus emergence Agro-ecological interface Indigenous virus Whole genomes Genetic diversity Recombination

a b s t r a c t Hardenbergia mosaic virus (HarMV), genus Potyvirus, belongs to the bean common mosaic virus (BCMV) potyvirus lineage found only in Australia. The original host of HarMV, Hardenbergia comptoniana, family Fabaceae, is indigenous to the South-West Australian Floristic Region (SWAFR), where Lupinus spp. are grown as introduced grain legume crops, and exist as naturalised weeds. Two plants of H. comptoniana and one of Lupinus cosentinii, each with mosaic and leaf deformation symptoms, were sampled from a small patch of disturbed vegetation at an ancient ecosystem–recent agroecosystem interface. Potyvirus infection was detected in all three samples by ELISA and RT-PCR. After sequencing on an Illumina HiSeq 2000, three complete and two nearly complete HarMV genomes from H. comptoniana and one complete HarMV genome from L. cosentinii were obtained. Phylogenetic analysis which compared (i) the four new complete genomes with the three HarMV genomes on Genbank (two of which were identical), and (ii) coat protein (CP) genes from the six new genomes with the 38 HarMV CP sequences already on Genbank, revealed that three of the complete and one of the nearly complete new genomes were in HarMV clade I, one of the complete genomes in clade V and one nearly complete genome in clade VI. The complete HarMV genome from L. cosentinii differed by only eight nucleotides from one of the HarMV clade I genomes from a nearby H. comptoniana plant, with only one of these nucleotide changes being non-synonymous. Pairwise comparison between all the complete HarMV genomes revealed nucleotide identities ranging between 82.2% and 100%. Recombination analysis revealed evidence of two recombination events amongst the six complete genomes. This study provides the first report of HarMV naturally infecting L. cosentinii and the first example for the SWAFR of virus emergence from a native plant species to invade an introduced plant species. Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.

Hardenbergia mosaic virus (HarMV) (genus Potyvirus, family Potyviridae) is a recently described virus from the native perennial legume Hardenbergia comptoniana, a plant species endemic to the South-West Australian Floristic Region (SWAFR) (Hopper and Gioia, 2004; Webster et al., 2007; Coutts et al., 2011; Wylie and Jones, 2011). The SWAFR is a species rich global diversity hot spot with around 8000 indigenous plant species where there was no plant cultivation until Europeans arrived in 1829 (Myers et al., 2000; Hopper and Gioia, 2004). Coat protein (CP) gene nucleotide (nt) sequencing placed 30 HarMV isolates into eight clades with up to 21% nt differences between sequences (Webster

∗ Corresponding author at: Crop Protection Branch, Department of Agriculture and Food Western Australia, Bentley Delivery Centre, Perth, WA 6983, Australia. Tel.: +61 9368 3333. E-mail address: [email protected] (M.A. Kehoe). http://dx.doi.org/10.1016/j.virusres.2014.02.012 0168-1702/Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.

et al., 2007). A ninth clade was suggested after the CP sequences of six additional HarMV isolates were added and compared with the initial 30 (Coutts et al., 2011). When their complete genomes were sequenced using Illumina GAIIx technology, two HarMV isolates found co-infecting a single H. comptoniana plant differed by 18% at the nt level (Wylie and Jones, 2011). The high degree of nt diversity over a small geographic range demonstrated by HarMV is characteristic of viruses that co-evolved with native plants locally over a long period of time (Spetz et al., 2003; Webster et al., 2007; Coutts et al., 2011). Potyviruses found in Australia fall into two groups, with roughly half of them being isolated from cultivated plants and found in other parts of the world. Potyviruses isolated from Lupinus spp. so far fall into this category. The other half constitute a potyvirus lineage found only in Australia which belongs to the bean common mosaic virus (BCMV) group (Gibbs et al., 2008; Coutts et al., 2011). Members of the Australian potyvirus lineage have only been

9564 8906 9659 9668

c

d

a

b

Three symptomatic plant samples were collected from a disturbed patch of native vegetation at the agro-ecological interface. Samples were sequenced on an Illumina HiSeq2000. Coded symptom descriptions: M, mosaic; LD, leaf deformation. Programs used for assembly of data: CLC, CLC Genomics Workbench 6.5 (CLC bio); Geneious 6.1.6 (Biomatters). Final sequences for MD2 and MD3 consisted of the consensus between de novo assembly and the mapped consensus. Final sequences for MD4A-D consisted of their de novo contigs only.

– – – – – – – – – – – – – – – – 292,188 17,738 33,716 29,120 9648 8975 9635 9600 KJ152154 KJ152155 KJ152156 KJ152157 12,538,704

12,239,175

1806

MD4-A MD4-B MD4-C MD4-D

2975 194 343 298

9647 352,433 3612 9726 NC015394 347,261 3516 9695 KJ152153 MD3 12,840,928 13,154,290

677

9647 522,746 5354 9751 NC015394 499,921 5072 9672 KJ152152 MD2 1691 17,182,534 17,616,843

1.Hardenbergia M, LD comptoniana 2.Lupinus M, LD cosentinii 3.H. comptoniana M, LD

Number of reads mapped to ref. sequence (Geneious) Average coverage Length of consensus (nt) Reference sequence used for mapping (Geneiousc ) No. of reads mapped to contig of interest (CLC) Average Contig length (nt) coverage Accession number Sample sequence ID No. of contigs produced (CLCc ) No. of reads after trimming No. of reads obtained Leaf symptomsb Plant/host ID

isolated from native plants or naturalised weed species introduced as potential pasture species, apart from passionfruit woodiness virus (PWV) from cultivated Passiflora spp. (Gibbs et al., 2008; Coutts et al., 2011). HarMV is the best studied member of this lineage. At the CP level, HarMV is most closely related to six other potyviruses found only in Australia: clitoria chlorosis virus (CliVY), hibbertia virus Y (HiVY), siratro 1 virus Y (S1VY), siratro 2 virus Y (S2VY), passiflora mosaic virus (PaMV) and PWV (Coutts et al., 2011). Lupinus spp. were introduced to the SWAFR around the early 1900s where they were used as sheep feed initially, and later domesticated as grain legume crops for rotation with cereals (French et al., 2008). L. angustifolius (narrow-leafed lupin), L. cosentinii (sandplain lupin), L. luteus (yellow lupin) and L. mutabilis (pearl lupin) became infected by HarMV experimentally in a glasshouse environment (Webster et al., 2007). Also, naturally occurring aphids spread HarMV from introduced H. comptoniana infector plants to L. angustifolius plants growing in experimental field plots (Luo et al., 2011). However, except within these field plots, HarMV has not been found infecting any Lupinus spp. or other introduced plant species naturally. We therefore investigated an introduced lupin-H. comptoniana interface scenario involving an ancient ecosystem (i.e. native Australian plants) and a recent agroecosystem (i.e. introduced species) in the SWAFR (Webster et al., 2007; Jones, 2009; Alexander et al., 2014; Vincent et al., 2014). As such, we report the first detection of HarMV infecting L. cosentinii naturally, and the first example of an indigenous virus effectively crossing the barrier between native and introduced species in the SWAFR. We also present four new complete and two nearly complete HarMV genome sequences, including one complete sequence from L. cosentinii and the remaining sequences from H. comptoniana. Leaf tissue from two H. comptoniana and one L. cosentinii plants all showing leaf mosaic and deformation symptoms, were collected at the agro-ecological interface from a patch (50 m × 5 m) of disturbed native vegetation surrounding experimental field plots at Medina near Perth, Western Australia (WA). Within the patch, the L. cosentinii plant sampled was growing as a naturalised weed in close proximity (4 m) to the sampled H. comptoniana plants. The samples were tested with generic potyvirus monoclonal antibodies (Agdia, USA) using the antigen-coated indirect ELISA protocol of Torrance and Pead, 1986. Absorbance values (A405 ) were regarded as positive when more than three times those of the healthy sap control. For testing by RT-PCR, total RNA was extracted using a Spectrum Plant Total RNA kit (Sigma-Aldrich, Australia) according to manufacturers’ instructions. Reverse transcription was performed with Improm-II reverse transcriptase (Promega, Australia) using the random primers provided according to manufacturer’s instructions. PCR was performed with the GoTaq green master mix (Promega, Australia). PCR primers for generic potyvirus identification were from Webster, 2008. Total RNA from each potyvirus positive sample was sent to the Australian Genome Research Facility (AGRF) for library preparation and barcoding (24 samples per lane) before 100 bp paired-end sequencing on an Illumina HiSeq2000. For each sample, reads were first trimmed using CLC Genomics Workbench 6.5 (CLCGW) (CLC bio) with the quality scores limit set to 0.01, maximum number of ambiguities to two and removing any reads with 30%. This is a much higher than the species demarcation borderline of 23–24% suggested for potyviruses at the nt level (Adams et al., 2005). Recombination is a very successful method of viral evolution with many well studied examples of economically important viruses, including potyviruses such as bean yellow mosaic virus (BYMV), potato virus Y (PVY) and turnip mosaic virus (TuMV) (e.g., Ohshima et al., 2002; Wylie and Jones, 2009; Karasev and Gray, 2013; Ohshima, 2013). Recombination analysis revealed recombination amongst HarMV whole genomes with patterns that matched their phylogenetic groupings. Following the addition of six new CP sequences to the sequences from Webster et al., 2007, Coutts et al., 2011 suggested that the HarMV phylogenetic clades IV and V should be combined into one clade. However, based on our recombination analysis, we suggest that they should remain separate because MD4-D (which represents the first genome sequence from clade V) was not a recombinant, whilst HQ161080 from clade IV was recombinant (Fig. 3). Moreover, as next generation sequencing techniques are becoming cheaper, more accessible and more widely used, the number of complete plant virus genomes available in public databases is rising rapidly. Given the readiness of potyviruses to undergo recombination both within species and with other species, it is becoming increasingly important to use recombination analysis to provide a thorough understanding of the genetic makeup and

phylogenetic placement of a potyvirus. Addition of further complete HarMV genome sequences to those studied here would allow other phylogenetic HarMV groupings defined from CP sequences to be confirmed for whole genomes. Although the recombination analysis conducted here only included HarMV and PWV, it would be interesting to include whole genomes of some of the more closely related Australian potyviruses such as PaMV, ClCV, HibVY, S1VY and S2VY, once they become available. Ongoing study of the evolution of such a geographically distinct group of viruses provides an exciting prospect! This study provides the first report of HarMV naturally infecting L. cosentinii, a lupin species that became naturalised after its introduction to the SWAFR. It also constitutes the first example for the SWAFR of an indigenous virus that has made a host species jump (Woolhouse et al., 2005), successfully crossing the ancient ecosystem-recent agrosystem interface. Luo et al., 2011 had previously demonstrated HarMV spread to L. angustifolius in an artificial field plot situation with deliberately introduced H. comptoniana infector plants. So far, the natural host range of HarMV is limited to H. comptoniana and H. violacea, with only the former being native to the SWAFR (Webster et al., 2007). So it seems that the nt and amino acid changes required to facilitate this jump may be few, given there were just eight nt and one amino acid differences between the HarMV sequence from L. cosentinii (MD3) and the closest HarMV sequence from H. comptoniana at the same site (MD2). Little is known regarding the feeding preferences associated with the natural aphid vectors of HarMV. It is possible they have a feeding preference for H. comptoniana, or that their transmission efficiencies are lower on their non-preferred hosts (e.g. L. cosentinii). Further experimentation would provide insight and understanding regarding the limited natural host range for HarMV. Its artificial host range includes species in four additional families (Webster et al., 2007; Coutts et al., 2011; Vincent et al., 2014). Our research provides the first example for the SWAFR of virus emergence from native plants to invade an introduced plant species. The symptoms in the L. cosentinii plant infected with HarMV were leaf mosaic and leaf deformation, but more severe symptoms such as plant death and stunting were recorded with experimental HarMV infection of L. cosentinii (Webster et al., 2007). The natural distribution of H. comptoniana in the SWAFR covers much the same area as the distribution of L. cosentinii. Moreover, this region borders a larger area of south west Australia where most of the grain legume crop species L. angustifolius is produced (Western Australian Herbarium, 1998). Further studies are

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warranted to determine whether HarMV infection is cause for concern for the grain legume industry. Viruses of native plant communities are sometimes ignored or poorly researched, even though viruses co-evolved with wild plants well before plant domestication and these communities are likely to contain potentially damaging viral pathogens. Here we have further evidence that as new contact between native plants and introduced crops or weeds increases due to mans’ activities and climate change, the threat of emerging viruses from indigenous plants to introduced plants is set to increase (Cooper and Jones, 2006; Webster et al., 2007; Jones, 2009; Jones and Barbetti, 2012; Alexander et al., 2014; Vincent et al.,2014). Acknowledgements This research was funded by an Australian Postgraduate Award (APA), and an Australian Grains Research and Development Corporation (GRDC) Studentship, Project number GRS10039. It was undertaken using the facilities at the Department of Agriculture and Food Western Australia. This study forms part of a PhD project by the first author at the University of Western Australia. References Adams, M.J., Antoniw, J.F., Fauquet, C.M., 2005. Molecular criteria for genus and species discrimination within the family Potyviridae. Arch. Virol 150, 459– 479. Alexander, H.M., Mauck, K.E., Whitfield, A.E., Garrett, K.A., Malmstrom, C.M., 2014. Plant-virus interactions and agro-ecological interface. Eur. J. Plant Pathol. 138, 529–574. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Boni, M.F., Posada, D., Feldman, M.W., 2007. An exact nanoparametric method for inferring mosaic structure in sequence triplets. Genetics 176, 1035–1047. Cooper, I., Jones, R.A.C., 2006. Wild plants and viruses: under-investigated ecosystems. Adv. Virus Res. 67, 1–40. Coutts, B.A., Kehoe, M.A., Webster, C.G., Wylie, S.J., Jones, R.A.C., 2011. Indigenous and introduce potyviruses of legumes and Passiflora spp. from Australia: biological properties and comparison of coat protein nucleotide sequences. Arch. Virol 156, 1757–1774. French, B., Shea, G., Buirchell, B., 2008. Introduction and history. In: White, P., French, B., McLarty, A. (Eds.), Producing Lupins, Department of Agriculture and Food Western Australia Bulletin 4720. , 2nd ed. Department of Agriculture and Food, South Perth, Western Australia. Gibbs, M.J., Armstrong, J.S., Gibbs, A.J., 2000. Sister-scanning: a Monte Carlo procedure for assessing signals in recombinant sequences. Bioinformatics 16, 573–582. Gibbs, A.J., Mackenzie, A.M., Wei, K.-J., Gibbs, M.J., 2008. The potyviruses of Australia. Arch. Virol 153, 1411–1420. Hopper, S.D., Gioia, P., 2004. The southwest Australian floristic region: evolution and conservation of a global hot spot of biodiversity. Annu. Rev. Ecol. Evol. Syst 35, 623–650. Jones, R.A.C., 2009. Plant virus emergence and evolution: origins, new encounter scenarios, factors driving emergence, effects of changing world conditions, and prospects for control. Virus Res. 141, 113–130.

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