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Citation for published version Fogell, Deborah J. and Kundu, Samit and Roberts, David L. (2019) Genetic homogenisation of two major orchid viruses through global tradebased dispersal of their hosts. Plants, People, Planet . pp. 1-7.

DOI https://doi.org/10.1002/ppp3.46

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Received: 23 January 2019 | Revised: 10 April 2019 | Accepted: 1 May 2019 DOI: 10.1002/ppp3.46

BRIEF REPORT

Genetic homogenisation of two major orchid viruses through global trade‐based dispersal of their hosts

Deborah J. Fogell1,2 | Samit Kundu3 | David L. Roberts1

1Durrell Institute of Conservation and Ecology, School of Anthropology Societal Impact Statement and Conservation, University of Kent, Orchid viruses are capable of causing flower deformities and death, which can se‐ Canterbury, UK verely impact the horticultural industry and wild orchid conservation. Here we show 2Institute of Zoology, Zoological Society of London, London, UK how two of these quickly evolving viruses display few genetic differences since their 3Human & Life Sciences, Canterbury Christ first emergence, across countries and host plants. This is concerning as, despite bios‐ Church University, Canterbury, UK ecurity regulations to control the movement of orchids and their related pathogens, Correspondence these patterns are suggestive of rapid and regular international movement of horti‐ Deborah J. Fogell, Durrell Institute of cultural material. Poor biosecurity practices could threaten the orchid horticultural Conservation and Ecology, School of Anthropology and Conservation, University industry and result in the accidental translocation or reintroduction of infected plant of Kent, Canterbury, Kent, UK. material intended to recover wild populations. Email: [email protected]

KEYWORDS Cymbidium mosaic virus, CymMV, horticulture, Odontoglossum ringspot virus, Orchidaceae, ORSV, phytosanitary, trade

1 | INTRODUCTION orchids, though their presence in natural populations is currently un‐ known (Umikalsum, Bakar, Khairun, & Faridah, 2006; Zettler et al., With an estimated 28,484 species (WCSP, 2017), orchids are among 1990). the most speciose families of flowering plants (Joppa, Roberts, CymMV is a member of the potexvirus family with a single‐ Myers, & Pimm, 2011). One of their most outstanding features is stranded RNA (ssRNA) positive sense genome of approximately their diversity in floral form, resulting in a sizeable international hor‐ 6.3 kb (Wong et al., 1997). Signs of disease include flower necro‐ ticultural industry where orchids consistently appear among the top sis, chlorotic or necrotic patches on the leaves, and growth retar‐ horticultural commodities in terms of pot plants (Hinsley et al., 2017; dation (Ajjikuttira et al., 2002; Pearson & Cole, 1991; Zettler et al., Royal Flora Holland, 2015; United States Department of Agriculture, 1990), though asymptomatic hosts can occur (Ajjikuttira et al., 2002; 2016), as well as comprise approximately 10% of the cut flower in‐ Zettler et al., 1990). Although the virus appears not to have a natural dustry (De, Pathak, Rao, & Rajeevan, 2015; Hinsley et al., 2017). vector for transmission (Namba & Ishii, 1971), it is stable and easily However, orchid viruses significantly affect flower quality and spread through contaminated horticultural tools, media, and pollen yield, and have the potential to cause substantial economic losses (Lawson & Brannigan, 1986; Moraes, Krause‐Sakate, & Pavan, 2017). (Khentry, Paradornuwat, Tantiwiwat, Phansiri, & Thaveechai, 2006; ORSV, a tobamovirus, also has a positive sense ssRNA genome of Wong, Chng, Lee, Tan, & Zettler, 1994). Whilst more than 50 dif‐ around 6.5 kb (Chng et al., 1996). ORSV infection can result in flower ferent viruses have been documented to infect orchids, Cymbidium malformation and color breaking, and ringspots on the leaves. As mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) with CymMV, the transmission of ORSV appears to be by means of are the most prevalent (Wong et al., 1994; Zettler, Ko, Wisler, Elliott, mechanical inoculation (Cánovas, Ballari, & Nome, 2016; Hu et al., & Wong, 1990). Both viruses have a global distribution in cultivated 1994; Zettler et al., 1990).

This is an open access article under the terms of the Creat​ive Commo​ns Attri​bution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors, Plants, People, Planet © New Phytologist Trust

 wileyonlinelibrary.com/journal/ppp3 | 1 Plants, People, Planet, 2019;00:1–7. 2 | FOGELL et al.

Whilst both of these viruses are currently only known from cul‐ genera (62% for CymMV and 74% for ORSV), with Cymbidium being tivated stock or domestic orchid collections, to date little emphasis the most prevalent (28% and 30% respectively). has been placed on the dangers of viral spill‐over into wild orchid Geneious 8.1.7 (Kearse et al., 2012) DNA editing software was populations through activities such as reintroduction and, conse‐ used to align and edit all DNA sequences. The programme jMod‐ quently, what impacts this may have on biodiversity loss. We are elTest 2.1.7 (Posada, 2008) was used to infer the best fit nucleotide aware of only a single study from Australia, in which several exotic substitution model across each dataset. A TN93 transition model viruses were described from wild Diuris orchid populations (Wylie, (Tamura & Nei, 1993) with gamma distributed rate variation was Li, Dixon, Richards, & Jones, 2013). The global spread of pathogens favoured for CymMV and an HKY85 transition and transversion poses an increasing threat to biodiversity (Daszak, Cunningham, & model (Hasegawa, Kishino, & Yano, 1985) was favoured for ORSV. Hyatt, 2000) and has been linked to the collapse of a number of Evolutionary rates were determined using the programme Beast wild populations and multiple species extinctions (Anderson et al., v1.8.2 (Drummond, Suchard, Xie, & Rambaut, 2012) using only those 2004). Most orchids are naturally rare and threatened with extinc‐ sequences available with a confirmed associated year of sampling tion through habitat loss and over‐collection from the wild (Roberts (n = 116 for CymMV and n = 101 for ORSV). The Bayesian skyline & Dixon, 2008). For example, Paphiopedilum vietnamense was de‐ coalescent demographic prior was used as it allows for temporal scribed new to science in 1999 only to be declared extinct in the changes in population size (Drummond, Rambaut, Shapiro, & Pybus, wild five years later through over‐collection for the horticultural 2005). Tracer v1.6 was used to ensure thorough model mixing and trade (Averyanov, 2004). Due to the threat from trade, all orchids that a reasonable effective sample size (ESS > 200) had been reached were placed on Appendix II, or higher, from the inception of the for all parameters. Two runs, each consisting of ten independent Convention on International Trade in Endangered Species (CITES) Monte Carlo‐Markov chains (MCMC) were implemented for 100 mil‐ (Koopowitz, 2001; Roberts & Solow, 2008), and currently comprises lion generations each, with trees sampled every 10,000 generations. over 70% of species listed on CITES (Hinsley et al., 2017). Within the The evolutionary rate and time to most recent common ancestor orchid horticultural community there is a level of resentment toward (TMRCA) were estimated under an uncorrelated relaxed molecular CITES, as it is perceived to negatively impact on trade by restrict‐ clock (Drummond, Ho, Phillips, & Rambaut, 2006; Lemey, Suchard, ing access and inhibiting the flow of plant material into the hobby & Rambaut, 2009). LogCombiner v1.8.2 was used to combine runs, (Hinsley et al., 2017; Koopowitz, 2001; Zelenko, 2005; Roberts pers. TreeAnnotator v1.8.2 was used to obtain the tree with the highest obs.). Along with national phytosanitary regulations, these regula‐ clade credibility (Drummond et al., 2012) and FigTree v1.4.2 was tions have the potential to limit the spread of orchid viruses across then used to visualise and edit the consensus tree (Rambaut, 2009). international borders by potentially reducing the movement of their Additionally, for greater analytical strength, maximum‐likelihood hosts. However, phytosanitary certificates are not always required, (ML) phylogenies with 1,000 bootstrap replicates were generated as in the case of free movement of materials within economic unions for both genes to infer geographic relationships between all avail‐ such as the European Union (European Commission, 2000), and in able sequences using PhyML 3.0 (Guindon et al., 2010). GENEIOUS some cases infected but asymptomatic plant material (Khentry et al., v8.1.7 (Kearse et al., 2012) was used to generate the consensus tree 2006) may be transported if prior screening is not carried out. prior to visualisation and editing in FigTree. Here we investigate the molecular evolution of the two most prevalent orchid viruses, CymMV and ORSV, using datasets repre‐ senting their global distribution in cultivated stock. We ask whether 3 | RESULTS the considerable international trade of cultivated orchids has effec‐ tively “homogenised” the genetic diversity of the viruses or if their Figure 1 shows the ML phylogeny of the CymMV capsid protein high mutation rate, as expected for RNA viruses (Duffy, Shackelton, sequences depicting their biogeographic association. The tree com‐ & Holmes, 2008), and regulations on international movement has led prises isolates from 29 host genera across 16 different countries to geographical and/or chronological differentiation. or overseas territories, with numerous small clades displaying lit‐ tle apparent geographical or host genus pattern to their clustering. The Chinese and South Korean isolates, which are particularly well 2 | MATERIALS AND METHODS represented in this study, form several distinct groups indicating the presence of multiple viral genotypes within these regions. The rate 2.1 | Sampling and data analyses of evolution calculated in the Bayesian analysis conducted over the All available nucleotide sequence data for the capsid protein of dated subset of isolates (Figure S1) was determined to be 1.58 × 10−3 CymMV and ORSV were obtained from the Genbank database (95% HPD 7.32 × 10−4–2.60 × 10−3) substitutions per site (Table 1). (Accessed 8 January 2018), along with any associated data regard‐ The TMRCA was determined to be 43.24 (95% HPD 15.86–75.58) ing host species from which they were isolated and sampling date years before the most recent confirmed sampling event in 2015 where available (n = 211 for CymMV, Table S1, and n = 146 for ORSV, (Table 1). Whilst, the clades in the Bayesian phylogeny appear to Table S2). Both viral isolate datasets were dominated by four host have slightly more structure with regards to their geographic origin FOGELL et al. | 3

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than with the ML tree, the analysis similarly suggests no host speci‐ the dated subset of isolates (Figure S2) was determined to be ficity (Figure S1). 1.93 × 10−3 (95% HPD 1.17 × 10−3–2.72 × 10−3) substitutions per Figure 2 shows the ML phylogeny of the ORSV capsid pro‐ site (Table 1). The ORSV TMRCA was determined to be 21.92 tein sequences comprising sequences isolated from 12 host gen‐ (95% HPD 21.01–23.41) years before the most recent confirmed era across 14 countries. As with CymMV, both South Korean and sampling event in 2015 (Table 1). The Bayesian analysis has clus‐ Chinese isolates are highly represented in this dataset and lit‐ tered the majority of South Korean isolates into four monophyletic tle geographical, or host genus clustering is observed. The rate clades but there was still no evidence of host specificity amongst of evolution calculated in the Bayesian analysis conducted over sequences (Figure S2).

TABLE 1 Estimated substitution rate and TMRCA (along with their 95% HPD intervals) of the capsid protein of CymMV and ORSV inferred from an uncorrelated relaxed clock model

95% lower Marginal Mean substitution 95% lower 95% upper TMRCA HPD interval 95% upper HPD Virus Likelihood rate (site/ year) HPD interval HPD interval (years) (TMRCA) interval (TMRCA)

CymMV −3,902.52 1.58 × 10−3 7.32 × 10−4 2.60 × 10−3 43.24 15.86 75.58 ORSV −1,261.73 1.93 × 10−3 1.17 × 10−3 2.72 × 10−3 21.92 21.01 23.41 4 | FOGELL et al.

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FIGURE 2 Maximum likelihood phylogenetic tree denoting relationships between Odontoglossum ringspot virus (ORSV) capsid protein sequences obtained from cultivated orchid hosts. Branches are labeled according to host genus and colored according to bootstrap support. Labels are colored according to country of sampling, as denoted in the key

4 | DISCUSSION Our analyses date the CymMV common ancestor to around 1972 (95% HPD 1939–1999), while the date for ORSV is much younger at This study suggests that there is little geographical and temporal dif‐ around 1993 (95% HPD 1992–1994). The broader confidence inter‐ ferentiation in global isolates of two of the most prevalent orchid vals surrounding the TMRCA for CymMV is likely due to a higher viruses, CymMV and ORSV. There is also no indication that different proportion of isolates from more diverse geographic regions, as op‐ genotypes of these viruses infect particular host species. This is in posed to the heavily biased ORSV dataset comprising predominantly line with previous studies (Ajjikuttira et al., 2002; Moles, Delatte, Chinese and South Korean isolates used in the Bayesian analyses Farreyrol, & Grisoni, 2007; Moraes et al., 2017) and suggests that (Figures 1, 2 and S1, S2). While the early history of orchid cultiva‐ there has been significant global transmission of these viruses. tion up until the early 1900s is well‐known (see Reinikka & Romero, While acknowledging that only a partial fragment of the genome has 1995), little has been documented regarding the expansion of and been analyzed to determine the TMRCA, and that all but three of changes within the industry over the last 50 years. As such, under‐ the sequences with confirmed sampling dates were obtained within standing the significance of the TMRCA for both viruses, as it relates a decade of the 2017 limit of this study (Table S1, S2), both viruses to the orchid industry, is problematic. However, according to Motes appear to have ancestral genotypes that originated within the last (pers. commun.), the cloning of orchids, beginning in the 1950s, 50 years. This finding is in line with the period of mass expansion of was a well‐established technique by the 1970s. Many older plants trade in ex situ propagated orchids. were used in mass production through these micropropagation FOGELL et al. | 5 techniques, some of which are likely to have carried viruses. During identified the presence of additional sub‐groups (Figure 1), but the 1970s, Singapore, Malaysia, Thailand, Hawaii, California, and with little differentiation based on geographic origin or host. The the Netherlands were rapidly expanding their orchid sectors, par‐ observed homogenisation is remarkable given the apparent lack ticularly for the cut flower industry. As expected in a host‐pathogen of a known natural vector. Interestingly, this has occurred despite system, the frequency of contact to facilitate pathogen transmission stricter international trade regulations for orchids under CITES, and and, therefore, the probability and persistence of infection increases phytosanitary regulations enforced by many countries (Khentry et when the host population reaches high densities (Lloyd‐Smith et al., al., 2006; Lawson & Brannigan, 1986). As CITES aims to ensure that 2005). the international trade in wild animals and plants does not threaten The early 1990s, corresponding with the TMRCA of ORSV, saw their survival, it does not act primarily as a means to enforce phy‐ the advent of mass production of Phalaenopsis, one of the most tosanitary regulation and biosecurity. However, it does represent widespread orchid genera in the industry (Hinsley et al., 2017; Motes a significant constriction on the movement of plant material across pers. commun.). After the controversy surrounding the use of the international borders (Koopowitz, 2001; Roberts & Solow, 2008; fungicide Benlate, that devastated the foliage growers in Florida Zelenko, 2005). Even so, the level of restriction on the movement and Hawaii (Geyelin, 2001; Motes pers. commun.), horticulturalists of material caused by CITES does not appear to have resulted in any looked to new crops. This came at a time when the Taiwanese gov‐ geographic separation of either virus. Homogenisation is only likely ernment were subsidising the growth in Phalaenopsis production and to increase further with the changes to CITES regulations which now became the source of much of the stock for the United States and, allow the movement of large consignments of specific orchid hybrids potentially, growers in other regions such as Europe (Motes pers. from Cymbidium, Dendrobium, Miltonia, Odontoglossum, Oncidium, commun.). This also saw the decline in then small growers, with the Phalaenopsis and Vanda without the need for CITES permits (see monopolisation of the industry by large horticultural corporations CITES, 2007). (Motes pers. commun.). Charles Darwin once speculated that “… the great grandchil- At approximately 10−3 substitutions per site per year we esti‐ dren of a single [orchid] plant would nearly … clothe with one uniform mated very high evolutionary rates for both viruses, but well within green carpet the entire surface of the land throughout the globe”, and that estimated for other RNA viruses of 10−5 and 10−2 substitutions noted “minute seeds within their light coats are well fitted for wide per site per year (Duffy et al., 2008). Whilst we have only analyzed dissemination…” (Darwin, 1862). While the multi‐billion‐dollar in‐ a portion of both genomes (~10%), studies of other positive sense ternational trade in orchids has certainly helped disperse orchids ssRNA plant viruses also document evolutionary rates very similar to through the horticultural community, it has also dispersed their those we have estimated here for CymMV and ORSV (e.g. Fargette associated viruses, resulting in a homogenised, or rather “uniform… et al., 2006; Wu et al., 2011). This high evolutionary rate may be ex‐ carpet” of CymMV and ORSV. While regulations are in place, such pected to result in divergence over time and geographic distribution, as plant health certification, that should, and others that could particularly in light of the restrictions on international trade imposed (i.e. CITES), potentially limit the dispersal of the virus, this clearly by national phytosanitary regulation and CITES. Even within the rel‐ does not seem to be the case. It is also possible for virus‐free plant atively short period estimated for the emergence of these viruses, material stocks for propagation and export to be readily gener‐ we would have expected to see some evidence of geographically ated with the application of heat and chemical treatments (Lim, specific divergence within isolated populations. However, the phy‐ Wong, & Goh, 1993; Wylie et al., 2013). The rapid global disper‐ logenetic patterns seen in Figures 1 and 2 suggests that the rate sal of viruses not only has the potential to impact this lucrative of dispersal of these viruses is high enough to result in any new horticultural industry, it also threatens orchid species in the wild variation being spread rapidly through the global distribution of cul‐ through poor biosecurity practices resulting in the reintroduction tivated orchids. Indeed, it is clear from our results that numerous of infected stock or management of wild populations with infected introductions of both of these viruses have occurred globally. We horticultural tools. frequently observe close phylogenetic relationships between iso‐ lates obtained from geographically distinct regions. For example, the ACKNOWLEDGMENTS well supported clade of CymMV isolates obtained from 12 different genera across eight countries (Figure 1). This rapid viral transmission We would like to thank Dr Martin Motes of Motes Orchids for his is aided in part by their stability (Hu et al., 1994), ease of transmission extremely useful discussions of the history of the orchid trade over through contaminated horticultural tools and media, and the pres‐ the last 50 years. ence of asymptomatic infections (Ajjikuttira et al., 2002). However, this is probably not enough to cause the global genetic homogenisa‐ AUTHOR CONTRIBUTION tion that we observe. In a previous study of 85 viral isolates Moles et al. (2007) showed DR and SK conceived the study, DF and SK managed the genetic the presence of two sub‐groups of CymMV. With the substantial data and ran the analyses, DF created the figures and tables, DR increase in data (both in terms of the number of isolates and the compiled information relating to the orchid trade, all authors wrote geographical, temporal and host species coverage) our study has and edited the manuscript. 6 | FOGELL et al.

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