First detection of orchid fleck virus in orchids in Mexico

Abstract

For the first time, an isolate of the dichorhavirus orchid fleck virus (OFV, family Rhabdoviridae) was found infecting an orchid plant in Mexico. The infected sample of Epidendrum veroscriptum was collected in a nursery in Lagunillas, municipality of Zihuateutla, Edo. Puebla. Mites gathered on this plant were analyzed by light and scanning electron microscopy, which consistently indicated the presence of adults of the species Brevipalpus californicus, the common vector of OFV. Viral identification was based on symptoms, cytopathology, and reverse transcriptase-PCR/sequencing of genome fragments of the RNA1 and 2 molecules. Since isolates of OFV causing citrus leprosis have been previously detected in the Mexican states of Chiapas, Querétaro, and Jalisco, we promote a pertinent discussion and thought-provoking questions regarding the epidemiology and putative evolution of OFV.

Orchid fleck dichorhavirus, the type member of the genus Dichorhavirus, family Rhabdoviridae, was first described in Japan in 1969 [1–4]. Since then, many isolates and strains of orchid fleck virus (OFV) have been found dispersed worldwide infecting a large range of orchid genera, possibly associated with the intense either legal or illegal international trade of these plants [5–10], and further ornamentals such as lilyturf (Liriope spicata Thumb.), cordyline (Cordyline sp.) and common hollyhock (Alcea rosea L.) [11–13]. Other isolates of OFV were found naturally infecting Citrus species in Mexico, Colombia, and South Africa causing the citrus leprosis (CL) disease [14–16]. In several of these infections, mites of the species Brevipalpus californicus Banks were identified as the natural viral vector [17, 18].

Mainly spread in the Americas, CL is considered the most serious viral disease affecting the citrus industry in Brazil, the largest sweet orange producer and orange juice exporter of the world [19, 20]. The application of acaricide for the control of CL vector mites notably impacts the São Paulo state citrus sector demanding up to US$ 54 million per year, a value representing almost 5% of the management cost of the orchards [21]. In Mexico, a country among the tenth world´s largest producers of oranges and the second of limes [20], several Citrus species are affected by CL [15], and due to its close geographic proximity to the USA, the disease is a potential threat to the country’s citrus industry [22].

Under natural conditions, viruses inducing CL, as well as all known Brevipalpus-transmitted viruses (BTVs), cause characteristic non-systemic diseases in their hosts [23]. In the infected plants, BTVs remain restricted to chlorotic spots or necrotic lesions commonly observed on fruits, leaves, and young branches. These typical lesions are the result of plant defense mechanisms in response to these viruses including the production of reactive oxygen species, the activation of hormone-mediated defensive pathways, and the induction of cell death, which are part of a hypersensitive-like response [24–26].

Apart from OFV, the dichorhaviruses citrus leprosis virus N (CiLV-N) and citrus chlorotic spot virus (CiCSV), and the cileviruses citrus leprosis virus C and citrus leprosis virus C2 (genus Cilevirus, family Kitaviridae), also cause CL [18, 27, 28]. Cileviruses are enveloped bacilliform viruses with a bipartite single-stranded (+) RNA genome, and represent the prevalent causal agents of CL in the Americas. Dichorhaviruses have naked rod-like shape particles containing a bi-segmented genome made up of single-stranded (-) RNA molecules [4]. Most of the genetic information contained in RNA1 (≈ 6.4 kb) of dichorhaviruses codifies for structural proteins from ORFs arranged in the order 3′-N-P-MP-M-G-5′, whilst the RNA2 (≈ 6.0 kb) only codifies the RdRp protein, also known as the L protein, responsible for the replication and transcription processes of the viral genome [4].

In this study, we investigated the putative causal agent of necrotic ringspot symptoms on leaves of an orchid plant, identified as Epidendrum veroscriptum Hágsater (Fig. 1a), detected during a survey of ornamental-infecting viruses in a nursery in Lagunillas, municipality of Zihuateutla, Edo. Puebla (20°22′54″N 97°94′55.6″W). Suspecting a possible infection by a dichorhavirus due to distinctive symptoms, leaf samples were taken and further analyzed by transmission electron microscopy and RT-PCR assays. Additionally, mites detected in this plant were collected and fixed in absolute ethanol for taxonomic identification using two different approaches.

Fig. 1.

a Ringspot-type lesion on leaves of the orchid Epidendrum veroscriptum found in a nursery in Lagunillas, municipality of Zihuateutla, Ed. Puebla, Mexico. b Transmission electron micrograph of a section through the leaf lesion tissue of E. veroscritum. A spongy parenchyma cell reveals a nucleus (N) with a large electron-lucent viroplasm (*) and short rod-like particles (arrows) interspersed in it. Rod-like particles, putative OFV virions, organized radially associated with the endoplasmic reticulum membrane, forming the so-called “spoke wheel” structure (SW). In the nucleus, part of the paracrystalline array (Pc) of the rod-like particles can be seen. c Enlargement of the nucleolus (Nu) and Pc areas observed in (b). Ch chloroplast, CW cell wall, M mitochondrion, and Vc vacuole. Arrows indicate longitudinal sections of viral particles

For the analysis by light microscopy, mites were mounted in Hoyer medium and examined using a Zeiss Axioskop Imager D2 microscope (Carl Zeiss AG, Jena, Germany) equipped with differential interference contrast (DIC) system. For scanning electron microscopy, the specimens were dehydrated in a critical point drier (Leica CPD 300, Vienne, Austria), mounted on aluminum stubs, gold-coated in a Baltec SCD 050 sputter coater (Liechtenstein), and examined in a JEOL JSMIT300 (Akishima, Japan). The detection of the following morphological features, palp with 3 segments and 3 setae on the terminal segment, presence of f2 setae on dorsum, two solenidia on leg II, characteristic reticulation pattern of dorsum and venter (Fig. 2a–c), and their subsequent comparisons with those described for the type specimens of Brevipalpus mite species [29, 30], indicated the presence of B. californicus. Further evidence of B. californicus identity was obtained with the morphological analysis of the mite spermatheca (Fig. 2d).

Fig. 2.

Micrographs of morphological characteristics from adult Brevipalpus californicus mites found infesting E. veroscriptum. a–b Light microscopy images in differential interference contrast (DIC) of the dorsal and ventral view of the reticulations, respectively. c Scanning electron micrograph of the dorsal view. d Light microscopy DIC image of the spermatheca vesicle

For the cytopathology analysis of the affected plant tissues, leaf fragments were fixed and processed as described previously [31]. Parenchymal cells of the lesions exhibited typical cytopathic effects of infection by dichorhavirus [4], i.e. electron-lucent inclusions in the nucleus, interspersed with short rod-like particles, which occasionally formed paracrystalline arrays (Fig. 1b). These particles were also found in the cytoplasm associated with membranes of the endoplasmic reticulum, in some instances arranged radially, forming a typical configuration known as “spokewheel” (Fig. 1c).

Molecular detection and characterization of the putative causal agent included the extraction of the total RNA from the affected tissues and the preparation of its corresponding cDNA as previously described [32]. A set of specific and degenerate primers were used for the detection of all known OFV strains by PCR tests [4]. Only those reactions using the specific (CiLV-N-NPF/CiLV-N-NPF) [14] and degenerate (L-DC_Fwd/L-DC_Rev) [28] primers tested positive. Amplicons of the expected sizes and corresponding to fragments of genes N (RNA1) and L (RNA2) of OFV_Mex1, were purified from the gel and sequenced in each direction using an ABI Prism 3730 automated sequencer (Applied Biosystems, CA, USA). After nucleotide BLAST and ClustalX analyses, N gene fragment of OFV_Mex1 (681 bp, GenBank accession number MT497517) showed the closest sequence identity (> 96%) with its cognates in the OFV isolates Br (MK522806.1) and Cym07 (BAX37186.1). Comparisons using the fragment derived from gene L (362 bp, GenBank accession number MT497518) indicated values higher than 97% nucleotide sequence identity with the L gene of OFV isolate So (NC_009609.1) and CL (MK522805.1). Phylogenetic analysis using genomic fragments from OFV_Mex1 and their cognates from other OFV strains showed well-defined and detached branches from those comprising the other citrus-infecting dichorhaviruses CiLV-N and CiCSV (Fig. 3). Within the OFV branch of each tree, closely related OFV isolates were distributed regardless of the host, but distribution differed between the N gene and L gene trees, most likely revealing the genome reassortment previously detected between OFV isolates [33, 34].

Fig. 3.

Phylogenetic reconstruction for OFV_Mex1 and viruses of the genus Dichorhavirus. Midpoint-rooted maximum likelihood phylogenetic trees were based on fragments of nucleotide sequences of N and L genes, a–b, respectively. Bootstrap support values (1000 iterations) of branches are indicated near nodes. Nucleotides sequences were aligned using MAFFT, and the phylogenetic relationships were inferred by using PhyML3.1. The trees were edited and visualized using Interactive Tree Of Life (iTOL) v4. Solid purple and green circles in the leaves compressing OFV indicate the host of collection of each isolate. Sequences of the dichorhaviruses citrus leprosis virus N (CiLV-N), citrus chlorotic spot virus (CiCSV), and Clerodendrum chlorotic spot virus (ClCSV) were used as outgroup

The detection of orchid-infecting OFV isolates in Mexico raises pertinent concerns and thought-provoking questions regarding the epidemiology and putative origin of the OFV-citrus strains in the country. The detection of B. californicus mites on the orchid plant is significant since they were previously shown as the vector of OFV-citrus in Mexico [17]. On the other hand, a recent report of orchid-citrus OFV spillover, likely involving B. californicus mites, occurred in the Eastern Cape region, South Africa [16]. Thus, the detection of OFV isolates and B. californicus mites in citrus orchards [15, 24, 34] and orchid nurseries in Mexico are signals indicating the need for an extensive evaluation of the OFV prevalence in orchids and the determination of the role of these plants as viral reservoirs.

Except for the worldwide distribution of OFV, which seems to be rather a consequence of anthropogenic actions, most BTVs have primarily been found from Argentina to the USA, suggesting that the Americas are the most plausible origin of these viruses [4, 23]. Orchids are ubiquitous worldwide and Mexico harbors more than 1200 species from which 40% are endemics, including E. veroscriptum [35, 36]. In contrast, Citrus spp., natives of Southeast Asia, were introduced in the American continent after the fifteenth century [37]. In this context, despite the lack of direct evidence supporting the American origin of OFV, the detection of OFV strains infecting native orchids in Mexico hints at the coevolution of an ancestral dichorhavirus and endemic plants from which current OFV strains originated. If our hypothesis is correct, new supportive evidence might be obtained from field surveys in Colombia where sweet orange (Citrus sinensis), Dieffenbachia sp., and Swinglea glutinosa plants were also found infected by OFV-citrus [18], but the orchid infection by OFV has not been formally reported yet.

Altogether, our results are a call to action stressing the requirement for new epidemiological studies and the obtainment of the complete genome of OFV_Mex1. Besides revealing the degree of identity between OFV isolates infecting citrus and orchids, genomic data might help to describe a plausible scenario of OFV evolution, among them, for instance, that current isolates could have arisen from (i) a recent orchid-citrus OFV spillover, as just detected in South Africa [16], (ii) host-associated speciation after a past spillover process, or (iii) extinguished variants of dichorhaviruses, e.g., OFV-N0, also known as CiLV-N0 [38].

Authors' contributions

PLRG and EWK conceptualized and wrote the original draft; GOC, PLRG, CCJ, ADT, and EWK worked on the formal analysis; PLRG, JFA, and EWK supervised the study; JFA and EWK were responsible for funding acquisition. PLRG, CCJ, ADT, and EWK worked on the investigation and methodology. All authors reviewed the final manuscript.

Funding

This research was funded by Fundação de Apoio à Pesquisa do Estado de São Paulo- FAPESP, grants 2014/08458-9 and 2017/50222-0. CCJ and ADT have scholarships from FAPESP, respectively 2016/01960-6 and 2018/12252-8. In addition, the study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior- Brasil (CAPES) (001. PNPD20132154-33141010001P4-PNPD-IBSP).

Data availability

Nucleotide sequences of fragments corresponding to the N and L genes of OFV_Mex1 obtained in this study were deposited in the GenBank accessions MT497517and MT497518, respectively.

Declarations

Conflict of interest

The authors have no conflict of interest.

Ethics approval

This article does not contain any studies with human participants or animals performed by any of the authors.