Passion fruit green spot virus: host range, experimental indicator plants, latency and incubation periodsABSTRACT
Yellow passion fruit (Passiflora edulis f. flavicarpa) is the most cultivated passion fruit species in Brazil, the world's largest producer. Several viral diseases have been reported to infect the Passiflora species, including those caused by passion fruit green spot virus (PfGSV, Cilevirus passiflorae). PfGSV was originally detected in the state of São Paulo, Brazil, and later in several other Brazilian states, Paraguay, and Colombia. In addition to Passiflora spp., the virus has also been found naturally infecting several other plant species. The PfGSV genome was recently fully characterized, leading to its identification as a member of the genus Cilevirus, family Kitaviridae. Despite this progress, detailed information on pathology, epidemiology, and disease control strategies for remains lacking. In the present work, we assessed PfGSV's natural and experimental host range using 162 plant species and/or cultivars belonging to 51 families. The eighteen plants tested were infected by viruliferous Brevipalpus yothersi mites, a vector of PfGSV, whereas four became infected after mechanical inoculation. A survey of plants naturally infected with PfGSV detected the virus in five additional species, in addition to the four previously reported. Following infestation with viruliferous mites, PfGSV could be detected seven days after inoculation (dai) in still asymptomatic yellow passion fruit plant leaves. The fabaceous lablab-bean plants (Dolichos lablab L.) showed small necrotic lesions five dai, indicating its suitability as a test plant for PfGSV mite-transmission assays.
Keywords:
Cilevirus passiflorae; Kitaviridae; Brevipalpus-transmitted viruses; Dolichos lablab
Introduction
Passion fruit green spot virus (PfGSV, Cilevirus passiflorae, family Kitaviridae) is the causal agent of the disease known as passion fruit green spot, which causes green spots to appear on ripe fruits, senescent leaves, and necrotic lesions on the stems of passion fruit plants. In severe cases of infection, girdling of stems by the coalescence of numerous necrotic lesions may ultimately result in plants death (Kitajima et al., 1997; Kitajima et al., 2003; Ramos-González et al., 2020). The disease was first described in 1994, in the state of Bahia, Brazil, and was initially called passion fruit sudden death (Ramos-González et al., 2020), though the causal agent was not identified. In 1997, similar symptoms were observed in Vera Cruz, in the state of São Paulo, Brazil, where the disease became known as passion fruit green spot, causing severe losses in small orchards of yellow passion fruit (Passiflora edulis Sims f. flavicarpa Deg.) (Kitajima et al., 1997). Later, it was found that both diseases were caused by the same etiological agent, PfGSV (Ramos-González et al., 2020). Since then, the virus has been found in several other Brazilian states, as well as in Paraguay and Colombia, and has commonly caused minor losses (Kitajima et al., 2003; Ramos-González et al., 2020; Roy et al., 2023).
PfGSV is transmitted by Brevipalpus yothersi Baker mites [formerly identified as B. phoenicis (Geijskes)] (Beard et al., 2015; Tassi et al., 2017; de Lillo et al., 2021). The virus has a positive-sense, bisegmented ssRNA genome. RNA molecules are 3’-polyadenylated and contain a "cap" structure in the 5’-termini. RNA1 contains two open reading frames, while RNA2 has five, encoding seven potential proteins, showing significant similarity with the cilevirus citrus leprosis virus C2 (CiLV-C2, Cilevirus colombiaense) (RNA1 of ∼72 % and RNA2 of ∼64 %) (Roy et al., 2013; Ramos-González et al., 2020). Although its complete genome has recently been sequenced, its biology and epidemiology remain limited.
In the present study, the experimental and natural hosts of PfGSV were assessed through mite and mechanical inoculation. Additionally, for diagnostic purposes, trials were conducted to determine the minimum time required to detect PfGSV by reverse transcription polymerase chain reaction (RT-PCR) in yellow passion fruit leaves after inoculation mediated by viruliferous mites. Furthermore, a search for a suitable indicator plant for PfGSV in mite-transmission assays was conducted to facilitate future diagnostics.
Materials and Methods
Colonies of B. yothersi
For PfGSV mite-transmission assays, an isoline colony of B. yothersi, collected from privet (Ligustrum sinense Lour.) and previously identified morphologically (Beard et al., 2015), was used. The mites were maintained under controlled conditions (14-h photoperiod, 25 °C temperature, and 60-70 % relative humidity) on detached leaves of jack bean [Canavalia ensiformis (L.) DC], which were placed on a moistened spongy pad. A mixture of sand, gypsum, and wheat (1:1:1) was applied along the leaf veins to provide refuge and oviposition sites for the mites (Salinas-Vargas et al., 2016).
Virus source
Infected privet leaves, exhibiting typical chlorotic spots or ringspots, with the characteristic symptoms of this virus, served as the inoculum source for PfGSV. PfGSV infection in these privet leaves was confirmed by RT-PCR using specific primers and further validated by transmission electron microscopy (TEM) (see below), and the amplicons were also sequenced to verify viral identity.
Detection of PfGSV by RT-PCR
To confirm the presence of PfGSV in experimentally or naturally infected plants, total RNA was extracted from symptomatic samples using TRI Reagent (Sigma-Aldrich) according to the manufacturer's protocol. To minimize contamination with phenolic or protein residues, the extracted RNA underwent purification according to the protocol established by Green and Sambrook (2020). For the detection of PfGSV by RT-PCR in the foliar lesions of the test plants exhibiting limited lesion development, as well as in mites used in the experiments, total RNA extraction was performed with the PureLink Viral RNA/DNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer's recommendations.
After extraction, approximately 500 ng of total RNA was used for cDNA synthesis using random hexamer primers and the "GoScript™ Reverse Transcriptase" kit (Promega). Subsequently, PCR amplification was performed using 5 µL of "GoTaq Master Mixes" (Promega), 2.6 µL of nuclease-free water, 2 µL of the cDNA obtained, and 0.2 µL of each primer (10 µM). A specific pair of primers targeting a fragment of the PfGSV movement protein gene was used: PfGSV_MPF: 5’-CGATATTTGATCAATCCGTT-3’ and PfGSV_MPR: 5’-CACCTTAAAATTCGAGGGTT-3’ (Ramos-González et al., 2020). Amplification of the 245 bp fragment occurred under the following conditions: 94 °C for 3 min; 30 cycles of 94 °C for 30 s, 56 °C for 35 s, 72 °C for 45 s; and 72 °C for 5 min. The amplicons were separated on a 1 % agarose gel and visualized with SYBR™ Safe DNA Gel Stain (Invitrogen).
Amplicons from the inoculum source were purified with the Wizard SV Gel and PCR Clean-up System kit (Promega). The purified amplicons were Sanger sequenced in both directions. Sequencing was performed at the Unidade Laboratorial de Referência em Biologia Molecular Aplicada of the Instituto Biológico de São Paulo. The consensus nucleotide sequences were compared with other sequences deposited in GenBank using the BLASTn program (http://www.ncbi.nlm.nih.gov/blast), to confirm the identity of PfGSV.
Cytopathic analysis of symptomatic tissues
Leaf tissues from both experimentally and naturally infected plants were examined using TEM. Tissues were fixed in a glutaraldehyde/paraformaldehyde mixture, post-fixed with OsO4, dehydrated in acetone, infiltrated, and embedded in low viscosity Spurr's epoxy resin (Kitajima and Nome, 1999). The blocks were sectioned in an ultramicrotome (Leica EM UC6) equipped with a Diatome diamond knife. Sections were mounted on 300-mesh copper grids, stained with uranyl acetate and Reynold's lead citrate, and examined using a JEOL JEM 1011 TEM, and images were digitally recorded.
Search for plants naturally infected by PfGSV
A continuous field survey was conducted in residential gardens and public parks in the city of Piracicaba, in the state of São Paulo, Brazil, and surrounding cities, to identify plants exhibiting symptoms typically associated with Brevipalpus-transmitted viruses (BTV), such as localized chlorotic/necrotic spots or ringspots. When symptoms were observed, samples were collected in sealed plastic bags and analyzed in the laboratory by TEM and RT-PCR to confirm PfGSV infection (Table 1).
Experimental host range of PfGSV by mites and mechanically
To determine the experimental host range of PfGSV, 162 plant species, from 51 families (Table 2), were maintained under greenhouse conditions prior to inoculation with viruliferous mites.
List of plants assayed for the susceptibility to passion fruit green spot virus (PfGSV-Cilevirus passiflorae), inoculated with viruliferous Brevipalpus yothersi mites.
B. yothersi mites, previously fed on symptomatic leaves of privet for 48 h to acquire the virus, were transferred in batches of 20 mites to the test plants. After inoculation, the plants were kept in a greenhouse for approximately two months and monitored daily for the development of lesions associated with PfGSV infection. Although the inoculation access period for this pathosystem is known to be about 12 h (unpublished data), in this experiment, the viruliferous mites were left on the plants throughout the entire experimental period. Symptomatic leaves were first examined to verify the presence of inoculated mites, in order to remove viruliferous mites before extracting total RNA from the leaves. Subsequently, they were analyzed by TEM and RT-PCR, using specific primers to confirm the presence of PfGSV in the lesions. Two plants of each species were inoculated. Four randomly selected leaves were inoculated into each plant, ensuring an even distribution of 20 viruliferous mites throughout the plant. For control purposes, one plant from each species was infested with 20 aviruliferous mites.
To assess the mechanical transmissibility of the virus, leaf extracts of PfGSV-infected privet or yellow passion fruit plants were used as the source of inoculum. The extracts were diluted in 0.01M phosphate buffer + 0.005M Dieca + 0.001M EDTA + 0.005M Na-thioglycolate; pH 7.2, as previously described for citrus leprosis virus C (Colariccio et al., 1995). Extracts were applied manually to the leaves of test plants dusted with Celite powder to facilitate penetration. The following test plants were assessed: Antirrhinum majus L., Chenopodium amaranticolor Coste and Reyn., Chenopodium quinoa Willd., Datura stramonium L., Gomphrena globosa L., Hibiscus cannabinus L., L. sinense, Nicotiana glutinosa L., Nicotiana tabacum L., P. edulis f. flavicarpa, Phaseolus vulgaris L. cv. ‘UNA’ and Vigna unguiculata (L.) Walp. Three plants from each species were used, being produced and maintained under greenhouse conditions. Symptoms were assessed every two days for three weeks. Plants that developed localized lesions were subjected to RT-PCR and TEM-based cytopathic analysis to confirm the presence of PfGSV. Assays were conducted in duplicate. Three plants from each species were inoculated into each replicate. C. quinoa plants were used as a positive control for the inoculations on account of its known susceptibility to PfGSV in preliminary tests and its short incubation period for the virus.
Indicator plants for mite-transmission studies
To identify a suitable indicator plant for PfGSV mite-transmission assays, various leguminous plant species were evaluated: P. vulgaris, Vicia faba L., V. unguiculata, Dolichos lablab L., Mucuna pruriens (L.) DC, Cajanus cajan (L.) Millsp., and P. vulgaris var. ‘Jalo’. The plants were grown and maintained in a greenhouse until the first pair of leaves was fully expanded. At this stage, the leaves were detached and placed in Petri dishes lined with moistened filter paper. To prevent mites from escaping, a moistened cotton barrier was placed along the leaf margins.
A group of approximately 500 B. yothersi mites from the isoline colony was transferred to detached, symptomatic PfGSV-infected privet leaves, which were maintained on a moistened spongy pad surrounded by moistened cotton. The mites remained on these infected leaves for 72 h. After this period, groups of 15 viruliferous mites were transferred to the detached leaves of assayed plants. The infested leaves were kept under laboratory conditions and evaluated for symptom appearance over seven days, the maximum period during which the leaves remained viable after detachmented. After this period, the mites were removed from the leaves. PfGSV infection in leaves was evaluated in terms of the presence of local chlorotic or necrotic lesions. Symptomatic leaves were analyzed using a Hirox high-resolution stereoscopic microscope (Hirox-USA Inc.) to verify the presence of even small lesions, and all observed symptoms were digitally recorded. Leaves were photographed with a digital camera, and for symptomatic samples, small tissue sections adjacent to lesions were excised, stored at −80 °C, and subsequently processed for total RNA extraction. RT-PCR assays were performed following the methodology described in the section on the detection of PfGSV by RT-PCR. To confirm viral presence, RT-PCR was performed on a pooled lesion excised from leaves exhibiting symptoms of the viral infection. Small fragments of lesion tissue were processed for cytopathological observation by TEM to confirm infection by PfGSV. Additionally, total RNA from non-viruliferous mites was extracted as an experimental control to confirm the absence of PfGSV in the mite colony. This experiment was carried out in triplicate, with 15 leaves per replicate and 15 mites per leaf.
Incubation and latency periods
To determine the incubation and latency periods of PfGSV, yellow passion fruit seedlings were used when the first pair of true leaves was fully expanded, approximately 40 days after planting. Two fully expanded seedling leaves were inoculated with five mites each. These mites had been previously fed on PfGSV-infected, symptomatic privet leaves for 72 h, to acquire the virus. To prevent mite movement to other parts of the plant, a barrier of entomological glue was applied onto the petiole of each inoculated leaf. A mixture of sand, gypsum, and wheat (1:1:1) was applied along the leaf veins to facilitate the colonization of mites. After inoculation, the plants were kept under controlled conditions (14 h photoperiod, 25 °C and 60-70 % relative humidity). Every seven days, for approximately two months, two leaves were randomly selected and collected per repetition. Immediately after collection, the mites were carefully removed from the leaves, which were then photographed with a digital camera and stored at −80 °C for subsequent total RNA extraction. RT-PCR assays using specific primers for PfGSV detection were performed.
The experiment was carried out in duplicate, with eight seedlings per repetition. At each evaluation point, two inoculated leaves were collected per repetition, yielding 32 leaves analyzed for PfGSV by for the experiment, total RNA was extracted from asymptomatic, non-inoculated leaves to confirm the absence of natural PfGSV infection and from non-viruliferous mites to verify that the used mites were not carrying the virus. Confirmation was done by RT-PCR using specific primers for PfGSV.
Statistical analysis
All experiments were conducted using a completely randomized design. Data on indicator plants were analyzed using a generalized linear model (GLM) with a negative binomial distribution to account for overdispersion. Treatment means were compared using the Tukey test (p < 0.05). Statistical analyses were performed using the R software (version 4.1.1).
Results
Virus source
The nucleotide sequence of the virus present in the leaves of L. sinense used as inoculum in the experiments was obtained after sequencing a fragment of the PfGSV movement protein gene, which was deposited in GenBank (accession number: PV239572). Sequence analysis revealed a high identity (99.18 %) with the PfGSV BSB1 isolate previously described by Ramos-González et al. (2020), which confirmed the identity of the virus present in the privet symptomatic leaves.
Host range
Field survey
RT-PCR and cytopathological analyses confirmed the presence of PfGSV in all nine symptomatic field plant species suspected of infection by Brevipalpus-transmitted viruses: Allamanda cathartica L., Cestrum nocturnum L., Clerodendrum thomsoniae Balf., Hibiscus schizopetalus (Mast) Hook, Lantana camara Linn., Ligustrum japonicum Thunb., Ligustrum lucidum Ait., Passiflora alata Curtis, and Schefflera arboricola Hayata (Table 1 and Figure 1A-I).
Symptoms caused by passion fruit green spot virus in naturally infected plant species. Chlorotic local lesions on leaves of A) Passiflora alata, B) Ligustrum lucidum, C) Clerodendrum thomsoniae, D) Cestrum nocturnum, E) Hibiscus schizopetalus, and F) L. japonicum; G) Green spots or dots on leaves of Schefflera arboricola; H) Local necrotic lesions surrounded by a yellow halo on leaves of Allamanda cathartica; I) Local necrotic lesions on leaves of Lantana camara.
A survey of plants naturally infected with PfGSV added five new PfGSV-infected species (A. cathartica, C. nocturnum, C. thomsoniae, H. schizopetalus, and L. camara (Table 1) to previously described species (P. edulis f. flavicarpa, P. alata Dryland, L. sinense, L. lucidum, L. japonicum, Hibiscus rosa-sinensis L., Hibiscus syriacus L., Lonicera japonica Thunb., Barleria cristata L. Catharanthus roseus (L.) G. Don., Brunfelsia uniflora (Pohl) D. Don., Dendrobium sp., Epidendrum sp., and Schefflera actinophylla (Endl.) Harms.) (Chagas and Colariccio, 2004; Kitajima et al., 2010; Costa-Rodrigues et al., 2022; Saito, 2022).
Experimental host range by mites
A total of 162 plants from distinct species or cultivars were mite-inoculated to assess the experimental host range of PfGSV. Out of these plant species infested with viruliferous B. yothersi, only 18 exhibited symptoms of viral infection, which was confirmed through cytopathological and molecular analyses (Table 2; Figure 2A-C and 3A-L).
Transmission electron micrographs of leaf lesions caused by passion fruit green spot virus in naturally or experimentally infected plants, revealing cytopathic effects characteristic of those induced by cileviruses (electron-dense vacuolated cytoplasmic viroplasm, and short, bacilliform particles contained in the cisternae of the endoplasmic reticulum). A) Ligustrum sinense; B) Passiflora edulis f. flavicarpa; C) Nicotiana tabacum. V = viral particles; *Viroplasm.
Symptoms caused by passion fruit green spot virus in plant species experimentally mite-inoculated. A) Green spot or dots on leaves of Passiflora edulis f. flavicarpa, B) Capsicum annuum, C) Lonicera japonica, D) Hibiscus rosa-sinensis, E) Cataranthus roseus, and F) Pelargonium sp. G) Chlorotic local lesions on leaves of Ligustrum sinense, H) Malvaviscus arboreus, I) Hedera helix, J) Hoya carnosa, and K) Brunfelsia uniflora. L) Necrotic local lesions on leaves of Nicotiana megalosiphon.
The symptomatic plants displayed a range of viral-induced lesions, including localized necrotic or chlorotic spots and green islands (Figure 3A-L). Cytopathic analysis of symptomatic tissues infected by PfGSV consistently revealed short, enveloped bacilliform particles (50-70 nm × 100-120 nm), contained in elements of the endoplasmic reticulum, and electron-dense viroplasms in the cytoplasm typical of cileviruses (Kitajima et al., 2003) (Figure 2A-C). In addition to cytopathic analysis, the presence of PfGSV in symptomatic plants was further confirmed by RT-PCR. None of the plants infested by aviruliferous mites developed symptoms.
Mechanically inoculated experimental host range
Four out of 12 plant species mechanically inoculated with PfGSV from symptomatic leaves of yellow passion fruit, i.e., A. majus, C. quinoa, D. stramonium, and N. tabacum, were infected and identified as experimental hosts for the virus (Table 3). Symptoms appeared at different time points post-inoculation. C. quinoa exhibited chlorotic lesions as early as five days after inoculation (dai) (Figure 4A). In contrast, symptoms in A. majus, D. stramonium, and N. tabacum developed approximately 15 dai and were only visible when the inoculated leaves entered the senescence process. Similar to what was observed in passion fruit, PfGSV infection in these new experimental hosts resulted in the formation of green spot islands (Figure 4B-D). When analyzed under a TEM, viral particles and viroplasm, typical of cilevirus, were observed in cells from symptomatic areas.
List of plants mechanically inoculated with leaf extract of Passiflora edulis f. flavicarpa infected with passion fruit green spot virus (PfGSV), to determine the host species of this virus.
Symptoms caused by passion fruit green spot virus on mechanically inoculated plant species. A) Chlorotic local lesions on leaves of Chenopodium quinoa; B) Green spots or dots on leaves of Datura stramonium, C) Nicotiana tabacum, and D) Antirrhinum majus.
No plants were mechanically infected when privet leaves were used as an inoculum source.
Incubation and latency periods
Yellow passion fruit seedlings were evaluated weekly for the presence of symptoms associated with PfGSV after inoculation with viruliferous mites. Symptoms became visible only from the fifth collection (35 dai) when green spots and specks were observed on the yellow leaves (Figure 5A-H), indicating a long incubation period. However, PfGSV became detectable by RT-PCR in all 32 mite-inoculated leaves as early as the first collection (seven dai) revealing a much shorter latency period.
Yellow passion fruit (Passiflora edulis f. flavicarpa) leaves collected over time after inoculation of passion fruit green spot virus with viruliferous mites. A, B, C and D) green leaves, asymptomatic, collected at 7, 14, 21 and 24 days after inoculation, respectively; E, F, G and H) senescent leaves, with symptoms of passion fruit green spot virus infection (green spots/spots), collected at 35, 42, 49 and 56 days after inoculation, respectively. The white layer on the leaves refers to the mixture of sand, gypsum, and wheat (1:1:1) used to facilitate mite colonization.
Indicator plants for mite transmission studies
Leguminous plants were selected for testing based on previous studies showing that common beans serve as a reliable indicator plant for another cilevirus, the citrus leprosis virus C (Groot et al., 2006; Garita et al., 2013).
RT-PCR assays and leaf symptom expression indicated that all plants tested, except V. faba and Vigna radiata (L.), became infected with PfGSV by mite-inoculation (Table 4; Figures 6A-H and 7A-C). Small localized necrotic lesions, observed on the leaves of V. faba and V. radiata that were negative for the presence of the virus by RT-PCR, were the result of mite feeding damage rather than viral infection, as demonstrated by infestation of aviruliferous mites (Figure 6B and E).
Percentage of symptomatic leaves of test plants used to determine the indicator plant for passion fruit green spot virus infection.
Lesions observed on the leaves of test plants, used to determine a good indicator plant for infection caused by passion fruit green spot virus, seven days after inoculation with viruliferous mites. A) Local necrotic lesion in the vein of Phaseolus vulgaris cv. ‘UNA’; B) very small necrotic lesions in the vein of Vicia faba; C) local necrotic lesion in the vein of Vigna unguiculata; D) local necrotic lesion in the vein of Dolichos lablab; E) local necrotic lesion in the vein of Vigna radiata; F) local necrotic lesion in the vein of Mucuna pruriens; G) local necrotic lesion on the leaf blade of Cajanus cajan; H) local necrotic lesion in the vein of Phaseolus vulgaris var. ‘Jalo’. Arrows: eggs of Brevipalpus yothersi.
Transmission electron micrograph of sections from necrotic leaf lesions on leaves of lablab bean (Dolichos lablab) caused by mite inoculation of passion fruit green spot virus (Cilevirus passiflorae). A and B) Electron dense viroplasm (*), characteristic of cilevirus infection in the cytoplasm of palisade parenchymal cells; C) Presumed passion fruit green spot virus virions (V) in a membrane-bounded cavity in a spongy parenchymal cell; ER = endoplasmic reticulum; Cl = chloroplast.
PfGSV transmission rates ranged from 0 % to 100 % (Table 4). While D. lablab, P. vulgaris, V. unguiculata, and P. vulgaris var. ‘Jalo’ showed high infection rates (≥ 91 %), M. pruriens and C. cajan exhibited intermediate levels (38-58 %), V. faba and V. radiata remained uninfected (0 %). Except for C. cajan, which showed localized necrotic lesions in the leaf interveinal area, all other infected species displayed local necrotic lesions along the veins as a symptom of the viral infection (Figure 6G). While plants were monitored for seven days post-inoculation, only lablab-bean developed symptoms within five days. Lablab-bean was identified as a suitable indicator plant for the PfGSV assays. Further confirmation of its infection was obtained through cytopathological analysis, which revealed cell alterations typical of cilevirus infection, i.e., the presence of electron-dense viroplasms in the cytoplasm and enveloped viral particles of bacilliform morphology (50-70 nm × 100-120 nm) contained in cavities of the endoplasmic reticulum (Figure 7A-C).
Discussion
Experimental mite inoculation of PfGSV showed that 18 of 162 assayed plant species were susceptible to infection. However, certain plant species that did not develop symptoms in these experiments are known to be naturally infected in the field or to have been successfully infected through mechanical inoculation (Kitajima et al., 2010; Saito, 2022). These discrepancies may have been caused by of the failure to acquire the virus during feeding, individual variation in the mite's ability to transmit the virus, the unpalatability of some test plants to this mite species, making them unsuitable hosts for feeding, or stress caused by manipulation. Additionally, certain Brevipalpus species other than B. yothersi may act as vectors of PfGSV and require further investigation (de Lillo et al., 2021). As regards the field survey, our research added to those already described five new host species of PfGSV: A. cathartica, C. nocturnum, C. thomsoniae, H. schizopetalus, and L. camara.
It should be noted that PfGSV does not have an extensive experimental host range as CiLV-C (citrus leprosis virus C, Cilevirus leprosis) (Garita et al., 2014) or CiLV-C2 (León et al., 2023), but it does have a wider spectrum of naturally infected hosts. To date, 13 non-Passiflora species have been reported as being naturally infected by PfGSV, whereas CiLV-C has been found in only eight non-citrus hosts, including Swinglea glutinosa (Blanco) Merr., Commelina benghalensis L., Alocasia macrorrhiza (L.) G. Don, Dieffenbachia sp., Canna indica L., Cycas spp., H. rosa-sinensis, and Eriobotrya japonica (Thunb.) L.) (León et al., 2006; Nunes et al., 2012; León et al., 2023). However, PfGSV has a much narrower natural host range than CiLV-C2, which has been reported to infect 37 plant species (León et al., 2023).
Understanding the natural host range of a pathogen is essential to epidemiological studies and the development of effective disease management strategies. All host plants have the potential to serve as virus reservoirs, thereby contributing to disease outbreaks in susceptible crops. Orchid fleck virus (OFV, Dichorhavirus orchidaceae), also transmitted by mites of the Brevipalpus genus, exemplifies this dynamic. OFV has a wide host range and has been detected in several regions of the world and can infect both ornamental plants and citrus species. In this context, orchids may serve as a transmission link for the virus to citrus plants, as demonstrated in South Africa (Cook et al., 2019; Barro et al., 2024). PfGSV has a more restricted geographic distribution. It has been reported in several parts of Brazil and Colombia, infecting passion fruit and other plant species, especially ornamentals (Kitajima et al., 2003; Ramos-González et al., 2020; Roy et al., 2023), and causing green spots in hibiscus in Paraguay (Costa-Rodrigues et al., 2022). However, both OFV and PfGSV have expanded their distribution range, possibly through the movement of infected ornamental plants, which act as transmission vehicles. Therefore, knowing the natural hosts of these viruses becomes relevant to avoiding their spread, thereby preventing their introduction into new areas and protecting susceptible crops.
Though PfGSV-infected privet leaves served well as a source of inoculum in mite-transmission experiments, they were unsuitable for mechanical transmission assays, even at high virus titer. This may be due to inhibitory substances present in privet leaf extracts, and only a suitable extraction buffer might be able to reverse this. C. quinoa appeared to be an excellent indicator in PfGSV mechanical transmission assays of PfGSV, reacting with local lesions at approximately five dai. However, in mite-transmission tests, C. quinoa was not infected, possibly because Brevipalpus mites do not feed on this host.
A. majus, C. quinoa, D. stramonium, and N. tabacum were not infected by PfGSV, showing no symptoms after inoculation with viruliferous mites. However, mechanical inoculation confirmed that these species are hosts of the virus, indicating that they are not suitable hosts for the mite vector, a finding corroborated by the absence of reproduction and the high mortality of the mite population on the inoculated leaves. This finding aligns with the results of Garita et al. (2014), which show a failure to transmit the cilevirus CiLV-C to N. tabacum and C. quinoa via viruliferous mites, reinforcing the hypothesis that B. yothersi mites do not feed on this plant and are thus unable to transmit viruses. In contrast, the same study reported successful transmission of CiLV-C to D. stramonium, suggesting that although the mite can feed on this plant, it does so with difficulty, thereby characterizing it as a poor host. On the other hand, L. sinense, P. edulis f. flavicarpa, P. vulgaris cv. ‘UNA’ and V. unguiculata exhibited symptoms of PfGSV infection after inoculation with mites, while no symptoms were observed after mechanical inoculation of this virus. This suggests that these test plants are suitable hosts for the vector and highly susceptible to mite inoculation, but much less susceptible to mechanical inoculation.
RT-PCR assays demonstrated that PfGSV could be detected in inoculated leaves as early as seven days after mite inoculation, in asymptomatic yellow passion fruit leaves before symptoms appeared, which took more than a month, indicating that the virus is available for acquisition and dispersion by vector mites. Therefore, unnoticed PfGSV infection in nurseries may result in the virus spreading via mites in the field, or allow its introduction into still virus-free regions. In this context, the development of tests for reliable, rapid detection of PfGSV in yellow passion fruit nurseries might be relevant to preventing its spread into the field or into virus-free areas.
The prolonged incubation period (∼35 dai) before symptom development in yellow passion fruit plants makes them unsuitable as indicator plants for studies of the virus-vector relationship. To address this limitation, we searched for an indicator plant that would consistently exhibit PfGSV-induced symptoms within a short period after mite-inoculation. Among the plant species tested, lablab-bean was highly susceptible and showed vein necrosis as early as five dai with viruliferous mites. Other plants tested showed symptoms after seven days or more, or did not show symptoms after inoculation. Lablab-bean is easy to cultivate, has a rapid growth rate, and can be used for experiments approximately ten days after planting. Mite populations established well in this host, as evidenced by their rapid colonization of the leaves. The eggs observed on the leaves (Figure 6A-H) used for the test took approximately ten days to hatch at the temperature used in the experiments, eliminating the possibility that the progeny would interfere with the observed results. Cytopathic effects, characteristic of cilevirus infection (Kitajima et al., 2003), were observed in the necrotic lesions caused by PfGSV on lablab-bean leaves (Figure 7A-C). However, since the infected cells seem to undergo rapid necrosis, the presence of the virus must be confirmed by molecular assays.
Certain plants have been designated as being suitable for studies involving BTVs. Arabidopsis thaliana (L.) Heynh has been successfully used as an assay plant for virus-host interaction experiments with CiLV-C (Arena et al., 2017), but it is difficult to handle and requires special and expensive climate chambers for growth in tropical areas. P. vulgaris cv. ‘UNA’ is an easily cultivated, non-care-demanding, rapid growth plant, and was suitable for CiLV-C (Garita et al., 2013). However, the visible lesions in response to PfGSV infection did not appear for a week (Table 4). Despite this, it is also suitable as an alternative assay plant to replace lablab bean, in studies with PfGSV, with a transmission rate of 96 % (Table 4). Its genetic uniformity ensures consistent responses to virus inoculation, which makes it a reliable alternative for experiments that prioritize reproducibility. Nevertheless, for studies aiming to rapidly observe viral symptoms and vector-host interactions, lablab-bean remains the most suitable host, due to its rapid symptom development and efficient support of mite populations.
In summary, this study expands understanding of the natural and experimental host range of PfGSV, and identifies new susceptible species. Early detection of the virus in infected, yet asymptomatic, yellow passion fruit and the identification of efficient indicator plants, such as lablab-bean, are important contributions to further studies aimed at controlling PfGSV and preventing its spread. Finally, quarantine services in PfGSV-free countries must be aware of the potential introduction of this virus through symptomless Passiflora spp. and ornamentals, as well as through viruliferous mites.
Declaration of use of AI technologies
Data availability statement
The data used to support the instructions for this study will be available from the corresponding author upon reasonable request.
Acknowledgments
This work is part of the M.S. dissertation of the first author, in the graduate program on Phytopathology and Nematology at Escola Superior de Agricultura Luiz de Queiroz of Universidade de São Paulo (USP/ESALQ), with a scholarship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-133829/2020-0) and Fundação de Estudos Agrários Luiz de Queiroz (FEALQ-90360). This research was part of a project on Brevipalpus-transmitted viruses supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP-2019/28078-9).
References
-
Arena GD, Ramos-González PL, Nunes MA, Chabi-Jesus C, Calegario RF, Kitajima EW, et al. 2017. Arabidopsis thaliana as a model host for Brevipalpus mite-transmitted viruses. Scientia Agricola 74: 85-89. https://doi.org/10.1590/1678-992X-2015-0380
» https://doi.org/10.1590/1678-992X-2015-0380 - Barro MP, Pereira LR, Castro ACLF, Ramos-González PL, Freitas-Astúa J. 2024. Could ornamentals be the trojan horse of citrus orchards? Challenges in controlling a mite-transmitted viral complex producing citrus leprosis. Indian Citriculture 2: 15.
-
Beard JJ, Ochoa, R, Braswell WE, Bauchan GR. 2015. Brevipalpus phoenicis (Geijskes) species complex (Acari: Tenuipalpidae): a closer look. Zootaxa 3944: 67. https://doi.org/10.11646/zootaxa.3944.1.1
» https://doi.org/10.11646/zootaxa.3944.1.1 - Chagas CM, Colariccio A. 2004. Pinta verde do maracujá doce (Passiflora alata Dryand) associada a partículas do tipo citoplasmático da leprose dos citros. Fitopatologia Brasileira 29: S148 (in Portuguese).
- Colariccio A, Lovisolo O, Chagas CM, Galletti SR, Rossetti V, Kitajima EW. 1995. Mechanical transmission and ultrastructural aspects of citrus leprosis disease. Fitopatologia Brasileira 20: 208-213
-
Cook G, Kirkman W, Clase R, Steyn C, Basson E, Fourie PH, et al. 2019. Orchid fleck virus associated with the first case of citrus leprosis-N in South Africa. European Journal Plant Pathology 155: 1373-1379. https://doi.org/10.1007/s10658-019-01854-4
» https://doi.org/10.1007/s10658-019-01854-4 -
Costa-Rodrigues M, Pereira LR, Ramos-González PL, Chabi-Jesus C, Tassi AD, Leão IG, et al. 2022. First report of Passion fruit green spot virus infecting hibiscus plants. New Disease Reports 45: e12080. https://doi.org/10.1002/ndr2.12080
» https://doi.org/10.1002/ndr2.12080 -
de Lillo E, Freitas-Astúa J, Kitajima EW, Ramos-González PL, Simoni S, Tassi AD, et al. 2021. Phytophagous mites transmitting plant viruses: update and perspectives. Entomologia Generalis 41: 439-462. https://doi.org/10.1127/entomologia/2021/1283
» https://doi.org/10.1127/entomologia/2021/1283 -
Garita LC, Tassi AD, Calegario RF, Kitajima EW, Carbonell SAM, Freitas-Astúa J. 2013. Common bean: experimental indicator plant for Citrus leprosis virus C and other cytoplasmic-type Brevipalpus-transmitted viruses. Plant Disease 97: 1346-1351. https://doi.org/10.1094/PDIS-12-12-1143-RE
» https://doi.org/10.1094/PDIS-12-12-1143-RE -
Garita LC, Tassi AD, Calegario RF, Freitas-Astúa J, Salaroli RB, Romão GO, et al. 2014. Experimental host range of Citrus leprosis virus C (CiLV-C). Tropical Plant Pathology 39: 43-55. https://doi.org/10.1590/S1982-56762014005000004
» https://doi.org/10.1590/S1982-56762014005000004 -
Green MR, Sambrook J. 2020. Precipitation of RNA with Ethanol. Cold Spring Harbor, New York, NY, USA. https://doi.org/10.1101/pdb.prot101717
» https://doi.org/10.1101/pdb.prot101717 - Groot TVM, Freitas-Astúa J, Kitajima EW. 2006. Brevipalpus phoenicis transmits citrus leprosis virus, cytoplasmic type (CiLV-C) to common bean (Phaseolus vulgaris) under experimental conditions. Virus Review and Research 11: 67-68.
- Kitajima EW, Rezende JAM, Rodrigues JCV, Chiavegato LG, Piza Jr. CT, Morozini W. 1997. Green spot of passion fruit, a possible viral disease associated with infestation by the mite Brevipalpus phoenicis. Fitopatologia Brasileira 22: 555-559.
- Kitajima EW, Nome C. 1999. Microscopia electronica em virologia vegetal. (Electronic Microscroscopy in vegetable virology), p. 59-86. In: Campo, DM, Lenardon SL. eds. Métodos para detectar patógenos sistêmicos (Methods to detect systemic pathogens). Instituto de Fitovirologia y Fisiologia Vegetal/Instituto Nacional de Tecnologia Agropecuaria, Córdoba, Argentina (in Spanish).
-
Kitajima EW, Rezende JAM, Rodrigues JCV. 2003. Passion fruit green spot virus vectored by Brevipalpus phoenicis (Acari: Tenuipalpidae) on passion fruit in Brazil. Experimental and Applied Acarology 30: 225-231. https://doi.org/10.1023/B:APPA.0000006551.74604.84
» https://doi.org/10.1023/B:APPA.0000006551.74604.84 -
Kitajima EW, Rodrigues JCV, Freitas-Astúa J. 2010. An annotated list of ornamentals naturally found infected by Brevipalpus mite-transmitted viruses. Scientia Agricola 67: 348-371. https://doi.org/10.1590/S0103-90162010000300014
» https://doi.org/10.1590/S0103-90162010000300014 -
León GA, Realpe CE, Garzon P, Rodriguez JA, Moreno MG, Childers CC, et al. 2006. Occurrence of citrus leprosis virus in Llanos Orientales, Colombia. Plant Disease 90: 682. https://doi.org/10.1094/PD-90-0682C
» https://doi.org/10.1094/PD-90-0682C -
León GA, Campos JC, Guevara YA, Roy A. 2023. Distribución, Plantas Hospederas y Ácaros Vectores del Virus de la Leprosis de los Cítricos en Colombia. Agrosavia, Bogotá, Colombia. https://doi.org/10.21930/agrosavi.investigation.7406429
» https://doi.org/10.21930/agrosavi.investigation.7406429 -
Nunes MA, Oliveira CAL, Oliveira ML, Kitajima EW, Hilf ME, Gottwald TR, et al. 2012. Transmission of Citrus leprosis virus C by Brevipalpus phoenicis (Geijskes) to alternate host plants found in citrus orchards. Plant Disease 96: 968-972. https://doi.org/10.1094/PDIS-06-11-0538
» https://doi.org/10.1094/PDIS-06-11-0538 -
Ramos-González PL, Santos GF, Chabi-Jesus C, Harakava R, Kitajima EW, Freitas-Astúa J. 2020. Passion fruit green spot virus genome harbors a new orphan ORF and highlights the flexibility of the 5’-End of the RNA2 segment across cileviruses. Frontiers in Microbiology 11: 1-16. https://doi.org/10.3389/fmicb.2020.00206
» https://doi.org/10.3389/fmicb.2020.00206 -
Roy A, Choudhary N, Guillermo LM, Shao J, Govindarajulu A, Achor D, et al. 2013. A novel virus of the genus Cilevirus causing symptoms similar to citrus leprosis. Phytopathology 103: 488-500. https://doi.org/10.1094/PHYTO-07-12-0177-R
» https://doi.org/10.1094/PHYTO-07-12-0177-R -
Roy A, Guilhermo LM, Nunziata S, Padmanabhan C, Rivera Y, Brlansky RH, et al. 2023. First report of Passion fruit green spot virus in yellow Passion fruit (Passiflora edulis f. flavicarpa) in Casanare, Colombia. Plant Disease 107: 2270. https://doi.org/10.1094/PDIS-09-22-2267-PDN
» https://doi.org/10.1094/PDIS-09-22-2267-PDN -
Saito MSG. 2022. Passion fruit green spot virus (PfGSV), isolated from Ligustrum: its relationships with the Brevipalpus vector mite. Dissertação (Mestrado). USP/ESALQ, Piracicaba, SP, Brazil. https://doi.org/10.11606/D.11.2020.tde-05052020-083432
» https://doi.org/10.11606/D.11.2020.tde-05052020-083432 -
Salinas-Vargas D, Santillán-Galicia MT, Guzmán-Franco AW, Hernández-López A, Ortega-Arenas LD, Mora-Aguilera G. 2016. Analysis of genetic variation in Brevipalpus yothersi (Acari: Tenuipalpidae) populations from four species of citrus host plants. PLoS ONE 11: e0164552. https://doi.org/10.1371/journal.pone.0164552
» https://doi.org/10.1371/journal.pone.0164552 -
Tassi AD, Garita-Salazar LC, Amorim L, Novelli VM, Freistas-Astúa J, Childers CC, et al. 2017. Virus-vector relationship in the Citrus leprosis pathosystem. Experimental and Applied Acarology 71: 227-241. https://doi.org/10.1007/s10493-017-0123-0
» https://doi.org/10.1007/s10493-017-0123-0
