The mountain papaya may be a possible reservoir of the Kashmir bee virus

Introduction

Kashmir bee virus (KBV) is a positive-sense, single-stranded RNA virus classified in the Dicistroviridae family within the Cripavirus genus (Mazzei et al., 2019). KBV primarily infects bee species such as Apis cerana, Apis mellifera, and bumblebees (Bombus spp) within the Apidae family. It has also been identified in wasps (Vespula germanica) from the Vespidae family (De Miranda et al., 2004). Notably, KBV can co-infect with closely related viruses, such as acute bee paralysis virus (ABPV) and Israel acute paralysis virus (IAPV), forming the AKI viral complex (De Miranda et al., 2004; Evans & Schwarz, 2011). KBV isolates have been reported globally, including Spain, New Zealand, South Korea, North America, Australia, and Chile (Berényi et al., 2006; Nanetti et al., 2021; Riveros et al., 2018; Tentcheva et al., 2004).

The KBV reference genome spans 9,524 base pairs and contains two open reading frames (ORFs) that encode nonstructural and structural proteins (De Miranda et al., 2004). The 5′ ORF encodes the non-structural polyprotein, comprising three domains corresponding to a helicase, a 3C protease domain, and eight RNA polymerase domains that includes an RNA-dependent RNA polymerase. The 3′ ORF encodes a structural polyprotein composed of two capsid protein domains belonging to the VP4 superfamily (De Miranda et al., 2004).

In honeybees, KBV primarily infects worker bees, but can spread throughout the colony under stressful conditions. Transmission occurs vertically from queen to offspring, and horizontally among bees through contaminated food, affecting individuals from the larval stage to adulthood (Meeus et al., 2014). Additionally, Varroa destructor, a common mite pest of bees, can be a vector and activator of KBV and other viruses (Brødsgaard et al., 2000; Shen et al., 2005). Survival of pathogenic KBV particles in mites can explain how different mite species are potential routes of KBV (Carreck, Ball & Martin, 2010; Tixier, 2018). Moreover, increased stress levels in mite-infected hives makes KBV transmission more likely (De Miranda, Cordoni & Budge, 2010). Although primarily infecting bees within the Apidae family, KBV has been found in other Hymenopterans, including the German wasp Vespula germanica and the Asian hornet V. velutina, both of which can prey on A. mellifera (Brenton-Rule et al., 2018; Felden et al., 2020; Mazzei et al., 2019). The transmission of KBV to V. germanica is thought to be horizontal by feeding on infected live or dead honeybees (Eroglu, 2023; Evison et al., 2012).

Plants serve as potential reservoirs of entomopathogenic viruses that affect pollinators through contamination of pollen and flowers. Recently the pollen virome has been explored (Fetters et al., 2022), revealing a wide diversity of plant and insect pathogenic viruses. The shared use of flowers by different pollinator species may contribute to horizontal transmission routes (Durrer & Schmid-Hempel, 1994), often through feces deposition (Figueroa et al., 2019). Recent research has emphasized the role of flowers (Alger, Burnham & Brody, 2019), nectar, and pollen in facilitating inter-species viral transmission among pollinators, suggesting that plants may act as reservoirs for entomopathogenic viruses (Mcart et al., 2014). KBV has been detected in honeybee feces (Hung, 2000), parasitic mites (Shen et al., 2005), and pollen (Singh et al., 2010). Similarly, the closely related ABPV and IAPV have been found in flowers. Transmission of viruses between managed bees, wild bees, and other pollinating insects is common (Gisder & Genersch, 2017; Mcmahon et al., 2015). In addition, plants serve as vector of entomopathogenic viruses that affect insect herbivores, such as aphid-infecting RNA viruses (Jones, 2018).

The mountain papaya (Vasconcellea pubescens), also known as Chamburo, Chilean papaya, or wild papaya (Salvatierra-González & Jana-Ayala, 2016) belongs to the Caricaceae family, V. pubescens is a perennial, herbaceous, and trioecious species capable of cross-pollination, occasionally exhibiting hermaphroditism (Chong-Pérez et al., 2018). The fruit is smaller in comparison to other papaya species (Briones-Labarca et al., 2015).

Well-described phytopathogenic viruses infecting Carica papaya include papaya ringspot virus, papaya leaf distortion mosaic virus, papaya lethal yellowing virus, and papaya mosaic virus, among others (Abreu et al., 2015; Adams, Antoniw & Beaudoin, 2005; Chávez-Calvillo et al., 2016; Razean Haireen & Drew, 2014; Yang et al., 2012). However, the virome of V. pubescens remains largely unexplored. Documenting viruses affecting this species is crucial due to their potential impact. Additionally, although not directly affecting papaya, the presence of entomopathogenic viruses could impact pollinating insects, such as bees, which interact with papayas (Badillo-Montaño, Aguirre & Munguía-Rosas, 2019). Therefore, establishing a viral library of mountain papaya could benefit both the agroindustrial and apiculture sectors.

In this study, we conducted transcriptome analyses of leaf, root, and fruit tissues to establish a virome library of the mountain papaya collected from fields in central Chile. This is the first report identifying the presence of KBV viral sequences in mountain papaya. The detection of KBV viral sequences in roots, leaves, and in vitro cultivated plants suggests that V. pubescens may often be a reservoir or vector for KBV infection.

Materials & Methods

Material collection

Plant samples were collected from V. pubescens orchards in Lipimávida (34°51′4.7″S, 72°8′27.6″W), Licantén (34°59′7.1″S, 71°59′4.9″W), Vichuquén (34°52′59.9″S, 71°59′36.2″W), Chanco (36°16′8.4″S, 72°42′57.2″W), Pelluhue (35°48′49.0″S, 72°34′26.4″W), Putú (35°12′47.9″S, 72°17′2.0″W), Iloca (34°55′57.7″S, 72°10′50.5″W), Duao (34°53′48.8″S, 72°10′46.6″W), Constitución (35°19′49.4″S, 72°24′33.1″W), and Curanipe (35°50′37.7″S, 72°38′21.1″W), located in the coastal areas of the Maule region, Chile (Fig. 1). In total, 100 orchards were sampled, with 15 plants selected from each orchard, and three samples taken per tissue type (leaves, roots, and fruits). Plant material was randomly selected using a zigzag pattern, covering the entire area of the orchard. Each tissue sample, including leaves, roots, and fruits, was collected in triplicate.

Results

Phylogenetic analysis

KBV has been previously reported in honeybee samples in Chile (Riveros et al., 2018). Phylogenetic analysis which revealed that KBV-Ch is similar to a KBV isolate from Spain, and sharing a clade with isolates from New Zealand and some samples from Chile (Fig. 2).

Detection of KBV in laboratory and field plants

Given the detection of KBV sequences in root and leaf tissues, suggesting potential vascular tissue incorporation of the virus, we investigated the presence of KBV in in vitro cultivated plants derived from V. pubescens explants collected from the field using RT-PCR. Examination of in vitro cultivated plants found a single, 209 bp, amplicon in one sample (Fig. 3).

Discussion

KBV was identified in the mountain papaya through transcriptome analysis of several tissues, and KBV sequences were found in greenhouse-grown plants. Through transcriptome analysis of leaf, root, and fruit tissues of the mountain papaya, V. pubescens, we successfully identified a near-complete genome sequence of the KBV, and detected KBV sequences in in vitro grown plants. The presence of KBV was notably higher in one replicate of fruit transcriptome, suggesting an occasional ocurrence of the virus in V. pubescens orchards. As a trioecious species capable of cross-pollination, V. pubescens produces male, female, and hermaphrodite plants (Carrasco et al., 2009). Pollination can occur via wind or insects, with self-pollination observed in commercial varieties of Carica papaya (Carrasco et al., 2022). Infected pollen presents a potential source of KBV particles in fruits. Some viruses can infect pollen, potentially transferring the infection to the plant ovule and resulting in virus-infected fruit. This infection in pollen can occur through various vectors, including abiotic factors (such as wind) and biotic factors (such as insects), suggesting a possible mechanism for virus transmission from pollen to fruit (Bhat & Rao, 2020). In addition, Figueroa et al. (2019) studied the mechanisms involved in pathogen deposition, persistence and acquisition in flowers. Bees, known to spend extended periods feeding on flowers, deposit and acquire feces from other pollinators, promoting transmission rates.

While reports on bee viruses in pollinated plants typically focus on flowers and pollen, other viruses from the Dicistroviridae family have been detected circulating in other plant tissues, among which most examples are from aphid viruses (Jones, 2018). For instance, the Rhopalosiphum padi virus (RhPV) spreads systemically in barley plants including roots, potentially infecting healthy individuals that consume RhPV-carrying plants (Ban et al., 2007). Similarly, the aphid lethal paralysis virus has been found in cucumber leaf transcriptomes, suggesting a comparable transmission route (Maina et al., 2017). We detected KBV RNA in roots and leaves, albeit in lower quantities compared to fruits, suggesting that KBV could circulate from contaminated sexual organs to other plant tissues. Despite rigorous surface cleaning of plant material before RNA extraction, residual KBV RNA could stem from superficial contamination with feces, corpses, or other insect sources carrying KBV particles. Importantly, KBV RNA was also detected in in vitro grown plants, which underwent stringent disinfection procedures, and only novel tissue was used for RNA extraction. Virus in in vitro plant cultures can originate from the source vegetative plant (Li et al., 2013). Such is the case of babaco plants (Vasconcellea x heilbornii), which are often propagated from cuttings without virus elimination protocols typically resulting in in vitro plants infected with viruses (Muñoz et al., 2023). Therefore, it is possible that KBV could systemically circulate through the plant as it was shown for other members of Dicistroviridae, utilizing pollinated plants as reservoirs and passive vectors. The phenomenon of horizontal transmission of entomopathogenic viruses by host plants as passive vectors remains understudied yet holds significant implications for pest control and conservation of wild pollinators (Jones, 2018).

While the exact mechanism of KBV introduction into V. pubescens populations in Chile remains uncertain, previous studies conducted by Chilean researchers have identified partial KBV sequences Apis mellifera populations across several regions in Chile (Riveros et al., 2018). Phylogenetic analysis of the KBV sequence obtained in our study reveals close genetic proximity to these previously reported Chilean isolates, as well as isolates from Spain and New Zealand, suggesting reintroduction of genetically different KBV isolates across continents. Furthermore, the interaction between bees and papaya trees is noteworthy (Badillo-Montaño, Aguirre & Munguía-Rosas, 2019). This interaction implies two key aspects: firstly, bees in the studied locations may act as vectors for KBV transmission, potentially disseminating the virus during pollination of V. pubescens; and secondly, there is a critical need to monitor bee populations in the coastal areas of the Maule Region for KBV presence. Such surveillance could benefit beekeepers and lay the groundwork for forthcoming investigations.

Conclusions

This study marks a significant advancement by documenting, for the first time, the detection of the “Kashmir Bee Virus Chile” within the transcriptomes of Vasconcellea pubescens, representing a milestone for the agro-industrial and apiculture sectors in Chile’s Maule region. Transcriptome analysis revealed the presence of KBV-Ch in various V. pubescens tissues, with a notable abundance in fruit tissues, suggesting a potential role as a reservoir or vector of infection. These preliminary results highlight the presence of KBV-Ch in V. pubescens, serving as a fundamental step for the development and validation of future hypotheses. Additional replication and testing in more plants are necessary. Future studies should focus on elucidating transmission mechanisms and assessing the virus’s impact on agriculture and beekeeping. These efforts are critical for developing strategies to control viral diseases affecting crops and pollinators.

Supplemental Information