Deciphering the virome of Chunkung (Cnidium officinale) showing dwarfism-like symptoms via a high-throughput sequencing analysis

Cnidium officinale (Korean name: chunkung), which is a flowering annual plant in the family Apiaceae [1, 2], has a long history of use in traditional medicine in Far East Asia, especially in Korea, China, and Japan. It has been used to treat various conditions, including pruritus, skin disorders, asthma, and erectile dysfunction [2‐6]. Chunkung plants are cultivated through their rhizomes and are used to relieve pain and treat menstrual disturbances [7], vitamin deficiencies [8], hypertension, and inflammation [9]. Active compounds, such as osthol [5], volatile alkyl phthalide derivatives, and polysaccharides, have also been identified in chunkung rhizomes. To date, 350 compounds have been isolated from chunkung and identified [5]. There are ongoing studies on the medicinal properties of chunkung as well as on the phytopathogens (e.g., viruses) that can infect chunkung [10].

Viruses, which are ubiquitous in the biosphere, can infect a wide range of hosts, including plants, animals, and microbes [11]. They can invade their hosts through a process called cross-species transmission or spillover [12‐14], which can adversely affect plant health, thereby decreasing the market value of cultivated plants [15‐19]. Viral species in agricultural systems differ significantly from those in natural ecosystems [12]. In the latter case, including wild plants and native vegetation, have a distinct relationship with their host plants and often have neutral effects or provide their hosts with slight advantages [20]. In agricultural systems, they can significantly affect plant growth, resulting in substantial yield losses [21‐23]. Plant viruses are naturally transmitted through a variety of pathways (e.g., pollen, seeds, and vectors) [24, 25]. High-density cultivation of plants can promote viral spread [26]. The diversity of viruses in ecosystems can be influenced by various factors [27]. These include changes in the genetic traits and composition of host populations, shifts in the ecology of both the host plant and the virus, and for vectored viruses, modifications in the vector’s ecology and genetics. Additionally, human practices also affect viral diversity. For example, monocultures, irrigation systems, extended growing seasons, the transport of seedlings, changes in land use, and the application of artificial soil amendments can alter ecological dynamics and favor the emergence and spread of specific plant viruses [20, 27]. Several economically important viral diseases affect chunkung [28, 29], and the emergence of novel viruses is increasingly being detected because of advances in high-throughput sequencing (HTS) technologies.

Predicting crop damage is difficult [30] due to multiple factors, such as geographic region, virus strain, host plant cultivar/variety, and timing of infection [19, 31]. Technological advances, surveillance efforts, and improved diagnostic capabilities have increased interest in plant virus research [32, 33]. The development of massively parallel sequencing, which is also known as HTS, has contributed to significant breakthroughs in virome research [20, 25, 30, 34, 35]. The diagnosis of viral diseases typically relies on specific techniques (e.g., ELISA, microarray, and PCR) [34, 36] that are relatively cheap/economic and can rapidly detect known viruses [37]. However, these methods may be inappropriate for examining samples co-infected with multiple viral agents [38, 39]. Alternatively, HTS can efficiently detect and identify multiple viruses, even in the absence of prior knowledge about viral sequences [21, 26]. Advances in nucleic acid isolation protocols and the availability of HTS technologies have enabled researchers to comprehensively study the viruses associated with a specific host [39, 40]. Until recently, our understanding of plant virus diversity was limited. However, over the past decade, the mainstream use of HTS in biodiversity surveys, has helped fill our knowledge gaps. As a result, full viromes [16] obtained by HTS approach may lead to significant breakthroughs by enabling inclusive viral genome analyses, facilitating metagenomic investigations [41, 42], expanding analyses to non-model hosts [43], identifying viral variants [16], and advancing our understanding of viral transcriptomics [32].

The objective of this study was to elucidate the viral diversity in chunkung via HTS. We conducted a comprehensive survey to identify viruses that may be responsible for putative growth abnormalities and complex infections, or that adversely affect chunkung plant growth. The results of this study offer valuable insights into the diversity of viral communities and their potential ecological interactions. Furthermore, the virus genomes that were annotated and classified in this study are valuable resources for future research on virus–host interactions and the development of disease management strategies applicable to chunkung (SRA accessions: SAMN39861745 and SAMN39861546 of the BioProject PRJNA1074493). To the best of our knowledge, this survey represents the first study of the chunkung virome.

The advancement of virome databases, powered by reliable and precise HTS techniques, has transformed virology and created new avenues for research opportunities. These include managing disease risks [54], tracking emerging diseases, and implementing new phytosanitary regulations [55]. Hence, HTS offers a significant advantage for virome characterization over conventional techniques such as ELISA, microarray, or (RT-) PCR detection because its efficacy is primarily dictated by the completeness of the reference databases [56], the depth of the data [57], and the accuracy of the base calls [58]. By analyzing viral genomes at a large scale [59], researchers can gain insights into factors such as transmission routes and epidemiology, host range, genetic diversity [6], and evolutionary dynamics [60].

In this study, we used HTS analyses to identify viral populations and their genome types in asymptomatic and dwarfed chunkugn plants (Fig. 1), which appear to be resistant and susceptible (Fig. 1) to the various viruses, respectively. Although chunkugn plants are susceptible to a wide range of RNA viruses, it was surprising that different DNA and RNA viruses could co-infect chunkugn plants. Here, we discuss the efficacy of HTS techniques in revealing a wide range of biologically significant breakthroughs. Due to its broad-spectrum approach, universality, and accurate pathogen profiling, HTS is capable of effectively detecting multiple viruses, both known and novel [61], in infected samples, even at minimal concentrations [56]. This holistic approach significantly reduces the risk of false negatives, enabling us to identify various co-infections (Fig. 10) and explore the untapped potential of viral diversity in chunkugn plants. In total, based on sequencing reads from 78 HTS-detected viruses, 61 viruses were collectively confirmed via (RT-) PCR in either of the collected samples (1D, 1A ~ 5D, 5A), as shown in Fig. 10. More specifically, the analysis confirmed the presence of 35 viruses in each sample, which is indicative of the multiple co-infections of individual plants. These viruses are broadly distributed and can infect and putatively persist in chunkung plants, regardless of the plant’s health status. According to our analysis, the viruses belong to no fewer than 21 genera (Fig. 5), representing 14 different families (Fig. 7). The strength of HTS techniques lies in their ability to identify potential chunkugn viruses (Fig. 10, Table 3), particularly in cases where disease symptoms are absent, ambiguous, or attributed to only a limited number of viruses, which may not be detectable using conventional methods. This capability is particularly crucial in the context of viral diseases, where early detection plays a pivotal role in implementing effective control strategies. In previous studies, researchers have investigated viruses infecting chunkugn plants [9, 28]. However, comprehensive studies on the chunkugn virome have been limited [3, 4, 9, 29], and there were no reports of DNA viruses. Our study filled this gap and identified the co-infection of chunkugn by four different virus genome types (i.e., ssRNA, dsRNA, ssDNA, and dsDNA) (Fig. 8). The interaction of different viruses can exacerbate disease symptoms in chunkugn plants and potentially spread them, indicating the possibility of further biodiversity and quasispecies development (different genome sequences within the same host) due to their rapid replication and mutation rates [38]. Therefore, chunkugn plant can serve as good model for investigating the co-infection caused by diverse viruses and their genetic variants.

According to this study, our analysis of the distribution of DNA and RNA viruses in chunkung plants indicated that RNA viruses were more abundant than DNA viruses (Additional file 2, Table S11). This prevalence can be explained by several factors [49]. First, compared with DNA viruses, RNA viruses have higher mutation rates [20], allowing them to rapidly adapt to changing conditions and infect plants. Second, RNA viruses have smaller genomes [37] that can be efficiently packaged and replicated within plant cells. Additionally, because of their relatively small genomes, RNA viruses are easily transmitted between plants and vectors [49]. Third, diverse genetic variants may be generated in RNA viruses via recombination [11, 62] and reassortment [11, 63]. Fourth, RNA viruses rely on the host plant cellular machinery for replication and protein synthesis [11], allowing them to establish infections more efficiently than DNA viruses. Finally, the mechanisms mediating the transmission of RNA viruses (e.g., insect vectors or pollen) enhance their dispersal among plant populations [24]. However, the occurrence and distribution of DNA viruses in plants can also be influenced by several factors. For instance, pararetroviruses (PRVs, family Caulimovirida) have wide geographical dissemination and invade a wide range of dicotyledonous and monocotyledonous plant species [64]. PRV infections can be both asymptomatic and symptomatic, and they can remain dormant in their host for an indefinite period, becoming active when confronted with specific stress [64].

In general, viruses are obligate parasites that tend to build symbiotic relationships with their hosts [65], creating a suitable environment for the co-existence of multiple viruses within a single plant. This co-infection of different viruses within the host could cause harm [38]. For instance, studies have shown that mixed infections in plants can lead to synergistic interactions [11], resulting in increased disease symptoms. Although viruses are considered primary pathogens, some may serve beneficial roles as symbiotic partners within the host plant [65]. In contrast, certain co-infected viruses may not be associated with host disease symptoms.

Despite its advantages, the HTS has limitations. For instance, while HTS analysis exclusively detected two viruses, LLDV and DMV (Fig. 10), in dwarfed plants, PCR data revealed that both viruses were also present in the asymptomatic plants. This discrepancy suggests that HTS alone may be insufficient for a thorough and accurate investigation of the complete virome [16, 41]. Furthermore, distinguishing between true viral sequences and background noise or contaminants introduced during sample preparation or sequencing is challenging [16, 66]. Future advancements may focus on reducing the cost and complexity of HTS workflows, increasing the speed and throughput of sequencing platforms, and improving bioinformatics tools for data analysis and interpretation. The evidence presented in this work can offer valuable insights for researchers developing control and preventive strategies [53], including the implementation of biosecurity protocols to curb the spread of plant viruses in the field. The significant value of HTS and bioinformatics in plant health management will bolster their effectiveness in combating viral diseases, promoting sustainable agriculture, and ensuring reliable virus diagnosis for both known and unknown viruses.

The data presented herein provide evidence that chunkung serves as an important reservoir of diverse plant viruses, which may be related to its limited genetic diversity. Results revealed an unexpectedly high co-infection rate. This raises intriguing questions regarding the mechanisms underlying the co-existence of several dozen viruses within individual plants. Additional research is needed to clarify how several dozen viruses can co-exist and interact with each other in a single plant. This study generated essential data for predicting viral outbreaks and for managing the potential risks related to the spread of phytopathogenic viruses in agroecological systems.