Viruses, Vol. 14, Pages 2718: A Novel Rhabdovirus Associated with the Idaho Population of Potato Cyst Nematode Globodera pallida

1. IntroductionGlobodera pallida, a potato cyst nematode (PCN), is a globally regulated plant parasitic nematode [1] that may reduce tuber yields up to 80% in infested potato (Solanum tuberosum) fields if left unmanaged [2,3]. G. pallida was first reported in the United States in Idaho in 2006 [4], and, due to its devastating economic impact on the potato industry, was immediately placed under strict quarantine and eradication programs [5]. Due to the high toxicity of chemical nematicides, such as methyl bromide, it is desirable to develop new, safer alternative strategies to eradicate or reduce nematode populations in the field. Restrictions on use of chemical pesticides have put studies on alternative methods of nematode control in the spotlight. Among safer alternatives are biological control agents (BCAs). Many examples show that viruses are viable as BCAs for limiting the impact phytopathogens, such as fungi, bacteria, insects, and even other viruses, have on crops [6].Viruses are obligate intracellular parasites found in all organisms that sometimes cause diseases in their hosts, and, if found in nematodes, could be explored as a potential tool for their control. One putative, positive-strand RNA virus was recently discovered among transcriptomics outputs from G. pallida, potato cyst nematode picorna-like virus (PcPLV) [7]. This transcriptomic library was generated from G. pallida and G. rostochiensis populations collected in Scotland [8,9], and the virus presence could not be confirmed experimentally in the US populations of these nematodes due to the quarantine status of G. pallida and G. rostochiensis in the US [7]. Hence, experimental evidence of the PcPLV virus presence in laboratory or field populations of G. pallida and G. rostochiensis is still lacking. Earlier this year, we published a transcriptomics study of G. pallida collected in the State of Idaho (USA) [10] and, in search of potential tools to manage G. pallida, decided to subject this extensive dataset to a bioinformatics analysis focused on discovery of viruses possibly present in the Idaho populations.Here, we report on a novel rhabdovirus genome assembled from the transcriptomics reads available from the database [10]. The presence of this virus, named potato cyst nematode rhabdovirus (PcRV), was validated and confirmed for the different life stages of the G. pallida populations maintained in the quarantine greenhouse in Idaho (USA), and the virus whole genome was re-sequenced using Sanger methodology. Another isolate of PcRV was identified among the publicly available transcriptomics outputs generated from the Scottish populations of G. pallida, with the genome sequence almost identical to the Idaho isolate of PcRV. Based on the phylogenetic position of PcRV, we propose to create a new genus with a tentative name Gammanemrhavirus, comprising two virus species, representing PcRV and ScRV, respectively. 4. DiscussionPlant-parasitic nematodes are heavy burdens on global food security. Use of microorganisms as biocontrol agents to manage plant parasitic nematodes is a promising approach, gaining more support over recent years. As obligate parasites, viruses can be viewed as a potential tool to control nematodes. For a long time, nematode viruses were neglected, primarily due to a lack of proper tools and approaches to address their presence. For example, in the late 1950s Loewenberg et al. (1959) first described the infection of root-knot nematodes with a virus-like pathogen supported by visible symptoms in nematodes, but no viral particles were visualized [27]. Although a number of electron microscopic studies reported virus-like particles in nematodes during the 1970s, due to a lack of biochemical or genomic analysis, these reports could not indisputably prove viral infections in nematodes [28,29,30]. It was not until the last decade that the advances in sequencing technologies and the reduction in costs for HTS have enabled and facilitated detection of naturally occurring viruses within free living nematodes, such as C. elegans and C. briggsae [31,32,33], as well as parasitic species of both animals [26,34] and plants (Heterodera spp., Globodera spp., and Pratylenchus penetrans) [7,22,35,36,37,38].Rhabdoviruses are single-stranded, negative-sense RNA viruses with genomes ranging between 10 and 16 kb in size. Rhabdoviruses have enveloped, bullet-shaped, or bacilliform virion morphology, although unenveloped rhabdoviruses with filamentous virions have also been described [21]. In the latest update of the rhabdovirus taxonomy [21,25], the family Rhabdoviridae included 20 genera and 144 species found in vertebrate, arthropod, and plant hosts. Two genera of rhabdoviruses included species found in nematodes, Alphanemrhavirus, and Betanemrhavirus. Phylogenetically, alphanemrhaviruses and betanemrhaviruses were placed into two different sub-families, Alpharhabdovirinae and an unassigned sub-family, based on the sequences of their replication-associated L-proteins [25] (see also Figure 3). In 2011, a rhabdo-like virus genome was described assembled from the HTS data from soybean cyst nematode samples, named soybean cyst nematode rhabdovirus (ScRV; [22]). Phylogenetically, ScRV did not fit into any established genus of rhabdoviruses, including alphanemrhaviruses and betanemrhaviruses, and was left as an unassigned member of Rhabdoviridae [25]. Now, we demonstrated that a newly found PCN virus, PcRV, has very similar genome organization to ScRV (Figure 1) and is closely related to ScRV phylogenetically (Figure 3). Given the topology of the phylogenetic tree for the L-proteins, we proposed both ScRV and PcRV to form a new genus, tentatively named Gammanemrhavirus.Detection of PcRV across different life stages, including eggs, preparasitic juveniles, and parasitic females, suggests vertical transmission as a possible route of infection. This observation is consistent with reports for other viruses of plant parasitic cyst nematodes [7,22,39] but not for root lesion nematode virus 1 (RLNV1) infecting the migratory species Pratylenchus penetrans, which was only detected in parasitic developmental stages, excluding eggs and juveniles [38]. Although vertical transmission has not been well studied for nematode viruses, this mode of transmission has been previously reported in insect viruses, such as the rhabdovirus Sigma virus infecting Drosophila [40]. Transovarial movement of the virus may be hypothesized to be an efficient means of transfer in cyst nematodes, whose life cycle is mostly sedentary except for the invasive J2 and adult male stages.Given that our RNA samples were extracted from cultured G. pallida research populations reared over a period of 5 years, the PcRV infection seems to be long lived and stable in the nematode populations. It is worth noting that, although we attempted to extract RNA from limited cysts collected directly from the originally infested field in Idaho [4], the biological material was too degraded to obtain sufficient quality and quantity of nucleic acid for RT-PCR amplification. The G. pallida 2013 rearing population represents the first generation of nematodes bred in our greenhouse conditions, and it is reasonable to speculate that PcRV most likely has already been present in the original G. pallida field population.Our RT-PCR testing confirmed presence of PcRV only in G. pallida but not in two other cyst nematode species capable of infecting potato, which are present in the US, G. rostochiensis and G. ellingtonae. In light of ScRV being detected in at least two nematode species from genera Heterodera, H. glycines, and H. schachtii, it was hypothesized that, due to a close phylogenetic relationship between these nematode species within one genus, viruses such as ScRV may have evolved within this subset of cyst nematodes before the species split from one another [39]. Thus, future studies examining the host status of Globodera spp. for PcRV are certainly warranted. Furthermore, picorna-like virus, PcPLV, identified previously in silico [7], was not detected with RT-PCR within our Globodera RNA samples. Notably, Ruark et al. [7] also failed in experimentally confirming the presence of this virus in G. rostochiensis RNA samples available to them. Interestingly, the HTS data used to identify the PcPLV genome were obtained from the Scottish population of G. pallida, where the PcRV genome was also discovered by us. Apparently, two identical or almost identical isolates of PcRV, PcRV-Id and PcRV-Sc, are circulating in the laboratory populations of G. pallida maintained in two geographically distant locations, Idaho and Scotland. Incidentally, the latter population of G. pallida maintained in Scotland is also infected with another virus, PcPLV [7], which is not present in the Idaho populations of PCN (Figure 5). This shows that viral infections of nematodes vary by geographic region and population. Alternatively, it is also possible that viral PcPLV titers in the Idaho populations are below detectible limits and the samples falsely appear virus-free.

Viral infections of plant parasitic nematodes prove to be a challenging research topic due to the ecological complexity within an agricultural system. Nematode virology represents a relatively new field, and this study further advances knowledge in this area. The effects of viruses on fitness of nematodes, viral tropism, and mechanisms driving viral transmission are largely unknown. Future research aiming at better understanding the effects of viruses infecting nematodes will help to explore potential use of PcRV as a novel management strategy for potato cyst nematodes.

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