Prevalence of the root lesion nematode virus (RLNV1) in populations of Pratylenchus penetrans from North America

Abstract Root lesion nematode virus 1 (RLNV1) was discovered in the migratory endoparasitic nematode species Pratylenchus penetrans. It was found in a P. penetrans population collected from soil samples in Beltsville, Maryland, USA. In this study, the distribution of the RLNV1 in 31 geographically distinct P. penetrans populations obtained from different crops was examined. The results demonstrate that RLNV1 is widespread in North American populations of P. penetrans and exhibits low genetic variability in the helicase and RNA-dependent RNA polymerase regions of the genome.

Presently, the number of viruses identified in the phylum Nematoda is limited. Considering the vast diversity of species within this phylum, more viruses naturally infecting nematodes are likely to be discovered. Recently, several new viruses were identified from wild populations of free-living (Félix et al., 2011;Franz et al., 2012;Frézal et al., 2019), animalparasitic (Shi et al., 2016;Williams et al., 2019) and sedentary plantparasitic nematodes (PPN; Bekal et al., 2011Bekal et al., , 2014Lin et al., 2018;Ruark et al., 2017Ruark et al., , 2018. We have recently discovered a new virus (the root lesion nematode virus, RLNV1) associated with the migratory nematode Pratylenchus penetrans (Vieira and Nemchinov, 2019). P. penetrans is an endoparasitic migratory PPN, which can infect a broad range of economically important crops (Castillo and Vovlas, 2007) and is among the top three most damaging species of PPN (Jones et al., 2013). Pratylenchus species were the most abundant PPN (69%) identified in 38,022 samples from the Pacific Northwest of North America by nematode diagnostic laboratories labs from 2012 to 2016 (Zasada et al., 2019).
The objectives of this study were: to determine the distribution of the RLNV1 in geographically distinct P. penetrans populations obtained from different North American cropping systems and to assess genetic variability of the virus by comparing sequence variations of the helicase and RNA-dependent RNA polymerase (RdRP) regions of identified RLNV1 isolates from the P. penetrans populations collected for this study.
Prevalence of RLNV1 in North America: Vieira et al.

Collection of Pratylenchus penetrans isolates
A total of 31 populations of P. penetrans were used in this study from different geographic locations across Canada and USA (Table 1). Their identification was confirmed by morphological and molecular markers available for this species (Castillo and Vovlas, 2007;Peetz and Zasada, 2016). Most of the nematode populations were initially collected from different crops and maintained in vitro on sterilized corn roots (Vieira et al., 2015). The P. penetrans isolate NL 10p RH, from which RLNV1 (GenBank accession MK138531) was first identified, was used as positive control (Vieira and Nemchinov, 2019). For each population, several hundred nematodes were extracted from roots under intermittent mist for 5 d (Ayoub, 1980), washed three times in distilled water, frozen in liquid nitrogen, and stored at −80°C until subsequent analyses.

Nematode RNA extraction
Total RNA (50 ng per sample) was extracted from mixed life stages (eggs, second-to fourth-stage juveniles (J2-J4), adult females and males) of P. penetrans using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer's instructions. RNA was treated with RNase-free DNase (QIAGEN) before reverse transcription. The quantity and quality of the extracted RNA was assessed using a ND-1000 NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) following the manufacturer's instructions. Primers were designed based on the available nucleotide sequence of the viral helicase (forward: 5´-GATCTCACGCGCTTTACCA-3´, pos. 4046-4064 and reverse: 5´-TCAGGTTCTGGAACAGGATTTC-3´, pos. 4978-4900) and RdRP (forward: 5´-CCCTATACAC AAATGGGAATAACAA-3´, pos. 7329-7353 and rev erse: 5´-ATGCTCTCAAACCAGTCACTAT-3´, pos. 8307-8328) and used for PCR amplification of the corresponding sequence regions. The presence of the nematode transcripts within each generated cDNA library was confirmed by amplification of the 18 S rRNA gene fragment of P. penetrans (forward: 5´-CGTAAGGGAA GAGCGCATTTA-3´ and reverse primers: 5´-CAGAT ACCCTACCATCGAAAGTT-3´). Reverse transcriptionpolymerase chain reaction (RT-PCR) was conducted using 1 µl of cDNA from each library and the following conditions: 2 min at 94°C; 38 cycles (30 sec at 94°C, 30 sec at 54°C, 60 sec at 72°C), and one cycle at 72°C for 10 min. The PCR reactions contained 1 × PCR buffer, 1 unit Taq Platinum polymerase (Invitrogen, Carlsbad, CA, USA) and 0.2 µM of each primer in a total of 50 µl of total solution. PCR products were separated by electrophoresis on a 1% agarose gel using TBE buffer (0.045 M Trisborate, 0.001 M EDTA, pH 8.0) and visualized using SYBR Safe DNA gel stain (Invitrogen, Carlsbad, CA, USA). The generated PCR products were then purified by PCR-purification kit (QIAGEN) and sequenced by Sanger sequencing using the corresponding forward and reverse primers by Macrogen Corp (Rockville, MD, USA).

Analysis of genetic diversity
Nucleotide sequences were aligned using MUSCLE program with default parameters incorporated into CLC Main Workbench software (V. 8). Predicted proteins sequences were obtained using CLC Main Workbench software (V. 8) and aligned using MUSCLE program with default parameters (Edgar, 2004). Pairwise genetic distances of both nucleotide and amino acid sequences (i.e. nucleotide differences and percent identity) were determined using CLC Main Workbench V. 8 (Qiagen, Hilden, Germany) software.

Geographic distribution of RLNV1
A total of 31 populations of P. penetrans were assessed for the presence of the RLNV1 by RT-PCR with primers derived from helicase and RdRP regions of the virus genome ( Fig. 1 and Table 1). These isolates were obtained from either established cultures or field collections of P. penetrans, originally collected from agricultural fields distributed throughout several areas of the USA and Canada (Table 1). Pratylenchus penetrans populations were collected mainly from potato and soybean fields, but were also collected from apple, cherry, corn, mint, and raspberry, which is consistent with the wide host range of this nematode species (Castillo and Vovlas, 2007).
PCR amplification resulted in amplicons of the expected sizes (855 bp for the helicase and 1,000 bp for the RdRP regions) from 14 out of the 31 P. penetrans populations (Fig. 1A, B). The presence of P. penetrans transcripts within each cDNA library was confirmed by amplification of a 150 bp fragment from the 18 S rRNA gene (Fig. 1C). The RLNV1 was found in P. penetrans populations collected from potato, soybean, mint, apple and corn fields, while the virus was not detected in P. penetrans populations  obtained from raspberry and cherry (Table 1). Overall, the RLNV1 was detected in 45% of the P. penetrans populations.

Sequence variability within RLNV1 isolates
The partial nucleotide sequences of the helicase and RdRP regions derived from 14 RLNV1 isolates associated with different P. penetrans populations were obtained and compared between each other (Tables S1 and S2). In one population (P. penetrans population R3784 collected from potato in New Brunswick, Canada), only the RdRP region was sequenced. Sequences exhibited a low level of nucleotide and amino acid variations in both regions of the viral genome. Pairwise comparisons of the nucleotide sequences in the helicase region ranged from 96.56 to 100%, while at the amino acid level the corresponding sequences had 98.89 to 100% identity among all the isolates (Table S1). For the RdRP fragment of the genome, the sequence identity ranged from 96.75 to 100% at the nucleotide level, and 98.11 to 100% at the amino acid level (Table S2). The Malek isolate originally collected from a potato field in Wisconsin displayed the highest genetic variability among all isolates.

Discussion
The main objective of this study was to characterize the extent of RLNV1 infection in P. penetrans populations collected from different plant hosts across North America. We conclude that the virus is widespread in the USA and Canada and affects P. penetrans populations collected from the diverse crop systems in North America. However, the virus was not found in P. penetrans collected from 11 geographic locations ( Fig. 1 and Table 1) and in the nematodes collected from raspberries and cherries (Table 1). These findings may potentially indicate the presence of virus-free or virus-resistant P. penetrans populations, especially in the case of positive and JOURNAL OF NEMATOLOGY negative results in nematodes collected from the same crop (potato and soybean) and in contiguous geographic locations. Negative results obtained with P. penetrans populations collected from raspberry and cherry may also suggest host-dependent susceptibility of P. penetrans to RLNV1. If true, this would likely to be related to the genetic variability among the nematode isolates rather than to the virulence of RLNV1, which exhibited considerable homogeneity in the two regions examined in this study.
The observed prevalence of RLNV1 in populations of P. penetrans may also imply that this virus could represent a new resource as a potential biological control agent. Viruses associated with C. elegans (Orsay virus) and C. briggsae (Santeuil Le Blanc and Melnik viruses) were shown to infect intestinal cells, were horizontally transmitted, and slowed host progeny, thus affecting host fitness (Félix et al., 2011;Félix and Wang, 2019). While the interaction of RLNV with P. penetrans and its impact on the nematode's viability and parasitism are not well understood, this approach may shed light on the potential avenue to reduce damage caused by P. penetrans to crop plants.