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

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´-CCCTATACACAAATGGGAATAACAA-3´, pos. 7329-7353 and reverse: 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´-CGTAAGGGAAGAGCGCATTTA-3´ and reverse primers: 5´-CAGATACCCTACCATCGAAAGTT-3´). Reverse transcription-polymerase 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.

Results

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).

Figure 1:

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.

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 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.