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

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.

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).

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.

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.

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.