Serendipitous identification of Pratylenchus curvicauda from the grainbelt of Western Australia

Soil samples containing a mixture of root lesion nematodes including P. curvicauda were obtained from four different locations in the wheatbelt region of Western Australia: Pingelly (32°32′2.4″S, 117°5′9.6″E), Williams (33°2′0″S, 116°53′0″E), Arthur River (33°20′19″S, 117°2′4″E), and Katanning (33°41′27″S, 117°33′19″E), with the help of staff at the Plant Pathology Section, Nematology Division of the Department of Primary Industries and Regional Development, Western Australia (Fig. 1). Nematodes were extracted from the soils using a misting apparatus described by Tan et al. (2013). A pure culture of P. thornei maintained on carrot disks at 23°C in our laboratory at the time of this experiment was used as a control during the morphological characterization. The partial 18S-ITS1-5.8S-ITS2-partial 28S and 28S-D3 expansion sequences of the P. thornei were also sequenced for the first time and used to differentiate the P. curvicauda reported in this study.

Figure 1:

Initial identification of the P. curvicauda was carried out using two important morphological features: the position of the vulva and the shape of the tail. The morphology of individual specimens was examined and photographed using a compound microscope (Olympus BX51). Before the examination, single nematodes were hand-picked using a fine feather and placed in a drop of water on a glass slide. The slide with nematode was quickly passed over a flame of a Bunsen burner to stop the nematode from moving. Morphological measurements which included nematode body length and the position of the vulva from the tail were measured from captured images using the scale bars of the image software package on the Olympus BX51 compound microscope. Based on the latter measurements, the percent distance of the vulva from the anterior end of the nematode body, V, was calculated. Comparative morphometric and light microscopy images of immobilized P. curvicauda from the four experimental locations and those of P. thornei were also conducted using a Zeiss Axioskop Upright microscope (Carl Zeiss Microscopy, LLC, USA) at the Center for Microscopy, Characterization and Analysis, University of Western Australia, Perth. Nematode specimens from Pingelly with typical P. curvicauda features were fixed in 4% formaldehyde solution and sent to an expert taxonomist in the UK for further characterization. The detailed morphometric measurements obtained were compared with those taken in Australia.

Scanning electron microscopy (SEM) was used to further characterize P. curvicauda. To do this, single nematodes were fixed in 3% glutaraldehyde in 0.025 M phosphate buffer (pH 7.0) overnight at 4°C followed by five washes in the same buffer. The specimens were then fixed with 1% osmium tetroxide (OsO₄) in 0.025 M phosphate buffer (pH 7.0) for 2 hr at room temperature in a fume hood, followed by five washes in the same buffer. The samples were dehydrated in a graded ethanol series (30%, 50%, 70%, and 90%), twice in each solution, for 15 min at a time. The 90% ethanol was then removed and replaced with 100% ethanol and then amyl acetate following the same regime. Specimens were then dried in a critical point dryer (FL-9496 BALZERS, Furstentum Liechtenstein). The nematodes were transferred to an SEM holder with conductive carbon tape and coated with a combination of 3 nm platinum and 10 nm carbon. The samples were then examined and images were taken using an SEM (Zeiss Ultra 55) at 5 KV.

Genomic DNA from individual adult female nematodes from the soil mixtures with typical P. curvicauda features and from pure cultures of P. thornei was extracted for PCRs using a modified protocol employing a worm lysis buffer (Wood, 1988). Each nematode was transferred onto a microscope slide and 10 μL of sterile water added. The nematode was then carefully diced with a fine scalpel blade after which the fragments were transferred into 50 μL of lysis solution (1% SDS, 50 mM EDTA, 100 mM NaCl, 100 μg/ml proteinase K, 1% 2-mercaptoethanol, 100 mM Tris-HCl pH 8.5) in a 1.5 mL centrifuge tube. The nematode lysate was then frozen at −80°C for 40 min followed by thawing to room temperature and then heating at 60°C for a further 40 min. The suspension was then centrifuged at 1,000 g for 2 min and the supernatant transferred to a fresh tube for extraction of nucleic acids using phenol-chloroform (PC:50:50). The PC-lysate emulsion was vortexed for a minute and centrifuged at 16,000 g for 2 min. The supernatant was added to one-tenth volume of 3 M NaOAc (pH 6.8) and 2.5 volumes of 100% ethanol, stored at −80°C overnight before being centrifuged at 16,000 g for 30 min to Analyses of sequenc pellet DNA. The pellet was washed twice with 400 μL of 70% ice-cold ethanol, dried in a fume hood and resuspended in 17 μL of nuclease-free water. The DNA was quantified using a spectrophotometer (Nanodrop ND-1000, Isogen Life Sciences) and stored at −20°C until use.

The DNA sequence of the partial 18S-ITS1-5.8S-ITS2-partial 28 S of the rDNA and part of the 28S-D3 expansion region were used to characterize and distinguish between P. curvicauda and other Pratylenchus species. The primer pair 18S-Int (5′-CGTAACAAGGTAGCTGTAGG-3′) and 26S-Int (5′-CCTCCGCTAAATGATATGC-3′) (De Luca et al., 2011) was used to amplify the partial 18S-ITS1-5.8S-ITS2-partial 28S region using the GoTaq^® Green Master Mix (Promega Corporation, Australia), a premixed ready-to-use solution containing 50 units/mL Taq DNA polymerase, 400 μM each of dATP, dGTP, dCTP, and dTTP with 3 mM MgCl₂ and 1 to 2 μL of the genomic DNA. The reactions were incubated at 95°C for 5 min; 35 cycles of 95°C for 30 sec, 55°C for 5 sec, and 72°C for 1.40 min followed by a final step of 72°C for 10 min. The 28S-D3 region of the rDNA was amplified with the primer pair D2-F (5′-GACCCGTCTTGAAACACGGA-3′) and D3-R (5′-TCGGAAGGAACCAGCTACTA-3′) (De Luca et al., 2004) using the same PCR reagents but with the following temperature profile: 94°C for 6 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and a final step of 72°C for 6 min. PCR products were observed on 1% agarose gel stained with SYBR Safe (Invitrogen Pty Ltd, Australia). The amplicons were cut out of the gel with clean sterile blades and the DNA purified using the Wizard^® SV Gel and PCR Clean-Up System (Promega Corporation, Australia). The DNA was sequenced using Sanger sequencing and cloned using the pGEM-T Easy vector system following the manufacturers’ protocol (Promega Corporation, Australia). Plasmid DNA was isolated using the Wizard^® Plus SV Minipreps DNA Purification System (Promega Corporation, Australia). Both strands of six randomly selected clones of amplicons from individual nematodes were sequenced using the Big Dye 3.1 dye terminator in an AB 3730 96 capillary DNA Sequencer (Applied Biosystems, Australia).

Sequence profiles of the clones of amplicons of the partial 18S-ITS1-5.8S-ITS2-partial and 28S-D3 expansion regions of P. curvicauda and P. thornei were edited using Geneious (V8.1.8) (Kearse et al., 2012). Consensus sequences were then made after alignment with both CLUSTAL O (1.2.3) (Mcwilliam et al., 2013) and Geneious. Computations in further sequence analyses were reduced by using representative/consensus sequences of the clones from nematodes isolated from the different locations. Phylogenetic relationships of the P. curvicauda with other Pratylenchus spp. were constructed using the generated rDNA sequences of P. curvicauda and P. thornei and those of similar regions of other Pratylenchus species retrieved from the National Center for Biotechnology Information (NCBI) databases using both keyword searches and the BLASTn tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analyses were done with MEGA7 (Tamura et al., 2013), using all the different parameters such as substitution models, rates and patterns, treatments of gaps/missing data, and tree inference methods employed by the Maximum Likelihood, the Neighbor-Joining and the Minimum-evolution approaches to determine the most consistent tree representing the relationship of the P. curvicauda samples with other Pratylenchus species. These trees were constructed using the bootstrap method as a test of Phylogeny with 1,000 replicates, where necessary they are presented with the bootstrap values. Representative sequences of Meloidogyne spp. and Radopholus similis, derived from similar regions of the rDNA, were used as outgroups for the phylogenetic analyses.

Differences between and within sequences of either the partial 18S-ITS1-5.8S-ITS2-partial 28S or the 28S-D3 regions of isolates of the Pratylenchus species studied were estimated using overall mean distances. The overall mean distance is an estimate of evolutionary divergence between any group of sequences and is a measure of the number of base substitutions per site between sequences compared. All such analyses were conducted using the Maximum Composite Likelihood model using MEGA7 where codon positions included were 1st+2nd+3rd+non-coding and all positions containing gaps and missing data were eliminated (Tamura et al., 2004; Kumar et al., 2016).

Sequences of clones of the partial 18S-ITS1-5.8S-ITS2-partial 28S and the 28S-D3 regions of isolates of the P. curvicauda and P. thornei have been deposited in the nucleotide database of NCBI with the GenBank accession numbers MN010380-MN010412 and MN006333-MN006351, respectively. The accession numbers are also appended to the clones shown in Figures 6 and 7.

Over 90% of nematodes isolated from the soil collected from the wheat and barley paddocks at Pingelly, Arthur River, Katanning and Williams, and previously stored at 4°C, were plant-parasitic nematodes. Under a compound microscope, features observed in adult females included the characteristic stylet, thick lips, a stylet knob and overlapping esophageal regions that clearly distinguished them as root lesion nematodes (Fig. 2I). Three important morphological features, namely, the tail shape, the body length, and the vulva position, V, were further used to distinguish P. curvicauda from the mixture of nematodes in these soils. The identification exercise was aided with comparisons to pure cultures of Western Australian isolates of P. thornei maintained on carrot discs in our laboratory, and published data on other common species, namely, P. neglectus, P. penetrans, P. quasitereoides, P. teres, and P. curvicauda. Description of the nematodes studied are presented below.

Figure 2:

Using the tail shape, body length and the V, P. curvicauda adult females were carefully isolated from nematode mixtures for further characterization. In total, 12 adult female nematodes from soil collected from Pingelly, 6 from Williams, 10 from Katanning, and 6 from Arthur River with the characteristic curvy tail were assessed further. The body length of all the P. curvicauda specimens varied from 441 μm to 768 μm with an average of 538 μm whereas the V ranged from 70 to 76, with an average of 74. The average body of adult female nematodes isolated from the Pingelly soil was 550 μm long (range 484-616 μm) with an average V of 74 (range 70-76). The respective average body lengths of the specimens from Williams, Arthur River, and Katanning soils were 520 μm (range 464-590 μm), 534 μm (range 478-636 μm) and 538 μm (range 441–768 μm) and the respective Vs for these nematodes were 74 (range 71-76), 73 (70-76), and 74 (72-77).

The morphology of isolated nematodes from the Pingelly soil conspecific to P. curvicauda (n = 12) was described in detail in both laboratories, in Perth, and in the UK by the renowned nematologist, the late Dr M. R. Siddiqi. The descriptions are below.

The en face of the head revealed a characteristic oral disc, which was slightly raised and divided (Fig. 3B,C). The head was rounded and was offset by a constriction, about 9 µm in diameter and 2.5 to 3 µm high with three distinct annules (Figs. 2IIa and 3IIB,C). The head framework was strongly sclerotised with its outer margins extending two annules into the body (Figs. 2IIa and 3IIB,C). These annules were set off from the body by a deep circular groove (cuticle constriction). Posterior to the circular constriction was an extremely wide first body annulus, which was evidently wider than the following body annules (Fig. 3B,C).

Figure 3:

The spear was strong, with the conus being 51 to 55 percent of the spear length (Fig. 2IIa). The basal knobs were rounded, 5 µm across and 2 µm high. The orifice of the dorsal esophageal gland was at 3 to 4 µm posterior to the basal knobs (Fig. 2IIb). Two ventrosublateral esophageal glands were evident, ventral to the intestine, whereas the dorsal esophageal gland was anterior-most (Fig. 2IIa,b).

The body of the nematodes was typically curved to a c-shaped with a maximum diameter of 20 µm (Fig. 2I,II). The lateral fields had four incisures, the outer ones were distinctly crenate, with five to six incisures seen in the vulval region (Fig. 2IIc). Some of the adult females studied may have been gravid as an anterior ovary with a single row of oocytes was visible near the esophageal glands. No sperm was visible in the spermatheca. The post vulval uterine sac was differentiated in most of the nematodes, with a few reduced cells of the posterior ovary, 1.5 to 1.7 times the vulval body width long (Fig. 2IIc,d). The vulva was slightly protruding (Fig. 3A).

The tail of the nematode was sub-cylindroid with the terminal fourth usually appearing lozenge-shaped, ventrally arcuate, terminus-rounded, and occasionally with an indentation (Figs. 2IId,e, 3E,F). The lateral field of the tail had four incisures in the anterior third, then with three incisures as it narrows down to the end just before the terminus. The phasmid was dot-like, usually with seven to nine annules or one-fourth of the tail length behind the anal level (Fig. 2IId,e).

No male nematode was identified from mixtures collected from any of the four locations, which is consistent with a Pratylenchus life cycle.

Seven detailed morphometric measurements of P. curvicauda (Pingelly) were taken and compared to similar published data for six Pratylenchus species including P. curvicauda, previously described in metropolitan Western Australia in 1991, P. teres and the recently re-described P. quasitereoides isolated from Katanning, Western Australia. Despite the overlapping morphometric features typical of Pratylenchus species, the ranges and averages of the parameters studied for the Pingelly samples were more similar to, and clearly indicated that the samples were conspecific to P. curvicauda (Table 1). The average body length of the P. curvicauda from Pingelly falls within the range reported for five Pratylenchus species except for the published P. neglectus which are generally shorter (Table 1). The a of the Pingelly specimens was similar to those of P. neglectus, P. penetrans, P. curvicauda, and P. teres, but depicts the species as significantly different from P. quasitereoides and P. thornei (Table 1). Similarly, despite the overlap in the range of b, the average values for the P. curvicauda species were similar to those for P. neglectus, P. penetrans, and P. thornei but could clearly distinguish it from P. quasitereoides and P. teres (Table 1). The average value of b′ for the Pingelly samples was similar to that of the published P. curvicauda, 3.37 and 3.4, respectively, and the other species except for P. quasitereoides where the range was outside of those for the other species and the average was almost twice as much for P. curvicauda from Pingelly. Whereas the c′ and the V for all the species in Table 1 were in range, the relative smaller value of c of the P. curvicauda distinguishes it from the other five species (Table 1).

Generally, characters that distinguished P. curvicauda from other Pratylenchus species were: the longer conus which was more than half of the spear length, the three annules in the lip region, the large rounded basal knobs, and the presence of phasmids in the anterior region of the tail which were usually about one-fourth the tail length behind the anus.

The body length and V was different from the Western Australia isolate of P. thornei cultured in our laboratory [P. thornei: n = 16, body length = 650 μm (range 557–809 μm), V = 76 (range 72–80)]. Also, the curved tail shape of P. curvicauda differed markedly from those of P. neglectus and P. penetrans: the tail of P. penetrans is generally rounded, and that of P. neglectus is conoid and for P. thornei the tail is conical with a round tip (Fig. 4).

Figure 4:

Several features of P. curvicauda distinguish it from P. quasitereoides. First, the lateral field of P. curvicauda is wider and has more incisures in the vulval region. Also, for the stylet, the conus is longer than the shaft including the basal knobs, and this appears to be larger. The tail of P. curvicauda is more arcuate ventrally and its terminus is smooth except for occasional single indentation, while that of P. quasitereoides was depicted as having a broadly rounded, finely crenate terminus tail. In addition, the tail pattern of P. curvicauda is also quite different since the inner incisures go past the phasmids which are at the end of anterior fourth of the tail, while they are behind the middle of the tail for P. quasitereoides.

The partial 18S-ITS1-5.8S-ITS2-partial 28S sequences from six randomly selected clones for each amplicon from 14 adult females of P. curvicauda were analyzed. They included two nematodes each isolated from soils of Pingelly, Arthur River and Katanning, four from Williams and four from soil previously isolated from wheat and barley fields of unknown location in the grainbelt region of Western Australia. The nematodes from the unknown location included in this study were among a mixture of root lesion nematodes from the wheat field and also had typical features of P. curvicauda. The amplicons were cloned because direct sequencing yielded ambiguous peaks that indicated they were of mixed templates. For maximum quality assurance, both strands of plasmid DNA of each clone were sequenced. The sequences of the 24 clones of the nematodes from Williams, the 12 from clones of nematodes from Arthur River and those from the Unknown location (UN) were 906 to 907 nucleotides (nt) long whereas the 12 clones of the nematodes from both Katanning (928-933 nt) and Pingelly (906-933 nt) were generally longer. Based on sequence homology, the 84 clones were grouped into 28 unique sequences which included six clones each from the nematodes isolated from Pingelly (PN), Arthur River (AR), Katanning (KAT) and the Unknown location with four for the 24 clones from the four nematodes in soils of Williams (WL). Representative sequences of clones from individual nematodes of particular locations were identified by symbols for the location, the nematode number and unique sequence group it represented. For example, the sequence WL2.1 was from nematode two (2) from Williams (WL) and represented the first unique group (1) of sequences of nematodes from Williams. The overall mean distance between the sequences was 0.013. Sequences of amplicons obtained from the individual nematodes had some degree of genetic variation; both in length and bases and confirmed the mixed sequence profiles from the direct sequencing of the amplicons. There was less sequence divergence in clones of the isolates from Arthur River, Unknown location, Pingelly with respective overall mean distances of 0.001, 0.002, and 0.012, whereas the sequences of nematodes isolated from Katanning and Williams were less identical to each other with overall mean distances of 0.021 and 0.020, respectively. These differences are clearly represented in the phylogenetic tree in which the more similar clones clustered together and those of Williams and Katanning were separated (Fig. 5A).

Figure 5:

Figure 6:

Figure 7:

Sequences representing the partial 18S, the ITS1, the 5.8S, the ITS2, and the partial 28S regions of the rDNA were delineated using characterized sequences of other nematodes. The partial 18S (62 nt) and 28S (37 nt) sequences of the amplicons were conserved among the P. curvicauda clones except for single nucleotide changes in three positions and a deletion in separate clones in the partial 18S sequences. There was, however, more heterogeneity in the 330 nt long 5.8S region with 24 base changes and 11 positions with insertions/deletions. As was the case for the full sequence analysis, the intra-specific heterogeneity in the ITS1 (311-392 nt) and ITS2 (268-342 nt) regions distinguished the clones from Katanning from those of the four other locations (Fig. 5B,C). Generally, there was more heterogeneity in the ITS2 sequences (overall mean distance of 0.025) than the ITS1 (overall mean distance of 0.010) (Fig. 5B,C).

The P. curvicauda sequences were compared to those of similar regions of other Pratylenchus nematodes. Redundancy in 919 sequences of the 18S RNA or the partial 18S-ITS1-5.8S-ITS2-partial 28S of Pratylenchus nematodes obtained from NCBI was reduced using the CD Hit suite (Huang et al., 2010), with a cut-off of 96% sequence homology, leaving 253 sequences. Of these, 134 were used for phylogenetic analyses and mean distance comparisons with the P. curvicauda sequences; this set of sequences had both primers used to amplify the rDNA region of P. curvicauda samples so the sequences between the primers were directly compared. The 134 sequences represented 33 recognized and two unspecified Pratylenchus species, but there was no sequence for any isolate of P. curvicauda as no such sequence has been published or deposited in NCBI or any other sequence database. The representative species included 8 of the 12 reported in Australia. Notably, no sequence of this section of the rDNA used for this analysis for any Pratylenchus species originating from Australia was publicly available. So, we amplified a similar region of the rDNA of P. thornei, originally cultured from a single nematode and maintained in our laboratory (Nicol et al., 2012), cloned the amplicons, and added sequences of five representative clones (from a total of 10) from two nematodes to the analyses. These sequences, labeled as P. thornei_WA were each 826 nt long, relatively shorter than those of P. curvicauda. Phylogenetic analyses of the P. curvicauda sequences and among the 134 representative sequences of the 35 Pratylenchus species explored using the Maximum Likelihood, the Neighbor-Joining and Minimum-evolution approaches of MEGA 6.0 with 1,000 bootstraps indicated P. curvicauda formed a highly supported separate clade with P. bolivianus, the closest species. In all the molecular phylogenetic trees considered, all the sequences of the same species were grouped together and were clearly distinct from the P. curvicauda clones including the P. thornei_WA sequences which were in the same clade as the representative P. thornei sequence (KY424246.1). Figure 6 is a representation of the phylogenetic tree using representative sequences of 34 of the 35 species, excluding that for the unidentified Pratylenchus species. As expected, sequences of the Pratylenchus species, including those of P. curvicauda were clearly distinct from representative sequences of the two outgroups of the genera Meloidogyne and Radopholus.

The distinct grouping of the Pratylenchus species was supported by the intra-species mean distances in sequences of the species analyzed. The overall mean distance of the 134 Pratylenchus sequences with the 28 of P. curvicauda was 0.202, and was higher than the overall intra-species mean distances for sequences of each of the individual species. Notably, the mean distances for representative sequences of eight Pratylenchus species commonly found in Australia were mostly higher than that for the P. curvicauda clones (0.013): P. neglectus (0.051), P. penetrans (0.043), P. vulnus (0.051), P. brachyurus (0.059), P. coffeae (0.036), P. crenatus (0.068), P. goodeyi (0.311), and P. zeae (0.069).

Sequences of the 28S-D3 regions of the rDNA were also analyzed; the aim was to characterize and distinguish the P. curvicauda further from other Pratylenchus species particularly those present in Australia for which similar sequences were available. The 17 consensus sequences of clones of the amplicons from the 28S-D3 regions of P. curvicauda analyzed were between 314 and 318 nt long with an overall mean diversity of 0.085. The sequences of nematodes from Katanning were less diverse with an overall mean diversity of 0.003; those from Pingelly, Arthur River and Williams had higher overall mean diversities, respectively, 0.061, 0.059, and 0.039. Sequences from the Western Australia P. thornei isolate had a mean diversity of 0.014.

From 842 sequences at the NBCI databases for the 28S-D3 region of the rDNA of Pratylenchus species, a set of 118 was used to study relationships (i.e. to construct phylogenetic trees and estimate mean distances) after reducing redundancy (with a cut-off of 98% similarity) and delineating the sequences based on those available for isolates of P. quasitereoides (215 bp); the latter exercise resulted in the use of less than the full sequence of the amplicons of P. curvicauda in the phylogenetic analyses. The resulting sequences represented 39 recognized and 12 unspecified Pratylenchus species including sequences of Australian isolates; 10 for P. quasitereoides (Hodda et al., 2014) and 2 for P. thornei (Subbotin et al., 2008). All molecular phylogenetic trees constructed using Maximum Likelihood, the Neighbor-Joining and Minimum-evolution approaches of MEGA 7.0 with 1,000 bootstraps grouped P. curvicauda as a separate Pratylenchus species, distinct from all the other Pratylenchus species and the outgroups, Meloidogyne javanica, and R. similis. Figure 7 shows the relationship of the P. curvicauda sequences with representative sequences of 40 species of Pratylenchus including a representative sequence of the 10 available for P. quasitereoides. Notably, because of the conservation of the 28S-D3 sequences even between species of different genera, the M. javanica used appears to be more closely-related to some of the Pratylenchus spp. The overall mean distance of all the sequences of the Pratylenchus species including those of P. curvicauda and P. thornei_WA was 0.167 compared to 0.12 among the 17 sequences of the P. curvicauda alone. Contrary to features of the partial 18S-ITS1-5.8S-ITS2-partial 28S, the overall mean distances among both the full sequences of amplicons of the 28S-D3 of P. curvicauda and the portions used for the phylogenetic analyses, respectively, 0.085 and 0.12, were higher than the intra-species value for the Pratylenchus spp. with multiple sequences in the data set used. There was a relatively lower diversity in the 28S-D3 sequences than the partial 18S-ITS1-5.8S-ITS2-partial 28S of species commonly found in Australia, namely, P. neglectus (0.013), P. vulnus (0.009), P. brachyurus (0.025), P. crenatus (0.024), and P. zeae (0.055) except for P. penetrans (0.058), P. coffeae (0.055), and P. thornei (0.019) where the opposite was true. Also, the separate phylo-grouping of P. curvicauda from the Australian isolates of P. quasitereoides (overall mean distance of 0.023) is clearly supported by the differences in the diversity in the representative sequences of the two species.