An Update on Root Lesion Nematode Species Infecting Cereal Crops in the Southwest of Western Australia

Abstract

Root-lesion nematodes (Pratylenchus spp.) reduce the yield and quality of cereal crops in Australia. Eleven of the ~90 species characterised are present in Australia, with those determined as economic pests of broadacre agriculture costing an estimated AUD 250 million annually. Two species, P. curvicauda and P. quasitereoides, recently re-described, were isolated from fields located in the grainbelt of Western Australia, but little is known about their distribution in the region surveyed in this study. To investigate this and possible co-infestations with other Pratylenchus spp., we surveyed seven commercial wheat, barley, and oat farms near Katanning, Cancanning, Kenmare, Duranillin, Darkan, and a barley seed-bulk nursery near Manjimup, all in the southwest grainbelt of Western Australia. Morphological and molecular characterisation of Pratylenchus spp. extracted from soil and plant roots indicated all fields surveyed were infested. Both P. quasitereoides and P. curvicauda were present as single or mixed populations with P. penetrans and/or P. neglectus, although they were not found in the same field. Analyses of the D2–D3 sequences of the identified nematodes indicated that the species found in Australia were distinct, particularly P. quasitereoides and P. curvicauda. This work suggests P. curvicauda is likely to be present more widely in the WA grainbelt. Expanding molecular diagnostic testing for Pratylenchus species in the region to account for both nematodes is urgently needed so effective management can be implemented.

1. Introduction

Root-lesion nematodes (Pratylenchus spp.; RLNs) are migratory endoparasites of plant roots; their hosts include major staple crops consumed worldwide [1]. They are the third most significant plant-parasitic nematodes in terms of the economic losses they cause to grain and horticultural crops [2]. In Australia, they are the most significant nematode pests in broadacre agriculture, which is also impacted by other plant parasitic nematodes, including the Australian cereal cyst nematode Heterodera australis and root-knot nematodes (Meloidogyne spp.) on horticultural crops [3,4,5,6]. Of the >90 species of RLNs described worldwide, 11 have been identified in Australia, mainly in the broadacre cropping areas, where major cereals and legumes are grown [7,8,9,10,11,12]. Available data indicate that RLNs cost Australian broadacre agriculture over AUD 250 million annually, with P. thornei and P. neglectus being the most widespread (GRDC, 2018). In Western Australia (WA), P. neglectus and P. quasitereoides are the most common RLNs in broadacre cropping, often co-existing and damaging cereal crops like wheat, a major export crop contributing AUD 1.6 billion to the state’s economy in the 2023–24 cropping season [13,14]. Infectivity of RLNs varies depending on the nematode species, soil type, climatic factors, and the susceptibility of host crops. For example, some cultivars of canola are resistant to P. thornei, but susceptible to P. neglectus, P. quasitereoides, and P. curvicauda [5,13,15,16,17]. In addition, some cultivars of other crops grown in Australia, including triticale, legumes (e.g., black gram, field pea, and faba beans), oilseeds (canola, soybean), sugarcane, and forage brassicas and sorghum, also exhibit low tolerance to infection by Pratylenchus spp. [8,10,11,16,18,19]. Knowledge of the status of these economic pests through monitoring, for example, to identify the most prominent pests in particular fields enable the development of species-specific management strategies to reduce the damage they cause. Most research and variety screening in Australia has focused on losses caused by P. thornei and P. neglectus, with quantification mainly for wheat and barley, and less so for other crop hosts susceptible to Pratylenchus spp. [8,10,11,18]. Up to 65% of RLN yield losses of wheat have been attributed to P. thornei in the northern Australian grainbelt [20], whereas in the southern region, losses could amount to 28% of yield [21]. Losses attributed just to P. thornei infestation can be about 4.3% of total wheat production/year in the eastern Australian subtropical grain-growing region, a loss of about AUD 38 million [10,22].

In the last four decades, soilborne disease and nematode pest surveys in the WA grainbelt have established that eight Pratylenchus spp., namely, P. neglectus, P. thornei, P. zeae, P. brachyurus, P. penetrans, P. scribneri, P. quasitereoides and P. curvicauda, infest major broadacre crops grown in the region, [3,5,7,23,24,25,26,27]. The most recent additions are P. quasitereoides [26], originally described as ‘similar’ in morphology to P. thornei and later thought to be P. teres, and P. curvicauda [7]. Mixed populations of RLNs appear to be common, and the level of susceptibility of available hosts and environmental conditions determines which species is the most prevalent seasonally. This makes the correct identification of Pratylenchus spp. present in fields vital to designing effective management strategies to curb specific damage caused by the species. Most Pratylenchus-infested soils have a patchy distribution of nematodes, with a significant portion of fields having infestation levels above economic thresholds, high enough to cause over 10% yield losses [28]. For instance, the level of infestation of P. neglectus and P. quasitereoides was used to predict 7%, 15%, and 16% yield losses of wheat, barley, and canola, respectively, during a field trial [13]. In an infested field, the economic impact usually varies from season to season, depending on the soil type, the nematode species present, the susceptibility of the crops grown, and how the field is managed, including over summer, between cereal growing seasons in rain-fed crops. The economic damage can be significant in a mixed Pratylenchus-infested field where at least one species present can multiply on over-wintering weeds or winter crops, leading to a build-up and persistence of infection in the main cereal growing season [16,19,21].

Since the redescription of P. teres, originally isolated from Katanning as P. quasitereoides [26], this RLN has been reported infesting fields in other areas in the grainbelt of WA. When surveyed in 2016, up to 63% of National Variety Trial sites were infested with mixed or single populations of P. neglectus and P. quasitereoides [13]. P. curvicauda was first described on a clover species in metropolitan Perth in 1991 [29], but it was only in 2019 that it was identified from soils in broadacre cropping fields infested with P. quasitereoides [7]. It has since been shown that P. curvicauda can multiply on several major crops, weeds, and known break crops [7,8]. So far, P. curvicauda has not been identified morphologically or by molecular tests in mixed populations with P. quasitereoides.

The aim of this study was, therefore, to extend the investigation of infestations of Pratylenchus spp. in soil and roots collected from cereal fields in the southwest of the WA grainbelt, including fields near the towns of Katanning and Darkan, where P. curvicauda or P. quasitereoides had been identified previously. Molecular cloning and sequencing were used (1) to assess the presence of Pratylenchus spp. in the fields, and (2) to determine whether P. quasitereoides and P. curvicauda were present outside the areas where they had been identified previously and whether they co-existed in infested soils.

2. Materials and Methods

2.1. Field Sampling of Commercial Cereal Fields

Root and soil samples were collected from seven commercial cereal fields located near the towns of Darkan (−33.3389, 116.7416), Duranillin (−33.5986, 116.7168), Katanning (−33.5751, 117.1544), Kenmare (−33.5382, 117.1936), and Cancanning (−33.1149, 117.5190), in the SW grainbelt of WA (Figure 1). For each field, except at Darkan, 30 plants and 1 kg of soil from around the roots of the plants were collected in October 2020, during the cereal growing season. Each field was sampled randomly at ten sites, 200–300 m apart. The plants and soil were placed in plastic bags and transported the same day to the WA State Agricultural Biotechnology Centre (SABC) at Murdoch University, Perth, where they were kept at 4 °C until analysed. At the time of sampling, the fields were sown to wheat cultivars Scepter (at Duranillin and Cancanning), Rockstar (at Katanning), Mace (at Kenmare), the oat cultivar Bannister (at Katanning), and barley cultivar Spartacus (Cancanning). Nematodes from the Darkan site were isolated from soil provided by the nematology research team at the Department of Primary Industries and Regional Development, Western Australia (DPIRD).

2.2. Sampling of Seed Bulk-Up Nursery

Sampling at the seed bulk-up nursery near Manjimup (−34.2993, 116.1208), in the SW grainbelt of WA (Figure 1) was done during the irrigated planting season in March 2020. The nursery, a part of DPIRD Manjimup research station facility, had 18 barley germplasm being bulked in separate plots, with many plants exhibiting visual symptoms consistent with RLN infestation, such as stunted growth, chlorotic leaves, and poor root growth with potential lesions. The germplasm consisted of six commercial cultivars and twelve single seed descent (SSD)-derived fixed experimental lines. The commercial cultivars were Compass, Granger, Lockyer, La Trobe, Hindmarsh, and RGT Planet. Seven of the SSD experimental lines, namely, WBGA200379, WBGA201474, WBGA201372, WBGA201442, WBGA201465, WBGA201476 and WBGA201834 were derived from four-way crosses of Compass/Granger//Lockyer/La Trobe. The other five SSD lines, 16WBGAB1001, 16WBGAB1045, 16WBGAB1119, 16WBGAB1003, and 16WBGAB1230, were derived from a cross between RGT Planet and Hindmarsh. During sampling, the plots were subdivided into three equal sections, with each section ascribed high, moderate, and low infestation zones based on the severity of symptoms on the plants. For each section, 15 relatively healthy plants and 15 plants with suspected symptoms of nematode infestation (stunting and leaf chlorosis) were uprooted with soil, placed in plastic bags, transported to the SABC at Murdoch University, stored, and had nematodes extracted as described above.

2.3. Confirmation of Pratylenchus spp. Infestation

RLN infestation of all plants (except at Darkan) was initially assessed using the presence of necrotic, brown lesions on infected roots, followed by acid fuchsin staining of root sections for the presence of nematodes [31]. For samples from each commercial field, the roots of 25 plants representative of each sampling site were washed with tap water and examined by light microscopy (Nikon SMZ18 stereomicroscope, Nikon, Tokyo, Japan) for lesions or browning. For the samples from the Manjimup nursery, three plants, one from each of the three sections of the 18 plots, were washed and examined. Fragments of roots of five plants per field or plot were stained with acid fuchsin and examined by light microscopy.

2.4. Isolation and Quantification of Pratylenchus spp. in Roots and Soil Samples

Plant-parasitic nematodes were first extracted from soil samples and root sections of infected plants, except from the Darkan site. From the suspension of nematodes, the RLNs were isolated and counted, and the mean number per gram of roots and 25 g of soil was used to measure infestation. Nematodes were extracted from 2 cm pieces of root and 25 g of soil (processed as 5 g batches) using a custom-made misting apparatus that sprayed a fine mist of water directly onto the roots or soil for 10 s every 10 min for three days [32]. The nematodes were suspended in water and collected in 50 mL falcon tubes. Pratylenchus spp. were then identified using two key features: the presence of a stylet, and notable thick knobs posterior to the stylet. Nematode suspensions in four replicates of 500 µL were counted using an Olympus BX51 compound microscope (Olympus Corporation, Tokyo, Japan).

Because of work restrictions during the COVID-19 pandemic, the number of nematodes in the soil samples collected from the Manjimup plots could not be processed immediately after sampling. Instead, the nematodes from each plot were maintained on the susceptible wheat cultivar Calingiri as described below [7,8]. The soil samples from different sections of the plots were mixed well, and 500 g was added to 9.5 kg of potting mix in a 10 L pot with a coffee filter at the bottom. The pots were placed in trays to prevent the nematodes from escaping. The potting mix was made by combining three parts of pasteurised ‘Murdoch mix’ (composed of composted pine bark, coarse river sand, coco peat in a 2:2:1 ratio) and one part of Richgro^® Grain and Cutting Mix and thoroughly mixing in a cement mixer for 15 min. The fertilisers Grower’s blue^® (60 g) and Osmocote^® (60 g), 20 g of dolomite, and 15 g of calcium carbonate were added per 40 L of the mix. Soil from each plot was maintained in pots in triplicate, with each containing ten seedlings of cv. Calingiri. The plants were maintained in a glasshouse until the nematodes in roots and soil were isolated and characterised as described above, 60 days later.

2.5. Amplification of Partial rDNA of Pratylenchus spp.

Genomic DNA was isolated from bulks of ten or fifty nematodes depending on the availability of intact, adult stages with apparent morphological features typical of Pratylenchus species in each sample (soil and plants) collected from each sample site in the different fields. DNA was extracted using the modified method described by [33], which involved manually cutting nematodes into pieces, followed by proteinase K treatment and then direct PCR of nucleic acids in the supernatant. Two microlitres of the supernatant were used immediately as the template for PCR or stored at −80 °C until used. A D2-D3 fragment of the 28S rDNA gene of the nematodes was amplified using the primer pair D2-F (5′-GACCCGTCTTGAAACACGGA-3′) and D3-R (5′-TCGGAAGGAACCAGCTACTA-3′) [34]. PCRs were done with 10 µM of each primer using the GoTaq^® Green Master Mix (Promega Corporation, Alexandria, Australia) in 20 µL volumes following the manufacturer’s instructions in a thermocycler (Applied Biosystems 2720, Applied Biosystems, Carlsbad, CA, USA). The temperature profile for the PCRs was 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min and a final extension at 72 °C for 5 min. The amplicons were visualised and then purified from 1% agarose gels containing SYBR^TM Safe DNA gel stain (Invitrogen Pty Ltd., Mt Waverly, Australia) using the Wizard^® SV Gel and PCR Clean-Up System (Promega Corporation, Alexandria, Australia).

2.6. Cloning and Sequencing of Clones of D2-D3 Amplicons

The D2-D3 amplicons were cloned following the instructions for the pGEM^®-T Easy vector System II (Promega Corporation, Alexandria, Australia). Inserts in recombinant bacterial colonies were amplified using the Universal M13 forward and reverse primers (10 µM), and the amplicons were sequenced with the forward primer. The PCR reagents and conditions were as described above. The 5 µL DNA template used in the PCR was obtained by suspending individual recombinant colonies in 20 µL of nuclease-free water. The amplicons were isolated from 1% agarose gels using disposable sterile scalpel blades and placed on the filter unit of a 200 µL filter pipette tip with the end cut off to fit a 1.5 mL centrifuge tube. DNA was eluted by centrifuging the tubes containing the filter tips with gels at 16,000× g for 30 s. Five microlitres of the eluted DNA were then used for Sanger sequencing using the Big Dye 3.1 dye terminator. The sequence chromatograms were analysed using the FinchTV software (version 1.4.0) (Geospiza, Inc.; Seattle, WA, USA; A minimum of ten clones per amplicon were sequenced at first, and when there was more than 3% variation in the sequences, more clones were analysed to account for the diversity.

2.7. Analyses of Sequences and Phylogenetic Relationships

Phylogenetic relationships were established between the sequences of the clones and similar sequences of other Pratylenchus spp. retrieved from the National Center for Biotechnology Information databases (NCBI, https://www.ncbi.nlm.nih.gov). Sequence diversity and phylogenetic trees were constructed using MEGA X (Kumar et al., 2018) employing the Maximum Likelihood, Maximum Parsimony, and Minimum Evolution approaches with the Kimura 2-parameter substitution model with 1000 bootstraps; the most consistent of the trees are presented. The sequence of Pratylenchus vulnus (Genbank accession no. LT985479) from Italy was distant enough to be used as an outgroup during the tree construction when those isolated in this study were compared. The sequences were aligned using MUSCLE [35] and Multalin [36] to visualise variable regions and to identify indels and substitutions.

2.8. Statistical Analysis

Significant differences in the means of the number of Pratylenchus spp. isolated from replicated roots or soil samples were assessed using Student’s t-test (Microsoft Excel toolkit) with p < 0.05 as the threshold for significance.

3. Results

3.1. Root-Lesion Nematodes Were Present in All Fields Surveyed

The root and soil samples from all seven commercial cereal fields at Duranillin, Katanning, Kenmare, Cancanning, Darkan, and the Manjimup nursery contained plant-parasitic nematodes, based on the presence of prominent stylet in the nematodes collected from the misting apparatus. The nematodes collected from Darkan had previously been identified as Pratylenchus species by DPIRD using molecular and morphological tools. For all other sites, root systems of 80 of the 154 plants assessed had localised pale brown to brown lesions, characteristic of infestation by Pratylenchus spp. The presence of nematodes in the acid-fuchsin-stained roots of these plants confirmed that all the cultivars of wheat, oats, and barley in the fields were infested with parasitic nematodes (Figure 2A–C). There were no observable galls on infected roots or cysts in the soil, indicating that root-knot and cyst nematodes were not present. In contrast, the lesions on infested roots and body length, distinct stylet and thick stylet knobs at the head region of the nematodes isolated from soil and roots strongly indicated they were Pratylenchus spp. (Figure 2D–F). The adult stages of the nematodes isolated from soil and roots from the commercial fields and the nematode suspension from the Darkan site were predominantly female except for those in the root and soil extracts from the Manjimup nursery, which also contained males (Figure 2F).

3.2. The Levels of Infestation in the Fields Differed Significantly

The levels of infestations in the fields varied with the fewest nematodes associated with the wheat cv. Scepter and barley cv. Spartacus in fields at Cancanning, with less than 100 nematodes per gram of roots or 25 g of soil (Figure 3). Significantly more nematodes were isolated from the roots and soil collected from the wheat fields at Kenmare, Katanning, and Duranillin (p < 0.05) (Figure 3).

There were significant (p < 0.05) differences in the mean number of nematodes in the roots of the barley germplasm at the Manjimup nursery (Figure 4). The levels of infestation arbitrarily assigned as high (H), moderate (M), or low (L) to the three sections of each barley plot based on the health of the plants during sampling generally correlated well with the calculated mean number of nematodes in roots (Figure 4A).

3.3. Molecular Confirmation of the Pratylenchus spp. Identified

Sequences of over 300 clones of the D2-D3 fragments amplified from nematodes collected from all the eight sites and morphologically identified as Pratylenchus spp. were analysed. A comparison of sequences of the clones with Pratylenchus spp. indicated that one or more species, P. neglectus, P. penetrans, P. quasitereoides or P. curvicauda, were present in roots and soil from the eight fields surveyed (Figure 5). All sequences of nematodes isolated from the Cancanning and Katanning sites were >98% identical to those of P. quasitereoides (Figure 5). In the other four fields there were mixed infections with two (Darkan, Kenmare and Manjimup) or three species (Duranillin), but none had all four species present (Figure 5). The nematodes from the Duranillin site were a mixed population of P. neglectus (20%), P. quasitereoides (20%), and P. penetrans (60%). Those identified in the Kenmare field comprised 54.5% P. penetrans and 45.5% P. quasitereoides, whereas those isolated from Manjimup were predominantly P. penetrans (~99%), with only ~1% being P. curvicauda. Most of the sequences of the nematodes obtained from the Darkan site were identified as P. curvicauda (92.6%), with 7.4% identified as P. penetrans (Figure 5).

The D2–D3 amplicons obtained from the nematodes ranged from 312 to 346 nucleotides (nt). The exceptions were the 240 nt long sequences, which matched P. curvicauda sampled at Manjimup, and the 192 nt long sequences of P. quasitereoides sampled at Duranillin, which were truncated manually because the 3′ nucleotides were of poor quality (

Table 1. The characteristics of sequences of the D2-D3 region of the 28S rDNA of nematodes identified in this study and Genbank accession numbers.

Sample Location	Clones Sequenced	Identified Pratylenchus spp.	Sequence Characteristics
			Unique Groups of Sequences Among the Population	Length (Nucleotides)	Positions with Insertions or Deletions	Positions with Substitutions	NCBI Genbank Accession Numbers
Darkan	26	P. curvicauda	7	312–316	8	83	PP203078-84
		P. penetrans	3	344	0	4	PP203085-87
Manjimup	141	P. penetrans	8	343–344	3	11	PP203088-95
		P. curvicauda	1	240	NA	NA	PP203096
Cancanning	15	P. quasitereoides	5	345	0	17	PP203097-101
Katanning	13	P. quasitereoides	4	344–346	2	12	PP203102-105
Kenmare	21	P. penetrans	1	344	NA	NA	PP203106
		P. quasitereoides	2	344–345	1	2	PP203107-108
Duranillin	40	P. penetrans	2	344	0	6	PP203109-110
		P. quasitereoides	1	192	NA	NA	PP203111
		P. neglectus	1	347	NA	NA	PP203112

).

The Genetic Distance (GD) among sequences of the same species was lowest for P. penetrans (GD = 0.008), followed by P. quasitereoides (GD = 0.043) and then P. curvicauda (GD = 0.159). Within sequences of a species from the same location, P. penetrans at Darkan and Manjimup were the least diverse (GD = 0.008 for both), whereas the sequences of P. quasitereoides at Katanning (GD = 0.021), Cancanning (GD = 0.012) and Kenmare (GD = 0.06) were more diverse. The GD among sequences of P. curvicauda at Darkan was 0.099. These differences are captured in the clear separation of the species into distinct clades in the phylogenetic tree in Figure 6, which compares representative sequences of nematodes identified from all locations with a P. vulnus sequence as the outgroup.

3.4. Phylogenetic Relationships of the Pratylenchus spp. Identified, with Reference to P. curvicauda and P. quasitereoides

Relationships of the nematodes identified from the cereal fields with other Pratylenchus spp. were established with 98 consensus sequences generated from 919 accessions of the 28S rDNA D2-D3 region of 52 known and 18 unclassified Pratylenchus spp. All phylogenetic trees constructed using the region of rDNA amplified and employing the Maximum Likelihood, the Neighbour-Joining and Minimum-evolution approaches of MEGAX with 1000 bootstraps indicated the species isolated in the study were closely related to the genus Pratylenchus. All three trees grouped the sequences in similar clades with those of Pratylenchus spp. identified in many other countries, further confirming the relatedness of the current isolates to Pratylenchus spp. A comparison of the amplified sequences with representative sequences of the eleven Pratylenchus species currently identified in Australia indicated that the four species identified from the cereal fields studied, particularly P. curvicauda and P. quasitereoides, were distinct. The mean distance between the 22 sequences identified as P. curvicauda (from Darkan and Manjimup) was 0.072. In contrast, the mean distance between the published P. curvicauda sequences previously identified from four locations in Australia was 0.110, indicating that within the clade, the recently identified sequences were more identical and distinct from representative sequences of the other eleven Pratylenchus spp. (Figure 7).

Similarly, all thirty-six P. quasitereoides sequences identified in this study grouped with the published P. quasitereoides sequences with very similar overall mean distances between the two groups of sequences, with a mean of 0.028 for those identified in this study and 0.029 for the published sequences (Figure 7). Among them, the two consensus sequences of nematodes isolated from Kenmare were almost identical (overall mean distance of 0.007) compared to those of the populations isolated from fields at Cancanning (0.025), Duranillin (0.026), and Katanning (0.028). The grouping confirms the closeness of the nematode species isolated from the fields and identifies them as a single species. The sequences of P. neglectus and P. penetrans grouped with all the publicly available sequences of the same species in the NCBI databases. These are the first published sequences of P. neglectus or P. penetrans from Australia, although the two species have been known to exist in Australia for decades.

4. Discussion

One aim of this study was to provide additional information on the infestation of Pratylenchus spp. in cereal fields in the SW grainbelt of WA. In particular, the study has extended understanding of the distribution of P. curvicauda, and possible co-existence with other common Pratylenchus species [8,13]. Overlapping morphological and morphometric features can make it difficult to distinguish between some Pratylenchus spp., so we used sequences of the D2–D3 region of the ribosomal DNA to identify Pratylenchus spp. in the seven commercial cereal and barley nursery fields surveyed. The results confirmed the presence of four Pratylenchus species, P. penetrans, P. neglectus, P. curvicauda, and P. quasitereoides.

Mixed infestations of P. neglectus and P. penetrans in some sites confirmed previous findings that both nematodes have been found in cereal crops in Australia [11,22]. In our study, P. curvicauda and P. penetrans were identified as mixed populations at Darkan and Manjimup, and P. penetrans with P. quasitereoides at Kenmare and Duranillin, with P. neglectus also present at Duranillin. In a previous survey, the presence of both P. quasitereoides and P. neglectus in canola fields was reported [13]. In Australia, where prevailing Pratylenchus spp. in an infested broadacre paddock have different virulence and can together infect a wider host range of crops or can survive in volunteer weeds or winter crops, the effect of mixed infestations and the economic damage they cause can be more pronounced over time, as their populations can build up and persist in the main cereal growing season [15,17,18]. However, we did not identify P. curvicauda and P. quasitereoides in the same fields.

One area highlighted by this work is the complexity of identifying Pratylenchus nematodes at the species level. For example, P. curvicauda and P. penetrans were identified by DNA sequencing of clonally derived RLN sequences from the field near Darkan. This site had been tested previously for the presence of RLNs over several seasons; these nematode assessments had not identified the presence of P. curvicauda and P. penetrans. Based on nematode morphology, single nematode sequencing and results provided by the commercial test ‘PredictaB’ (based on amplification of DNA sequences conducted by the South Australia Research and Development Institute, Australia), P. quasitereoides and P. neglectus were previously identified as the dominant species. Such differences in identification highlight the difficulty of identifying nematodes in complex soil ecosystems, even with increasing access to molecular analyses. There are several potential explanations for the differing Pratylenchus species being identified at one site over time. First, the distribution of nematodes in any landscape is inherently patchy by nature and sampling methods aim to gain an overall picture of nematode levels and distribution, but samples are often collected at a single time point. Therefore, sampling may not reflect the complete spectrum of nematode species present. In addition, over time, seasonal and crop variations can affect the populations of RLNs that are present. Second, complexity in nematode identification may result from the use of different extraction methods at different times and by different laboratories. Further, the commercial test for P. quasitereoides was developed before P. curvicauda had been identified in the region, and some refinement of the commercial test for Pratylenchus species is now needed to ensure the accuracy of results delivered. Nevertheless, the identification of P. quasitereoides in fields at Katanning, Cancanning, Kenmare, and Duranillin, and P. curvicauda at Darkan and Manjimup, indicates that the incidences of the nematodes may well be more widespread in the grainbelt of WA than previously thought. Current WA sowing guides only report resistance ratings for cereals against P. neglectus or P. quasitereoides [6]. This is because they are the prevailing species in the WA grainbelt. However, in the future, the presence of P. curvicauda, should be considered.

The diversity in the sequences generated from the nematodes collected in our study clearly distinguished the species and confirmed their relatedness to other Pratylenchus spp. Similar diversity in the rDNA sequences of many organisms has been used to discover and, in some cases, re-classify species, for example, P. parazeae, identified from sugarcane infested with P. zeae in China [37]. In the last decade alone, a new species of Pratylenchus has been described, re-discovered in different areas, or re-classified each year, including P. quasitereoides and P. curvicauda in WA, and, in most cases, the evidence is backed with rDNA sequences [7,26,37,38,39,40,41,42,43]. These examples highlight the changing trend in combining morphological and morphometric measurements with molecular tools to characterise new and known Pratylenchus spp. to confirm species attribution. Most Pratylenchus spp. in Australia were identified by morphometric features, and, surprisingly the rDNA sequences we have reported for P. penetrans and P. neglectus are the first to be published from Australian populations, although the transcriptomes of P. thornei and P. zeae have been published [44,45]. It is probable that additional Pratylenchus spp. will be identified in Australia, including the areas previously surveyed in WA, making the development of tools such as those used in this study and the sequences generated an important resource for characterising new species.

5. Conclusions

The results presented here expand the current knowledge on the existence and distribution of the known RLN pests P. penetrans and P. neglectus and the lesser-known and studied P. curvicauda and P. quasitereoides in the grainbelt of WA. Advances in Next Generation Sequencing and reduction in sequencing costs will enable single nematode identification, reducing the complexities currently associated with nematode characterisation, particularly RLN identification, to the species level. This is needed to establish management strategies and specific programmes for breeding crops with high levels of tolerance or resistance to specific RLNs, because plant responses to infection by Pratylenchus spp. are species-dependent. In turn, such techniques will enable the development of management strategies appropriate for each RLN species to be improved, so that actual and potential crop losses from Pratylenchus spp. infestations can be reduced [9,46]. Furthermore, while several aspects of the biology of P. quasitereoides and P. curvicauda have been studied [3,6,7], additional studies are needed to better understand the ecology of both species.