Pratylenchus species in pastures in the South East Region of South Australia

In southern Australia, root lesion nematodes are recognised as important pests in field crops (particularly Pratylenchus neglectus and P. thornei; Vanstone et al. 2008) as well as being the most numerous group of plant parasitic nematodes in pastures (Stirling and Lodge 2005). To assess the risk posed to crops by pre-plant populations of P. neglectus and P. thornei, their population densities can be estimated by real-time polymerase chain reaction (RT-PCR) using DNA extracted from soil (Ophel-Keller et al. 2008). However, when these assays, and an assay being developed for P. penetrans, were applied to 70 pasture soils across southern mainland Australia, few detections were made (I. T. Riley and A. C. McKay, unpubl. data). To reconcile this with the data of Stirling and Lodge (2005), it must be assumed that other species of Pratylenchus predominate in the southern pastures. In order to test this assumption, nematodes were extracted from 16 pasture samples (Fig. 1 and Table 1) collected in the South East Region of South Australia (SA) for species determination by morphology (Loof 1991) and RT-PCR (with TaqMan probes specific for P. neglectus, P. penetrans and P. thornei). Nematodes were extracted from roots (separated from soil and lightly rinsed) in a misting cabinet and from soil by the Whitehead tray method (Hollaway et al. 2003). A representative selection of adults were fixed and mounted for morphological examination. DNA was extracted from 30 individual pratylenchid nematodes from five sites for partial sequencing of the D3 expansion region of the 26S ribosomal gene. Soil samples (~500 g) were submitted to SARDI Diagnostics for soil DNA extraction and assay (Hollaway et al. 2003; Ophel-Keller et al. 2008).

Table 1.  Description of 16 pastures in the South East Region, South Australia in which Pratylenchus population densities were estimated by direct extraction and P. neglectus, P. penetrans and P. thornei were assayed by RT-PCR

Ten of the 16 sites sampled were found to contain Pratylenchus spp. (Table 1). Application of the DNA tests for P. neglectus, P. penetrans and P. thornei indicated that only three samples contained any of these species (two sites with P. neglectus and one with P. thornei). Morphological examination of the Pratylenchus from the other sites identified morphotypes with affinities to P. penetrans (Sites 1, 4, 8 and 9) and P. crenatus (Site 12). Moderate to good D3 sequences (~600 bp) were obtained from eight specimens. Six sequences had 88–99% similarities (BLAST analysis; Altschul et al. 1990) with P. crenatus (4), P. neglectus (1) and P. zeae (1). However, except for the single P. neglectus (99% similarity), these matches were not sufficient to provide confident identification to species level.

The data reconfirm that Pratylenchus is common in high rainfall pastures, as found by Stirling and Lodge (2005). It appears that there is greater species diversity in these pastures than occurs in the northern cropping areas of SA (Riley and Wouts 2001). Consistent with the current results, Riley and Wouts found P. penetrans in the Murray Mallee Region of SA and P. crenatus in western Victoria, and Hollaway and co-workers (2008) found these species in a small proportion of cereal crops at similar latitudes across southern Victoria. However, the negative results for the DNA assay for P. penetrans points to the possibility of cryptic or undescribed species occurring in the study region. Alternatively, that the genetic diversity in P. penetrans was not fully captured by the assay given that P. penetrans has been suggested to be a species complex (De Luca et al. 2004). Either proposition is consistent with the failure of the BLAST analysis to provide convincing species indications. Pratylenchus, even with many species reproducing asexually, appears to be a genetically variable group (Andrés et al. 2000; De Luca et al. 2004). Clearly there is a need to better understand the diversity and impact of Pratylenchus in high rainfall pastures, based on genetic diversity determined by molecular as well as morphological means.