The ryegrass cyst nematode Heterodera mani was first described parasitising several grasses in Northern Ireland, with perennial ryegrass Lolium perenne (Poaceae, Poales) designated as the type-host (Mathews 1971). Other known hosts include the grasses Alopecurus geniculatus, Dactylis glomerata, Festuca arundinacea, Festuca pratensis, Festuca rubra commutate, Festuca rubra rubra, Glyceria fluitans, Lolium multiflorum and Vulpia bromoides (Mathews 1971; Mowat 1974). Mathews (1971) and Mowat (1974) determined that H. mani does not reproduce on barley, oats or wheat.

Heterodera mani is a member of the Heterodera avenae species-group, differing from the other members in having a J2 stage with robust and deeply concave stylet knobs, as well as combinations of morphological and morphometric features (Subbotin et al. 2010). The species is also reliably distinguishable from other species of Heterodera using the cytochrome c oxidase I (COI) and internal transcribed spacer (ITS) gene regions (Subbotin et al. 2003, 2010, 2018; Huston et al. 2022).

In 2022, H. mani was reported in Australia for the first time, on a farm in north-western Tasmania (Jain et al. 2022). This nematode had previously been reported throughout Europe, from California in the USA and from South Africa (Subbotin et al. 2010).

During an ongoing study of Australian populations of Heterodera australis, a population of Heterodera mani was detected on a broadacre cropping property near Esperance in Western Australia. The sample was collected from an area in a paddock commonly cropped with a cereal and oilseed rotation where annual ryegrass Lolium rigidum is prevalent as a weed. This finding represents the first record of H. mani in Western Australia.

Soil samples were collected from a cereal field on a farm 45 km north-east of Esperance, Western Australia, with a history of infestation with annual ryegrass L. rigidum. Soil samples were placed in plastic bags and transported to the Australian National Insect Collection, in Canberra, Australian Capital Territory. Heteroderid cysts were extracted from soil using Cobb’s sieving and decanting method (Cobb 1918). Collection sites were initially screened for heteroderid species composition through extraction of DNA from crushed whole cysts. Following preliminary detection of H. mani at one collection site, 19 additional cysts were processed for dual molecular and morphological study. Cysts were placed in a drop of water and cut in half using a scalpel blade. Interior contents of cysts were carefully removed using fine dissecting forceps and needles. Eggs and other tissues were transferred directly to tissue lysis buffer and stored frozen until DNA extraction. Vulval cones were mounted on slides in a modified Kaiser’s glycerin jelly (see Dioni 2003), and photographs were taken using a ZEISS Axiocam 506 mono camera mounted on a ZEISS Axioscope light microscope. Measurements of vulval plates were taken using ZEISS Blue imaging software (ZEISS, Germany). When available, a subset of second stage juveniles (J2s) recovered from individual cysts were mounted on temporary slides in water and photographed and measured as above. These and additional J2s recovered were then killed in hot water, preserved in 4% formalin, processed to glycerol using the slow method (Hooper 1986) and mounted in glycerol on wax-ring slides for lodgement in the Australian National Insect Collection. Voucher specimens of vulval cones and J2s are lodged under the accession numbers: 8782–8801.

Genomic DNA was extracted from specimens using a DNeasy Blood and Tissue kit (Qiagen®) following the manufacturer’s instructions. Two molecular markers were targeted: the mitochondrial cytochrome c oxidase I (COI) gene region and the large subunit ribosomal RNA (28S rRNA). The COI region was amplified using the forward primer JB3 (5′-TTT TTT GGG CAT CCT GAG GTT TAT-3′) (Bowles et al. 1992) and reverse primer JB5 (5′-AGC ACC TAA ACT TAA AAC ATA ATG AAA ATG-3′) (Derycke et al. 2005). The 28S region was amplified using the forward primer D2A (5′-ACA AGT ACC GTG AGG GAA AGT TG-3′) and reverse primer D2B (5′-TCG GAA GGA ACC AGC TAC TA′) (Nunn 1992). PCR and clean-up followed Huston et al. (2023) and was the same for both gene regions. PCR products were sent to the Biomolecular Resource Facility, Australian National University, Canberra, for Sanger sequencing and resultant reads were assembled and edited using Geneious Prime® v2022.1.1 (Biomatters). Newly generated sequences were aligned with those of other members of the Heterodera avenae species group using MUSCLE (Edgar 2004) as implemented in MEGA 11 (Tamura et al. 2021). Sequences of Heterodera glycines and Heterodera schachtii were used as outgroups. Differences between taxa were examined in MEGA 11 with Neighbour-joining analyses (Saitou and Nei 1987) using the number of differences method with inclusion of transitions and transversions and complete deletion of gaps and missing data; nodal support was estimated with 500 bootstrap replications. Phylogenetic trees and photographic images were edited and annotated in Adobe Illustrator CS6.

Sequence data was successfully generated for 15 cysts collected. Initial BLAST (Altschul et al. 1990) results of COI sequences indicated that all 15 cysts represented H. mani. The 15 newly generated COI sequences and seven 28S sequences of H. mani lacked intraspecific variation, thus only a single sequence of each gene was submitted to GenBank (COI: OQ918095; 28S: OQ918098). The COI sequences of H. mani from Western Australia did not differ (i.e., 0% divergence) from those from Tasmania, Australia or those from Germany or the USA. 28S sequence data was not available for H. mani prior to the present study. The newly generated 28S sequences of H. mani differed from those of Heterodera aucklandica by 3 base positions (bp), from Heterodera avenae by 2 bp, from Heterodera filipjevi by 6–7 bp, from Heterodera hordecalis by 2–11 bp, from Heterodera latipons by 14–15 bp, and from Heterodera pratensis by 2 bp. Neighbour-joining analysis of the COI dataset placed the Western Australian sequence in a clade with those of H. mani from other regions with high support (Fig. 1). Analysis of the 28S dataset (Fig. 2) resolved H. mani as sister to a clade comprising sequences of H. avenae and a single sequence of H. aucklandica, H. hordecalis and H. pratensis.

Fig. 1
figure 1

Phylogram from the neighbour-joining analysis of the COI mtDNA dataset. Sequence of Heterodera mani generated in this study shown in bold. Bootstrap support values are shown at the nodes. The scale bar indicates the number of base differences

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Fig. 2
figure 2

Phylogram from the neighbour-joining analysis of the 28S rDNA dataset. Sequence of Heterodera mani generated in this study shown in bold. Bootstrap support values are shown at the nodes. The scale bar indicates the number of base differences

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Morphological specimens were consistent with the concept of H. mani. Cysts were bifenestrate with conspicuous bullae and an underbridge was not conspicuous (Fig. 3). Morphometrics of the vulval plate (Table 1) were within the range reported for this species (see Subbotin et al. 2010). Morphometric data obtained from J2s (Table 1) were also consistent with previous reports for this species (Subbotin et al. 2010; Jain et al. 2022). Stylet knobs of J2s were well-developed and exhibited the deeply concave anterior faces (Fig. 3) characteristic of this life stage in this species (Subbotin et al. 2010).

Fig. 3
figure 3

Heterodera mani second stage juveniles (a-e) and vulval plate (f). (b) and (d) are enlargements of (a) and (c), respectively; (e) hyaline region of tail. Arrows indicating the deeply concave stylet knobs characteristic of H. mani. Scale bars = 20 μm

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Table 1 Morphometrics of vulval region and second stage juveniles of Heterodera mani from Western Australia. Measurements are presented in the form: mean ± standard deviation (range). Abbreviations: Morphometric indices (a-c, b’, c’) follow Hooper (1986)
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Huston et al. (2022) showed that 28S gene sequences were generally not sufficient to distinguish between several members of the H. avenae species group and the 28S sequences of H. mani generated here differ by as little as 2 bp from some H. avenae group members. However, the present results indicate that 28S sequences can reliably delineate H. mani from related species. This is similar to the situation regarding sequences of the internal transcribed spacer region (ITS; comprising ITS1-5.8S-ITS2). Although ITS sequences of H. mani differ by only 2 bp from some sequences of H. avenae, these sequences of H. mani were found to consistently form a monophyletic clade in phylogenetic analyses (Huston et al. 2022).

The present finding of H. mani in Western Australia, coupled with the recent first detection of this species in Tasmania (Jain et al. 2022) suggests H. mani is likely widespread throughout Australia. Grass species of the genus Lolium are thought to be hosts for H. australis (McLeod 1992) but considering the subtle morphological differences between this species and H. mani (see Subbotin et al. 2010), it is possible that at least some reports of cyst nematodes from L. rigidum were H. mani rather than H. australis. Heterodera mani has been reported from Lolium multiflorum and Lolium perenne (Mathews 1971; Mowat 1974), but we are not aware of any previous records from L. rigidum. Because Heterodera mani is not known to parasitise cereals or oilseeds, it seems likely that L. rigidum is the host for the H. mani population discovered. However, confirmation of L. rigidum as a host will require observation of white females on the roots of this plant. Heterodera mani has probably been present in Australia for many years but had not been recognised as distinct from H. australis until recent molecular work.

Annual ryegrass is the most common ryegrass weed present in Australian dryland cropping systems (Jones et al. 2000). A cultivar of this species, cv. Wimmera, is sometimes sown in dryland areas for grazing purposes where other ryegrass species do not persist due to the Mediterranean climate (hot, dry summers) or where greater tolerance of soil salinity is required. Ryegrasses are typically inhabited by endophytic fungi which facilitate production of alkaloids that can have negative impacts on grazing insects and other animals (Popay et al. 2021; Karpyn Esqueda et al. 2017). Although endophytes have been reported to provide some protection against a variety of insect pests, including root-feeding species (Karpyn Esqueda et al. 2017; Popay et al. 2021), data on deterrence of plant parasitic nematodes has largely been inconclusive (Cook et al. 1991; Bell et al. 2009; Stewart et al. 1993; Eerens et al. 1998; Panaccione et al. 2006). Evaluating the potential for endophytic fungal-conferred protection against H. mani in ryegrasses may be warranted. The damage cyst nematodes cause cereal crops suggests investigations into the capacity of endophytic fungi to confer protection against these pests could be invaluable.