First report of ryegrass cyst nematode, Heterodera mani, in Tasmania, Australia

Cyst nematodes of the genus Heterodera are a major group of sedentary plant parasites causing a significant economic impact, restricting production and market access globally (Moens et al. 2018). The ryegrass cyst nematode Heterodera mani is in the Avenae group and is found predominantly in pastures and grasslands in Europe, California, and South Africa. It was first described by Mathews (1971) from Northern Ireland. Known hosts are grasses (family Poaceae), principally Lolium perenne (perennial ryegrass), but also Dactylis glomerata (cat grass) and Festuca pratensis (meadow fescue) (Subbotin et al. 2010). Mowat (1974) reported that H. mani causes negligible damage to the yield of L. perenne in pot trials; however, Maas & Brinkman (1982) determined that it may cause significant damage to spring and autumn-sown perennial ryegrass in field conditions. During a routine examination for potato cyst nematode from a farm near Mawbanna in north-west Tasmania, Australia, several pale to dark brown Heterodera cysts were extracted that were lemon shaped with the presence of a small vulval cone at the posterior end and a distinct neck. The J2 (n=20) stylet length ranged from 24-26 µm with round knobs deeply concave anteriorly, hyaline tail length was 37-42 µm, true tail length ranged from 59-68 µm and total body length varied from 526-559 µm. All the above characters match those described for H. mani (Subbotin et al. 2010). To verify this identification, DNA was extracted from five individual J2 juveniles from a single cyst using QIAamp DNA micro kit (Qiagen®), and two gene regions amplified: internal transcribed spacer region of ribosomal RNA (ITS-rRNA) with primer pair AB28 and TW81 and cytochrome oxidase 1 (CO1) with primer pair JB3 and JB5 (Bowles et al. 1992; Curran et al. 1994; Derycke et al. 2005). One PCR reaction contained 10 µM (1 µl each) of each primer, 12.5 µl of OneTaq® DNA Polymerase and 5 µl of DNA template with a final volume of 25 µl. PCR products were sent for purification and Sanger sequencing at Macrogen (Seoul, Rep. of Korea). All resulting sequences were trimmed, aligned, and analysed using Geneious Prime® 2022.0.1 (www.geneious.com). Five ITS sequences (accessions ON402852-ON402856) and five CO1 sequences (accessions ON402857-ON402861) were submitted to GenBank. These ITS sequences were very similar to each other and exhibited 99.16-100% similarity with that of H. mani isolate from Hamminkeln, Germany (AY148377) (Subbotin et al. 2018). The CO1