Cyst forming species of the family Heteroderidae are significant plant parasitic nematodes exhibiting highly specialised interactions with their host plants (Moens et al. 2018). Of the eight genera of cyst nematode species, only two, Heterodera and Globodera, contain economically important species known to cause serious yield losses in important food crops such as potatoes, cereals, brassicas, carrots, and sugar beet (Moens et al. 2018). Initially, species of the genus Heterodera were separated into three species groups, based on their vulval cone structure, however, morphological and molecular analysis later supported their reclassification into eight species groups, namely, Afenestrata, Avenae, Cardiolata, Cyperi, Goettingiana, Humuli, Sacchari, and Schachtii (Handoo and Subbotin 2018; Subbotin et al. 2010).

Heterodera humuli Filipjev 1934, commonly known as the hop cyst nematode is a plant parasite frequently observed on hop (Humulus lupulus) (Subbotin et al. 2010). There are several other plants that have been reported as hosts for H. humuli including peas, beans, vetch, mustard, clover, nettle, and Cannabis (Bernard et al. 2022; De Grisse & Gillard 1963; Sen & Jensen 1969; Tanha Maafi et al. 2003; Winslow 1954). Early reports of H. humuli association with hop considered it to be a variety or strain of the beet cyst nematode, Heterodera schachtii (Triffitt 1929; Voigt 1894). However, Filipjev (1934) believed that it differed from other known species of the genus Heterodera and named it as Heterodera humuli. It belongs to the Humuli species group and is morphologically similar to Heterodera ripae Subbotin et al. (2003) and H. vallicola (Eroshenko et al. 2001; Handoo and Subbotin 2018), the most conspicuous difference being that H. humuli has a longer J2 hyaline and true tail region in comparison to H. ripae and a longer fenestral region along with longer males in comparison to H. vallicola (Subbotin et al. 2010). In addition, ITS rRNA and CO1 mtDNA gene sequences are efficient molecular markers for species differentiation in the Humuli group (Subbotin et al. 2010; Subbotin et al. 2022).

The occurrence of H. humuli has been reported from Europe, North America, Asia, Africa, and Australia (Subbotin et al. 2010). Early hop plantations in Australia were established using imported seed, with James Squires being credited for Australia’s first brewery, founded in Sydney, New South Wales (Darling et al. (early access); Dodds 2017). In 1822, vegetative hop planting material was brought from England to Australia and these plants were established in Tasmania by an experienced hop grower and emigrant, William Shoobridge. By the mid-19th century the hop industry was well established in Tasmania, and since then hop has been commercially cultivated in the southern states of Australia, both in Tasmania and Victoria (Pearce 1976). The distribution of H. humuli in Australia was previously thought to be limited to only two hop gardens in Tasmania where it was reported to occur at high population densities (Hay and Pethybridge 2003). Experimental studies have shown significant reduction in plant height, dry weight of shoots and cones per string along with severe manganese and nitrogen nutrient deficiencies (Hafez et al. 2010; Hay and Pethybridge 2003).

In May 2021, five soil samples each were collected from Rostrevor Hop Gardens and Buffalo River Farm in Merriang (Victoria) and 16 samples from Bushy Park (Tasmania), Australia. Cysts were extracted based on the flotation and sieving techniques developed by Cobb (1918). Eight soil samples from Victoria and all 16 from Tasmania were positive for the presence of cysts. To determine species identity, morphological and molecular analyses were undertaken. Vulval cone regions were excised and mounted in a modified Kaiser’s glycerine jelly (Dioni 2003) and compared to vulval cones from the literature and specimens from a population of H. humuli cysts obtained from Washington state, USA. Photographs of fenestra were taken using a ZEISS Axioscope light microscope and a mounted camera Axiocam 506 mono. Twenty second-stage juveniles (J2s) were also extracted from cysts for temporary and permanent mounts. Temporary mounts were examined using Leica DM6 B (Leica Biosystems, Germany). Measurements and images of morphologically distinguishing characteristics such as the stylet, tail, and body length were taken using a mounted camera Leica DMC 4500 (Leica Biosystems, Germany) operated using the Leica Application Suite X (LAS X) software v3.6.0.20104 (Leica Biosystems, Germany). Genomic DNA was extracted from 4 Victorian cysts and 30 Tasmanian cysts using QIAamp micro-DNA kit (Qiagen®). Three gene regions were amplified: the internal transcribed spacer region of rRNA (ITS rRNA), the large subunit ribosomal RNA (28S rRNA) and cytochrome oxidase 1 (CO1). Detailed protocols for PCR amplification, sanger sequencing, and analyses are as described by Jain et al. (2022). Phylogeny reconstruction was performed using members of the Humuli group of the genus Heterodera. Bayesian Inference (BI) analysis was performed using MrBayes 3.2.6 (Huelsenbeck and Ronquist 2001) to infer the posterior probability support values.

Cysts from Tasmania and Victoria were dark brown and generally lemon-shaped, although occasionally spherical. The vulval cone was bifenestrate with a broad vulval bridge (Fig. 1) and bullae were absent. Cysts were indistinguishable from those obtained from Washington, USA. Vermiform second-stage juveniles (Fig. 2a) were observed along with the presence of a tapering tail and an irregularly shaped terminus (Fig. 2c). A robust stylet (Fig. 2b) had large basal knobs flattened but slightly concave anteriorly. Morphometrics of cysts and second-stage juveniles of H. humuli from Australia are shown in Table 1. All the morphometrics were consistent with those described for H. humuli (Subbotin et al. 2010). Microscopic slides of vulval cone sections and J2 juveniles (slide accession numbers 8741–8781) were deposited in the Nematology section of the Australian National Insect Collection, CSIRO, Canberra, ACT, Australia.

Fig. 1
figure 1

Photomicrographs of vulval region of Heterodera humuli cysts from two different states of Australia. a Tasmania b Victoria. Scale a and b = 20 µm

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Fig. 2
figure 2

Photomicrographs of second-stage juvenile (J2s) of Heterodera humuli from Victoria, Australia. a: second-stage juvenile b: head at 100x magnification c: tail region at 100x magnification. Scale bar a = 50 µm, b and c = 30.8 µm

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Table 1 Morphometrics of vulval region of cysts and second-stage juveniles of Heterodera humuli from Australia (all measurements are given in µm)
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In phylogenetic analyses Australian isolates clustered with H. humuli isolates from other parts of the world (Fig. 3). Eighteen identical ITS rRNA gene sequences were generated from Tasmanian cysts of which a single sequence fragment of 920 bp was deposited in GenBank under the accession number OP236520. The Australian isolates shared 100%, 99.78%, and 99.87% similarity with sequences from the USA, Iran, Kyrgyzstan (GenBank accession numbers MT804361, AF498384 and MT804360 respectively) (Subbotin et al. 2022). Two identical sequences were generated from Victorian cysts of which a single sequence of 932 bp was deposited in GenBank under the accession number OP236521 sharing 100% similarity with sequences from USA and Iran (GenBank accession numbers MT804361 and AF498384 respectively) (Subbotin et al. 2022).

Fig. 3
figure 3

Phylogenetic relationship between ITS-rRNA gene sequences of species belonging to the Humuli group of the Heterodera genus as inferred from Bayesian analysis. New sequences generated in this study are highlighted in bold type. Posterior probability support and bootstrap support values for Bayesian Inference (BI) and Maximum Likelihood (ML) are given for appropriate clades, respectively. Values below 70% are not indicated. GenBank accession numbers are taken from Subbotin et al. (2022). A sequence of Heterodera avenae is taken as the outgroup

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Twelve identical 28S rRNA gene sequences were generated from Tasmanian cysts of which a single sequence fragment of 738 bp was deposited in GenBank under the accession number OP236514. This sequence shared 100% similarity with a sequence of H. humuli from Iran (GenBank accession number MG598808) (Fatemy et al. 2017).

Twenty-seven identical CO1 mtDNA gene sequences were generated from Tasmanian cysts of which a single sequence fragment of 393 bp was deposited in GenBank under the accession number OP236516 sharing 99.48% similarity with that of an H. humuli isolate from Russia and Belgium (GenBank accession numbers MW279122 and MT808370 respectively) (Subbotin et al. 2022). Two identical CO1 gene sequences were also generated from Victorian cysts of which a single sequence fragment was submitted in GenBank under the accession number OP236517, sharing 99.40% similarity with H. humuli isolates from Belgium, Germany and Russia (GenBank accession numbers MW279114, MW279118, MW279122) respectively (Subbotin et al. 2022). Analysis of CO1 gene sequences of populations from Tasmania and Victoria (Australia) deposited to GenBank during this study suggests that they belong to a unique CO1 haplotype (Fig. 4).

Fig. 4
figure 4

Phylogenetic relationship between CO1 haplotypes of species belonging to the Humuli group of the Heterodera genus as inferred from Bayesian analysis. New sequences generated in this study are highlighted in bold type. Posterior probability support and bootstrap support values for Bayesian Inference (BI) and Maximum Likelihood (ML) are given for appropriate clades, respectively. Values below 70% are not indicated. GenBank accession numbers are taken from Subbotin et al. (2022). A sequence of Heterodera avenae is taken as the outgroup

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H. humuli are sedentary endoparasites that feed within the roots of the host plant and can be found adhering to the infested soil attached to the rhizome (Mahaffee et al. 2009). The first hop farms in Australia were established using seed in New South Wales in 1803, thus it seems most likely that H. humuli was first introduced to Australia via unchecked rhizomes imported by William Shoobridge to Tasmania from England in 1822 (Dodds 2017). Although Australian populations of H. humuli represent a unique haplotype, it would seem reasonable to expect some level of genetic divergence between the European source and the Australian founder population across 200 years of isolation, especially in quickly evolving genes such as ITS and CO1. Multiple, subsequent introductions are also possible. An alternative scenario to a European origin is natural dispersal of this nematode without human activity with plants of the genus Urtica that can also host hop cyst nematode (Subbotin et al. 2010).

H. humuli can parasitise several other crops with a rising concern that populations can build over time in perennial crops such as hop, with no effective control measures, and subsequently, unknown and known weed varieties (such as nettles, vetch, etc.) can potentially add to the problem (Darling et al. 2020; De Grisse and Gillard 1963; Sen and Jensen 1969; Tanha Maafi et al. 2003). Heterodera humuli has also been reported from hemp (Cannabis sativa), although there are conflicting reports and thus its ability to parasitise these plants is considered variable (Bernard et al. 2022). Consequently, H. humuli could potentially pose a threat to the recently established and rapidly growing Australian medicinal cannabis industry, impacting access to a significant global market, estimated to be worth $80 billion by 2024. This is of particular significance to some Australian states, including Victoria, that have fast developing facilities for medicinal Cannabis cultivation (Bernard et al. 2022; McPartland 2000; MTPConnect and Deloitte Access Economics 2021). Therefore, further experimentation is required to understand the pathogenicity of H. humuli and its impact on susceptible crops, including different Cannabis varieties.