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BACKGROUND: The nematode phylum includes many species key to soil food webs with trophic behaviours extending from feeding on microbes to macrofauna and plant roots. Among these, the plant parasitic cyst nematodes retain their eggs in protective cysts prolonging their survival under harsh conditions. These nematodes, including those from the genus Heterodera, cause significant economic losses in agricultural systems. Understanding of nematode diversity and ecology has expanded through application of genomic research, however, for Heterodera species there are very few available whole genome sequences. Sequencing and assembling Heterodera genomes is challenging due to various technical limitations imposed by the biology of Heterodera. Overcoming these limitations is essential for comprehensive insights into Heterodera parasitic interactions with plants, population studies, and for Australian biosecurity implications. RESULTS: We hereby present draft genomes of six species of which Heterodera australis, H. humuli, H. mani and H. trifolii are presently recorded in Australia and two species, H. avenae and H. filipjevi, currently absent from Australia. The draft genomes were sequenced from genomic DNA isolated from 50 cysts each using an Illumina NovaSeq short read sequencing platform. The data revealed disparity in sequencing yield between species. What was previously identified as H. avenae in Australia using morphological traits is now confirmed as H. australis and may have consequences for wheat breeding programs in Australia that are breeding for resistance to H. avenae. A multigene phylogeny placed the sequenced species into taxonomic phylogenetic perspective. Genomic comparisons within the Avenae species group revealed orthologous gene clusters within the species, emphasising the shared and unique features of the group. The data also revealed the presence of a Wolbachia species, a putative bacterial endosymbiont from Heterodera humuli short read sequencing data. CONCLUSION: Genomic research holds immense significance for agriculture, for understanding pest species diversity and the development of effective management strategies. This study provides insight into Heterodera, cyst nematode genomics and the associated symbionts and this work will serve as a baseline for further genomic analyses in this economically important nematode group.
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Genoma de los Helmintos , Genómica , Suelo , Tylenchoidea , Animales , Filogenia , Suelo/parasitología , Tylenchoidea/genética , Tylenchoidea/clasificación , Tylenchoidea/aislamiento & purificaciónRESUMEN
The potato cyst nematode Globodera rostochiensis originates from the Andean Mountain region in South America and has unintentionally been introduced to all inhabited continents. Several studies have examined the population genetic structure of this pest in various countries by using microsatellite markers. However, merging microsatellite data produced from different laboratories is challenging and can introduce uncertainty when interpreting the results. To overcome this challenge and to explore invasion routes of this pest, we have genotyped 22 G. rostochiensis populations from all continents. Within populations, the highest genetic diversity was observed in the South American populations, the European populations showed an intermediate level of genetic diversity and the remaining populations were the less diverse. This confirmed pre-existing knowledge such as a first introduction event from South America to Europe, but the less diverse populations could originate either from South America or from Europe. At the continental scale, STRUCTURE genetic clustering output indicated that North America and Asia have experienced at least two introduction events. Comparing different evolutionary scenarios, the Approximate Bayesian Computation analysis showed that Europe served as a secondary distribution centre for the invasion of G. rostochiensis into all other continents (North America, Africa, Asia and Oceania).
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Variación Genética , Repeticiones de Microsatélite , Solanum tuberosum , Tylenchoidea , Animales , Europa (Continente) , Solanum tuberosum/parasitología , Tylenchoidea/genética , Especies Introducidas , Teorema de Bayes , Genotipo , Enfermedades de las Plantas/parasitología , Genética de Población , América del SurRESUMEN
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 sequences exhibited 98.96-100% similarity with that of H. mani isolate from Washington, USA (MG523097) (Subbotin et al. 2003). Obtained sequences were mapped to reference sequences downloaded from NCBI GenBank and maximum likelihood phylogenetic trees were calculated. Due to the lack of further living nematode material, pot experiments were not performed. Such experiments are not feasible in Tasmania currently and transfer of live nematode material to the Australian mainland presents logistic and legal issues. However, morphological and molecular evidence for species determination of H. mani was unequivocal and contributes to the list of cyst nematode species present in Australia. This is the first detection of H. mani in Australia and is a range extension of the species from North America, Africa, and Europe to Australia. The nematode may cause damage to perennial ryegrass in Australia, however, impact on yield still needs to be investigated.
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The scope of this paper is limited to the taxonomy, detection, and reliable morphological and molecular identification of the potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis. It describes the nomenclature, hosts, life cycle, pathotypes, and symptoms of the two species. It also provides detailed instructions for soil sampling and extraction of cysts from soil. The primary focus of the paper is the presentation of accurate and effective methods to identify the two principal PCN species.
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Potato cyst nematodes (PCN) are damaging soilborne quarantine pests of potato in many parts of the world. There are two recognized species, Globodera pallida and G. rostochiensis, with only the latter species-the golden cyst nematode-present in Australia. PCN was first discovered in Australia in 1986 in Western Australia, where it was subsequently eradicated and area freedom for market access was reinstated. In Victoria, PCN was first detected in 1991 east of Melbourne. Since then, it has been found in a small number of localized regions to the south and east. Strict quarantine controls have been in place since each new detection. It has previously been speculated that there were multiple separate introductions of PCN into Victoria. Our study utilized a historic (years 2001 to 2014) PCN cyst reference collection to examine genetic variability of Victorian PCN populations to investigate potential historical origins and subsequent changes in the populations that might inform patterns of spread. DNA was extracted from single larvae dissected from eggs within cysts and screened using nine previously described polymorphic microsatellite markers in two multiplex polymerase chain reaction assays. Sequence variation of the internal transcribed spacer region of the DNA was also assessed and compared with previously published data. A hierarchical sampling strategy was used, comparing variability of larvae within cysts, within paddocks, and between local regions. This sampling revealed very little differentiation between Victorian populations, which share the same microsatellite allelic variation, with differences between local regions probably reflecting changes in allele frequencies over time. Our molecular assessment supports a probable single localized introduction into Victoria followed by limited spread to nearby areas. The Australian PCN examined appear genetically distinct from populations previously sampled worldwide; thus, any new exotic incursions, potentially bringing in additional PCN pathotypes, should be easily differentiated from existing established local PCN populations.