JOURNAL of NEMATOLOGY Serendipitous Identification Of
Total Page：16
File Type：pdf, Size：1020Kb
JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2019-046 e2019-46 | Vol. 51 Serendipitous identification of Pratylenchus curvicauda from the grainbelt of Western Australia Farhana Begum1, John Fosu-Nyarko1*, Shashi Sharma2, Bill Macleod2, Abstract 2 1 Sarah Collins and Michael G. K. Jones A Pratylenchus species identified during a survey of Pratylenchus 1Plant Biotechnology Research quasitereoides incidence at four locations of the grainbelt of Western Group, WA State Agricultural Australia is described. Morphological and morphometric features Biotechnology Centre, College of indicated the previously undescribed morphotypes in nematode Science, Health, Engineering and mixtures encountered were conspecific to P. curvicauda, and were Education, Murdoch University, clearly distinguishable from nine common Pratylenchus spp. Typical Perth, WA, 6150, Australia. features of P. curvicauda were its body length (415–540 µm), which 2Department of Primary Industries was curved to a c-shaped with a maximum body diameter of 20 µm, and Regional Development, South and the nature of its tail; 34 µm long, 2.8 µm wide at the anus and a Perth, WA, 6151, Australia. typical ventrally arcuate with a round terminus. Sequenced for the first *E-mail: J.Fosu-Nyarko@murdoch. time, the sequences of the partial 18S-ITS1-5.8S-ITS2-partial 28S (80 edu.au; [email protected] clones, 14 individual nematodes) and the 28S-D3 (17 clones) regions of the rDNA of P. curvicauda had overall mean distances of 0.013 and This paper was edited by Zafar 0.085, respectively. Phylogenetic analyses with sequences of both Ahmad Handoo. segments of the rDNA clearly showed the P. curvicauda isolates as Received for publication February monophyletic, distinct from ca 40 Pratylenchus species. Notably, it 17, 2019. was distinct from Pratylenchus species present in Australia including P. quasitereoides and a Western Australia isolate of P. thornei. Further research into the biology of P. curvicauda is needed to facilitate development of strategies for its management, if it is an important pest. Key words Internal transcribed spacer (ITS), Molecular phylogeny, Morphology, Nematode classification, Pratylenchus spp., Pratylenchus curvicau da, Root Lesion Nematodes, Scanning Electron Micros- copy (SEM), Taxonomy. Root lesion nematodes (Pratylenchus spp.) are one 2014) (previously P. teres Khan and Singh, 1974) of three economically important plant-parasitic nem- (Hodda and Nobbs, 2008; Jones and Fosu-Nyarko, atode pests of many crops worldwide (Jones et al., 2014). They are often present as mixed populations 2013). The over 77 species currently described are in infested soils. Seven of these species are known polyphagous and cause yield losses of major grain to be present in the southern and western grainbelts crops including wheat and barley and even more of Australia. These are P. neglectus (Rensch, 1924; significant damage under drought conditions (Taylor Filipjev and Stekhoven, 1941), P. thornei (Sher and et al., 1999; Castillo and Vovlas, 2007). In Australia, Allen, 1953), P. quasitereoides, P. penetrans (Cobb, about 12 economically important Pratylenchus spe- 1917; Filipjev and Stekhoven, 1941), P. zeae (Graham, cies have been described and these include the re- 1951), P. brachyurus (Godfrey, 1929; Filipjev and cently described P. quasitereoides (Hodda et al., Stekhoven, 1941), and P. scribneri Steiner in Sherbakoff © 2019 Authors. This is an Open Access article licensed under the Creative 1 Commons CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0/ Serendipitous identification of Pratylenchus curvicauda from the grainbelt of Western Australia and Stanley (1943). Whereas P. neglectus is the most species of Pratylenchus (Waeyenberge et al., 2009; common in this region, P. thornei is the main pest of Palomares-Rius et al., 2010). Also, the nucleotide se- wheat and barley in the northern grainbelt of the con- quences of the D2 to D3 regions of the large subu- tinent (Vanstone et al., 2008). A recent survey on the nit ribosomal genes (28S), which is thought to evolve distribution of Pratylenchus species in 360 paddocks slowly, have been used to examine the evolutionary in Western Australia indicated P. neglectus was the relationships among species of many genera includ- most prevalent (48% incidence) with 32% of the ing Pratylenchus (Al-Banna et al., 1997). paddocks surveyed estimated to have mixed infes- Pratylenchus teres, which was previously con- tations, usually with P. quasitereoides (Collins et al., sidered to be endemic to Western Australia, has re- 2017). Knowledge of plant-parasitic nematode spe- cently been re-described as Pratylenchus quasiter- cies present in any infested field is essential because eoides using traditional methods and sequences of their management could be undermined by a shift to the 28S-D3 region of the rDNA (Hodda et al., 2014). a predominance of species for which crops grown The latter species is reported to occur with P. ne- are not resistant (Jones and Fosu-Nyarko, 2014). This glectus in Katanning, Western Australia. In a recent shift emphasizes the need for accurate identification survey to study the prevalence of P. quasitereoides of the species present in an infested field for success- in four wheat and barley fields of the grainbelt of ful management of infestations. Western Australia, initial assessment of the morpho- Since the discovery and first description ofP. metrics of isolated nematodes indicated the features curvicauda in metropolitan Perth, Western Australia, of some nematodes did not conform to those of Pra- in 1991 (Siddiqi et al., 1991), there has been no fur- tylenchus spp. commonly reported. This study was, ther study on the nematode in Australia. As such, its therefore, undertaken to describe the species which pest status and existence in the grainbelt of Australia was prominent in the mixed population of Pratylen- is not known, and no specific management strate- chus species found: we report the use of morpho- gy is in place for this potential pest of wheat, barley, metric measurements, morphological features, and and other important crops. There is extensive ongo- genetic variation within the partial 18S-ITS1-5.8S- ing research on the identification and management ITS2-partial 28S and 28S-D3 regions of the rDNA to of root lesion nematodes in Western Australia, but characterise P. curvicauda and to distinguish it from none currently includes P. curvicauda (Collins et al., other Pratylenchus species including those com- 2015, 2017). One possible reason is the overlapping monly found in Australia. This exercise is an impor- morphological and morphometric features of root tant step in assessing the pest status and economic lesion nematodes that often make it difficult to dis- importance of the nematode to the grains industry in tinguish between species accurately (Castillo and Western Australia. Vovlas, 2007). Hence, molecular approaches and phylogenetic analyses tools have been combined to Materials and methods distinguish species, as together, they offer greater accuracy and reproducibility. These tools are also Nematode population adaptable for nematode diagnostics, as long as the original specimens used as standards were identi- Soil samples containing a mixture of root lesion fied correctly using traditional methods (Al-Banna et nematodes including P. curvicauda were obtained al., 1997; Subbotin et al., 2008; De Luca et al., 2011). from four different locations in the wheatbelt re- As for many other organisms, the genes encoding gion of Western Australia: Pingelly (32°32′2.4″S, the ribosomal RNA subunits have proved to be useful 117°5′9.6″E), Williams (33°2′0″S, 116°53′0″E), Ar- in taxonomic studies of nematodes. These genom- thur River (33°20′19″S, 117°2′4″E), and Katanning ic regions vary in their rate of evolution depending (33°41′27″S, 117°33′19″E), with the help of staff at on whether they encode functional products or not. the Plant Pathology Section, Nematology Division of They include those of the ribosomal small subunit the Department of Primary Industries and Regional genes which can be extremely conserved, or the Development, Western Australia (Fig. 1). Nematodes non-coding internal transcribed spacer (ITS) regions were extracted from the soils using a misting appa- which are much more variable between species of ratus described by Tan et al. (2013). A pure culture of the same genera (Mckeand, 1998). Sequencing of the P. thornei maintained on carrot disks at 23°C in our ITS regions has revealed species-specific variations, laboratory at the time of this experiment was used as which can be used as diagnostic markers, so ena- a control during the morphological characterization. bling accurate identification of species and studies The partial 18S-ITS1-5.8S-ITS2-partial 28S and 28S- on the phylogenetic relationships between and within D3 expansion sequences of the P. thornei were also 2 JOURNAL OF NEMATOLOGY Figure 1: Locations of the four wheat/barley paddocks in the Western Australian grainbelt where P. curvicauda samples were isolated. The four sites are circled; Pingelly, Williams, Arthur River, and Katanning (https://maps-australia.com/south-western-australia-map). sequenced for the first time and used to differentiate Nematode specimens from Pingelly with typical P. the P. curvicauda reported in this study. curvicauda features were fixed in 4% formaldehyde solution and sent to an expert taxonomist in the UK Morphological studies and morphometric for further characterization. The detailed morpho- metric measurements obtained were compared with measurements those taken in Australia. Initial identification of the P. curvicauda was carried out using two important morphological features: the Preparation of nematode samples for position of the vulva and the shape of the tail. The scanning electron microscopy morphology of individual specimens was examined and photographed using a compound microscope Scanning electron microscopy (SEM) was used to (Olympus BX51). Before the examination, single nem- further characterize P. curvicauda. To do this, sin- atodes were hand-picked using a fine feather and gle nematodes were fixed in 3% glutaraldehyde in placed in a drop of water on a glass slide.