JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2019-041 e2019-41 | Vol. 51 New data on known species of Hirschmanniella and Pratylenchus (Rhabditida, Pratylenchidae) from Iran and South Africa Ebrahim Shokoohi1,*, Joaquín Abolafia2, Phatu William Mashela1 Abstract and Nafiseh Divsalar3 Hirschmanniella anchoryzae from Iran and Pratylenchus hippeastri 1Green Biotechnologies Research from South Africa were recovered during a survey of plant-para- Centre of Excellence, University sitic nematodes belonging to the family Pratylenchidae. Both spe- of Limpopo, Private Bag, X1106, cies were studied using morphological and molecular techniques. Sovenga, 0727, South Africa. Hirschmanniella anchoryzae is identified based on the flattened head, short stylet (19–22 µm), excretory pore position (anterior to pharyn- 2Departamento de Biología Animal, Biología Vegetal y Ecología, go-intestinal junction), spicule length (27–30 µm), and existence of an Universidad de Jaén, Campus ‘Las axial mucro at the tail end. Phylogenetic analysis using 28S rDNA Lagunillas’ s/n. 23071-Jaén, Spain. showed monophyly of Hirschmanniella which Iranian H. anchoryzae placed close to H. halophila (EU620464; EU620465). This result was 3Department of Plant Protection, supported by the principal component analysis of Hirschmanniella Faculty of Agriculture, Shahid species. SEM observation of the South African population of P. hip­ Bahonar University of Kerman, peastri showed the presence of two annuli in the lip region. Mor- Kerman, Iran. phometric characters resembled those of specimens earlier reported *E-mail: [email protected] from South Africa. Hierarchal cluster using morphometrical criteria showed that the Floridian (USA) and South African populations form This paper was edited by Zafar a group. However, the principal component analysis showed varia- Ahmad Handoo. tion within this species. The molecular study of P. hippeastri popula- Received for publication March 26, tions using 18S, ITS, 28S rDNA, and COI of mtDNA showed that all 2019. P. hippeastri cluster in one group and confirmed the identification of the species using both morphological and molecular techniques. In addition, the results indicated that South African populations group close to the USA populations. Illustrations of both species including light and scanning electron microscopy observations for P. hippeastri are provided. Key words Iran, Morphometric, mtDNA, Phylogeny, Root-lesion nematode, rDNA, South Africa. The root-lesion nematodes belong to the family Praty- Hirschmanniella have been reported from Iran (Majd lenchidae and cause severe damage on various crops Taheri et al., 2013). Those species have been stud- and yield reduction (Perry and Moens, 2013). The ied by morphological characters except for two un- genus Hirschmanniella has been established by Luc knowns which have been studied by morphological and Goodey (1964). To date, three known species of and molecular DNA barcoding using 28S rDNA (Majd Hirschmanniella, namely, H. anchoryzae (Ebsary and Taheri et al., 2013). Root-lesion nematodes, Praty­ Anderson, 1982), H. gracilis (De Man, 1880; Luc and lenchus (Filipjev, 1936), are after root-knot and cyst Goodey, 1964), and H. oryzae (Van Breda de Haan, nematodes listed as the third economically most im- 1902; Luc and Goodey, 1964), and with two unknown portant genus that adversely affects crop production © 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/ New data on known species of Hirschmanniella and Pratylenchus (Rhabditida, Pratylenchidae) from Iran and South Africa worldwide (Castillo and Vovlas, 2007; Jones et al., Statistical analysis 2013). Pratylenchus hippeastri, the amaryllis lesion nematode, was first described by Inserra et al. (2007) Principal component analysis and the correlation of from roots of Hippeastrum sp. in Florida (USA). In- morphometric data using the Pearson method was serra et al. (2007) distinguished the species due to done by XLSTAT (Addinsoft, 2007). In total, 11 morpho- individuals having slender bodies, flat, plain, and metric traits obtained from fixed nematodes including smooth head regions with two lip annuli (some spec- ‘de Man’s indices’ (a, b, b′, c, c′, and V), body length, imens with an incomplete third annulus) of which the stylet length, pharynx length, tail length, and position second lip annulus is thicker than the first, ellipsoidal of the excretory pore were used for PCA analysis of stylet knobs, rectangular and empty spermathecae Hirschmanniella. The species, namely, H. halophi­ with large round cavities, and conoid tails with bluntly la (Germany: Sturhan and Hallmann, 2010), H. loofi pointed termini, usually with ventral constrictions or (The Netherlands: Sher, 1968; Germany: Bert and subhemispherical and smoothened. Three years Geraert, 2000), H. kwazuna (South Africa: Van den later, the male of this species was described by De Berg et al., 2009), H. pomponiensis (USA: De Ley Luca et al. (2010) from bromeliads in Florida. Posteri- et al., 2007), Hirschmanniella sp. (Iran: Majd Taheri et orly, Gu et al. (2014) and Wang et al. (2016) reported al., 2013), H. gracilis (Iran: Jahanshahi Afshar et al., the species from the rhizosphere of apple in Japan 2006), H. oryzae (India: Sher, 1968; Tiawan: Lin, 1970), and China, respectively. H. mucronata (India, the Philippines and Thailand: Sher, The main objectives of the present study were to 1968; Taiwan: Chen et al., 2006; Cambodia: Khun et (i) to identify the populations of Hischmanniella and P. al., 2015), H. belli (USA: Sher, 1968), H. santarosae hippeastri using morphology, morphometrics, and mo- (USA: De Ley et al., 2007), and H. anchoryzae (Can- lecular DNA barcoding; (ii) to study of morphological ada: Ebsary and Anderson, 1982; Iran: Pourjam et al., variations among different P. hippeastri populations, 2000, present study) were studied. Regarding Praty­ and (iii) to determine the phylogenetic position of H. an­ lenchus species, hierarchical clustering analysis was choryzae from Iran using 28S rDNA and P. hippeastri done using morphometric data and the Rstudio, pv- from South Africa using rDNA and mtDNA genes. clust package (Suzuki and Shimodaira, 2015). To per- form this analysis, 63 specimens of P. hippeastri were used. The averaged populations used for comparative Materials and methods purposes, hierarchical clustering as well as their mor- Nematode materials phometric data are available in the databases from the USA, two populations from Florida; for 32 specimens, In 2015, Hirschmanniella specimens were collected respectively (Inserra et al., 2007; De Luca et al., 2012), from the rhizosphere of Mentha aquatica in Royan 16 specimens from Japan (Gu et al., 2014), 10 speci- (Mazandaran Province, Iran) and Pratylenchus spec- mens from China (Wang et al., 2016), and 5 specimens imens were collected from rhizosphere soil samples from South Africa as presented in the current study. of Cape Willow trees (Salix mucronata) growing on the In total, 14 morphometric traits obtained from fixed banks of the Mooiriver in Potchefstroom (North-West nematodes were used for identification purposes and Province, South Africa) and extracted from soil using hierarchical clustering analysis: ‘de Man’s indices’ (a, the Whitehead tray method (Whitehead and Hemmings, b, b′, c, c′, and V), body length, pharynx overlapping 1965). Nematodes were fixed with a hot 4% formal- (distance from pharyngeal-intestinal junction to the dehyde solution and transferred to anhydrous glycerin end of overlapping), stylet length, DGO, MB (middle of (De Grisse, 1969). Measurements were done using an metacorpus to anterior end), post vulva sac length, tail Olympus CH-2 and Omax light microscope (Nematol- length, and position of the phasmid (Castillo and Vov- ogy Laboratory; University of Limpopo) equipped with las, 2007). Data on the morphometric measurements an ocular micro- and/or a curvimeter and drawing tube. of the populations were analyzed using the bootstrap method. The same morphometric characters except b' were used for PCA analysis of P. hippeastri. Scanning electron microscopy (SEM) Specimens preserved in glycerine were selected for ob- DNA extraction, PCR, and phylogenetic servation under SEM according to Abolafia (2015). The analysis nematodes were hydrated in distilled water, dehydrated in a graded ethanol-acetone series, critical point dried, DNA extraction was done using the Chelex meth- coated with gold, and observed with a Zeiss Merlin mi- od (Straube and Juen, 2013). Five specimens of each croscope (5 kV) (Zeiss, Oberkochen, Germany). species were hand-picked with a fine tip needle and 2 JOURNAL OF NEMATOLOGY transferred to a 1.5 ml Eppendorf tube containing 20 µ l Jiménez, 1963; Braun and Loof, 1966) (AF442189; double distilled water. The nematodes in the tube were FJ717817; JQ917439) based on the study of Shokoohi crushed with the tip of a fine needle and vortexed. In (2013) were obtained for comparison of 18S, ITS, and total, 30 µL of 5% Chelex® 50 and 2 µL of proteinase 28S rDNA. Rotylenchus macrosoma (Dasgupta et al., K were added to each of the microcentrifuge tubes 1968) (KY992847) was used as the outgroup for the COI that contained the crushed nematodes and mixed. of mtDNA analyses based on the study of Van Megen These separate microcentrifuge tubes with the nema- et al. (2009). The ribosomal and mitochondrial DNA se- tode lysate were incubated at 56°C for 2 hr, and then quences were analyzed