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78Russian Journal of Nematology, 2018, 26 (1), 29 - 41
Morphological variation and molecular study of the root lesion nematode Pratylenchus thornei
Nafiseh Divsalar1, Ebrahim Shokoohi2, Akbar Hoseinipour1, Hendrika Fourie3 and
Esmat Mahdikhani Moqaddam4
'Department of Plant Protection, College of Agriculture, Shahid Bahonar University of Kerman, 7616-9133, Kerman, Iran, 2Green Biotechnologies Research Centre of Excellence, University of Limpopo, Private Bag X1106, 0727, Sovenga, South Africa 3Unit for Environmental Sciences and Management, North West University, 2520, Potchefstroom, South Africa, ^Department of Plant Protection, College of Agriculture, Ferdowsi University of Mashhad, 91775-1163, Mashhad, Iran
e-mail: [email protected]
Accepted for publication 22 November 2017
Summary. During a survey of plant-parasitic nematodes in the northern part of Iran, six populations of Pratylenchus thornei were studied by means of morphological and molecular characterisation. The results showed that body length has a highly significant correlation with all morphometric data indexed in the measurement table, except for the c' index and the dorsal pharyngeal gland opening. However, the highest correlation was detected between body length and 'a' index (r = 0.805). Furthermore, stylet length had a significant correlation with body length (r = 0.511), tail length (r = 0.300) and V (r = 0.324). On the study of ratios, the results revealed that b and a are more reliable to evaluate the species. A molecular study of the P. thornei populations using 28S rDNA showed close relationships of the Iranian and USA, Morocco, Moldova, Spain and United Kingdom populations extracted from NCBI Genbank. The results indicated monophyly of P. thornei. Measurement table, illustrations and phylogenetic analysis for this species are given.
Key words: Iran, morphometric, phylogeny, 28S rDNA.
Root-lesion nematodes belong to the genus Pratylenchus Filipjev, 1936 and, after root-knot and cyst nematodes, are listed as the third economically most important nematode pest genus that adversely affect crop production worldwide (Castillo & Vovlas, 2007; Jones et al., 2013). These nematodes are distributed worldwide across different climatic zones and environments (Loof, 1991; De Goede & Bongers, 1998). This genus comprises 70 nominal species (De Luca et al., 2011), with Pratylenchus thornei Sher & Allen, 1953 being one of the most frequently reported species (Castillo & Vovlas, 2007; Yan et al., 2008; Smiley et al., 2014). Members of the genus Pratylenchus migrate within the root tissues and feed on the cortical parenchyma (Castillo & Vovlas, 2007) and may multiply to a very large number (10000-35000 individuals (g root)-1) (Loof, 1991). Thus, they cause severe damage and large necrotic lesions on roots, tubers or other below-ground parts of plants (Castillo & Vovlas, 2007; Jones et al., 2013). In addition, the genus Pratylenchus has been shown to have morphological variation (Loof, 1991; Ryss, 2002; Fayazi et al., 2012).
Accurate identification of Pratylenchus spp. is challenging because of limited diagnostic characters being available and the occurrence of intraspecific variability of morphological characteristics (Castillo & Vovlas, 2007). For example, lip region annuli of P. hippeastri was originally described with two incisures (Inserra et al., 2007); however, later it was reported with two to three (De Luca et al., 2010; Wang et al., 2015). Hence, new tools for species identification recognition is needed. Furthermore, the occurrence of cryptic species and divergence between populations have been listed due to the discovery of new species using phylogenetic and molecular analysis (De Luca et al., 2011). During the last decades, new methods such as molecular and phylogenetic studies have been provided for identification as well as for distinguishing between species of Pratylenchus (Subbotin et al., 2008; De Luca et al., 2011). Among the ribosomal DNA markers, the 28S rDNA gene has been mainly used to separate species of Pratylenchus (Carta et al., 2001; De Luca et al., 2004, 2011; Subbotin et al., 2005, 2008). Pratylenchus thornei can, for example, be easily distinguished from other closely related
© Russian Society of Nematologists, 2018; doi:10.24411/0869-6918-2018-10002
species using the partial D2-D3 segments of the 28S rDNA gene (Al-Banna et al., 2004).
The main objectives of the present study were: i) to identify accurately six different populations of P. thornei using partial D2-D3 segments of the 28S rDNA gene; ii) to study the morphological variation that occurs among the different P. thornei populations; and iii) to study the phylogenetic relationships of the studied P. thornei populations and related species using 28S rDNA.
MATERIAL AND METHODS
Nematode materials. In October 2015, Pratylenchus specimens were extracted from rhizosphere soil samples of wheat, blackberry, wild and herbaceous plants from six localities (Table 1) within the Iranian provinces of Golestan, Guilan and Mazandaran using the Whitehead tray method (Whitehead & Hemming, 1965). Extracted nematode individuals were fixed with a hot 4% formaldehyde solution and transferred to anhydrous glycerin using the method of De Grisse (1969). Measurements of different structures and organs of the fixed nematode individuals were done with an Olympus CH-2 light microscope equipped with an ocular micro- and/or a curvimeter and drawing tube.
DNA extraxtion, PCR and phylogenetic analysis. DNA extraction was done using the Chelex method (Holovachov et al., 2013). Twelve Pratylenchus specimens from each of the six localities sampled were hand-picked with a fine tip needle and transferred to a 1.5 ml tube containing 20 ^l double distilled water. The nematodes in the tube were crushed with the tip of a fine needle and vortexed. Twenty microliters of 5% Chelex® 100 and 2 ^l of proteinase K were added to each of the microcentrifuge tubes that contained the nematode specimens of the different localities, and mixed well with the crushed nematode material. These separate microcentrifuge tubes with the nematode solutions were incubated at 56°C for 2 h. Then, the solutions were incubated at 95°C for 10 min to deactivate the proteinase K (Holovachov et al., 2013). The supernatant was then extracted from each of the tubes and stored at -20°C. Following this step, the forward and reverse primers, D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B (5' -TCGGAAGGAACCAGCTACTA-3'), respectively (Subbotin et al., 2006), were used in the PCR reactions for partial amplification of the 28S rDNA region. PCR was conducted with 8 ^l of the DNA-extract from the nematode specimens of each tube added to 12.5 ^l of 2x PCR Master Mix Red (Pishgam Company), 1 ^l of each primers (10
pmol ^l-1) and ddH2O to comprise a final volume of 25 pl The amplification was processed using an Eppendorf master cycler gradient (Eppendorf, Hamburg, Germany), with the following programme: initial denaturation for 3 min at 94°C, 37 cycles of denaturation for 45 s at 94°C, extension for 45 s at 56°C and annealing for 1 min at 72°C, and finally an extension step of 6 min at 72°C followed by a temperature on hold at 4°C. After DNA amplification, 4 ^l of products from each tube were loaded on a 1% agarose gel in TBE buffer (40 mM Tris, 40 mM boric acid, and 1 mM EDTA) for evaluation of the DNA bands. The bands were stained with 50 mM ethidium bromide and visualised and photographed on a UV transilluminator. The amplicons of each population was next stored at -20°C. Finally, the PCR products were purified for sequencing with the primers used for amplification by the Pishgam Corporation. Available sequences for other Pratylenchus spp. were obtained from NCBI GenBank for comparison to the sampled species obtained during this study. Also, an outgroup, Zygotylenchus guevarai (Tobar Jiménez, 1963) Braun & Loof, 1966 (JQ917439) based on a study by Shokoohi (2013), was obtained for comparison. The ribosomal LSU sequences were analysed and aligned using BioEdit (Hall, 1999). The length of the alignment was 837 bp and the base substitution model was evaluated using jModeltest 0.1.1 (Posada, 2008). Phylogenetic trees were elaborated using the Bayesian inference method as implemented in the program Mr Bayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The analysis HKY + G model was selected using jModeltest 0.1.1 (Posada, 2008). The selected model was initiated with a random starting tree and run with the Markov chain Monte Carlo (MCMC) for 106 generations. The Bayesian tree was ultimately visualised using the TreeView program (Page, 1996). Genetic pairwise distance was estimated using options implemented in Mega V.7.0.14 (Kumar et al., 2016). The partial D2-D3 segment of 28S rDNA sequences of P. thornei obtained from the sampled populations as a result of this study were deposited in GenBank under accession numbers: KX 258735 (province of Golestan, Gorgan region), KX 258736 (province of Golestan, Ziarat region), KX 258737 (province of Guilan, Roodsar region), KX 258738 (province of Guilan, Langerood region), KX 258739 (province of Mazandaran, Nowshahr region) and KX 258740 (province of Mazandaran, Sari region) (Table 2).
Statistical analysis. Correlation of morphometric data was done using the two-tailed Pearson correlation. All analyses were done using the
Fig. 1. Pratylenchus thornei Sher & Allen, 1953. A: Neck. B: Female reproductive system. C: Anterior end. D: Entire female. E, F: Female posterior end. G: Lateral field. (Population code: IR5, Mazandaran, Nowshahr region, collected from wheat rhizosphere).
SPSS 13 (SPSS Inc., 2005) statistical package. Hierarchical Clustering analysis was done by using morphometric data and R studio, pvclust package (Suzuki & Shimodaira, 2015). According to the analyses, 19 specimens obtained as a result of this study belonged to P. thornei. The populations used for comparative purposes, hierarchical clustering as well as their morphometric data are available in the databases from USA (Sher & Allen, 1953), Russia (Ryss, 1988), Canada (Yu, 1997), Germany (Loof, 1960), Italy (D'Errico, 1970) and India (Khan & Singh, 1975). In addition, ten specimens from Iran and those previously reported (Pouijam et al., 1999; Mirzaipour et al., 2016) were analysed. The eleven morphometric traits used for identification purposes and hierarchical clustering analysis included: de Man's indices (a, b, c, c' and V), body length, pharynx length (distance from anterior to pharynx-intestinal junction), stylet length, post vulva sac length, tail length and position of the secretory-excretory pore from the anterior end (de Man, 1888). The data for these characters were obtained from the fixed specimens. Data on the morphometric measurements of the populations were analysed using the bootstrap method.
RESULTS
Pratylenchus thornei Sher & Allen, 1953 (Figs 1-3)
Material examined. Eight females from Nowshahr, in good state of preservation.
Measurements. See Table 1.
Females. Body 420-680 ^m long, slightly curved after fixation, cuticle finely annulated with 1.0-1.5 ^m wide at midbody. Maximum body diameter 1520 ^m. Lip region round to flat, with slightly depression, bearing three annuli, continuous with the body. Lateral field with four lines, two outer lines crenated, occupying about 30% of midbody diameter. Stylet length 14.0-17.6 ^m; basal knobs usually rounded and flatted. Dorsal pharyngeal gland opening (DGO) at 2-3 ^m posterior to stylet base. Median bulb oval to rounded, nerve ring located just after bulb, at 48-57% of the neck. Secretory-excretory pore at 70-100 ^m from anterior body, at 68-76% of the neck. Hemizonid one annuli anterior to secretory-excretory pore. Pharyngeal glands overlapped with intestine about 30-57 ^m. Pharynx 61-99 ^m long, body length about 5.5-9.9 times pharynx length. Ovary not reached to pharyngeal glands. Oocytes in one or two rows. Vulva occupied 71-82% of body length. Spermatheca not visible. Post-vulval uterine sac 20-
31 ^m long, vulva with a transverse slit. Tail terminus shows variation, round, truncate, smooth and in two populations slightly spatulate. Tail 19-32 ^m long, about 2.0-2.8 times anal body diameter. Phasmid located at posterior half of the tail, 45-57% of tail length.
Male. Not found.
Other material examined. The specimens studied were similar with those fixed and measured from the provinces of Gorgan, Guilan and Mazandaran. However, they differ in body length (427-688 vs 440-790 ^m) and 'a' index (5.3-8.9 vs 6-9.9). Also, the stylet length of the specimens from the Gorgan population was smaller (14-15 ^m) than those from the other populations (Table1).
Remarks. This species is distributed worldwide and reported by many scientists (Sher & Allen, 1953; Loof, 1960; D'Errico, 1970; Khan & Singh, 1975; Ryss, 1988; Yu, 1997; Pourjam et al., 1999; Castillo & Vovlas, 2007; Fayazi et al., 2012; Mirzaipour et al., 2016). All the populations studied fit well with those studied by Sher & Allen (1953); however, they differ in stylet length (14-17 vs 17-19 ^m). Compared to the Dutch population studied by Loof (1960), a shorter 'c' index (16-25 vs 18-29) was recorded. In comparison with the Italian population studied by D'Errico (1970), specimens of the Iranian populations studied differed in 'b' value (5-9 vs 4.8-7.8). Compared with the specimens studied by Khan & Singh (1975), the studied populations had a shorter 'C' value (11-21 vs 18-29). Ryss (1988) studied a population of this species having a longer stylet compared to the studied Iranian populations (14-17 vs 16-18 ^m) and compared to specimens studied by Yu (1997), these Iranian populations differed in tail shape (truncate and slightly spatulate vs round, conical round, truncate). In comparison with the material studied by Castillo & Vovlas (2007), the Iranian populations studied differed in post-vulva uterine sac length (2031 vs 33-46 ^m) and 'b' index (5.5-9.9 vs 4.7-8.7). Compared with Fayazi et al. (2012) there is no significant differences. They have indicated morphological variation in P. thornei as found in the present study.
According to the key presented by Castillo & Vovals (2007), P. thornei is similar to P. brachyurus (Godfrey, 1929) Filipjev & Schuurmans Stekhoven, 1941 and P. cruciferus Bajaj & Bhatti, 1984. The species is distinguished from P. brachyurus by the number of lip annuli (three annuli vs two annuli) and from P. cruciferus by labial shape (flat and slightly round vs flat) and hemizonid position (one annuli anterior to secretory-excretory pore vs 2-8 annuli anterior to secretory-excretory pore).
Table 1. Morphometrics of Pratylenchus thornei Sher & Allen, 1953 populations studied form different localities in
Iran. All measurements are in ^m.
Character Populations
Province Golestan Mazandaran Guilan
Locality of sampling Gorgan Ziarat Nowshahr Sari Roodsar Langrood
n 6ÇÇ 6ÇÇ 8ÇÇ 9ÇÇ 7ÇÇ 8ÇÇ
L 507±56.5 571±42.4 540±52.7 548±88.6 627±45.4 598±59.4
(465-567) (534-641) (486-623) (427-688) (548-683) (520-669)
a 31.2±3.8 29.8±2.2 31.5±3.7 30.1±3.4 33.1±1.8 33.3±3.4
(26.8-34.0) (27.1-33.2) (26.8-36.4) (26.4-34.9) (31.7-36.7) (29.0-37.0)
b 6.2±0.8 7.8±0.7 6.3±0.5 6.7±0.9 7.2±1.0 7.3±1.3
(5.3-6.7) (7.2-8.9) (5.5-6.9) (5.5-8.0) (6.0-9.2) (6.2-9.9)
c 22.5±0.9 21.0±1.9 26.0±2.4 22.9±2.3 28.3±3.0 24.5±3.2
(21.4-23.2) (18.1-23.1) (21.6-28.3) (18.9-26.4) (22.4-29) (21.2-29.3)
2.4±0.3 2.4±0.2 2.3±0.3 2.3±0.3 2.0±0.1 2.3±0.1
c (2.0-2.6) (2.0-2.6) (2.0-2.8) (1.9-2.7) (1.8-2.3) (1.9-2.5)
V 76.5±3.1 (74-80) 76.9±5.6 (71-81) 76.2±6.0 (70-82) 77±2.5 (71-78) 77±3.1 (73-81) 78±2.5 (74-81)
Lip height 7.3±0.2 (7-7.5) 7.9±0.7 (7.2-8.6) 7.8±0.5 (7.2-8.1) 7.7±0.4 (7.2-8.1) 7.9±0.3 (7.5-8.2) 7.9±0.1 (7.8-8.1)
Lip region diameter 2.8±0.2 (2.7-3.1) 2.8±0.2 (2.7-3.2) 2.9±0.2 (2.7-3.2) 2.7±0.2 (2.5-3.1) 2.7±0.4 (2.3-3.2) 2.6±0.4 (2.2-3.1)
Stylet length 14.8±0.4 (14-15) 15.5±1.3 (14-17) 15.5±1.2 (14-17) 15±1.1 (14-16) 15.8±1.1 (14-17) 15.8±1.3 (14-17)
Conus length 7.9±0.9 (7-9) 8.4±1.1 (7-10) 8±1.5 (7-10) 8±0.6 (7-9) 8±0.6 (7-9) 8.1±1.0 (6.4-9.0)
Pharynx length 86±2.1 (83-87) 73±6.6 (61-78) 84±4.2 (79-90) 81±8.5 (72-99) 87±10.2 (68-95) 83.1±8.8 (67-93)
Pharyngeal 125.7±6.3 111.8±6.8 118.7±7.3 123.1±11.9 133.4±122.8 129.8±10.3
glands (121-134) (103-119) (109-129) (99-134) (110-145) (119-146)
Excretory pore 80.6±2.2 (78-82) 81±4.4 (75-87) 84.5±12.9 (70-109) 83.8±13 (69-97) 82.9±11.5 (61-93) 81.2±4.6 (74.7-86.5)
MB 41.5±0.8 42.8±2.6 42.4±1.9 (39-44) 41.7±2.3 40.3±4.3 40.3±3.0
(41-42) (38-44) (38-46) (34-46) (36-43.7)
PVS 21.5±2.5 (22-26) 29.1±3.6 (24-32) 22.6±1.1 (21-24) 22±1.9 (20-24) 22.7±2.3 (20-26) 23.3±1.3 (22-25)
Max. body diameter 17.7±1.1 (16-19) 19.1±0.7 (18-20) 17.2±0.9 (16-18) 18.1±1.5 (16-20) 18.9±0.9 (17-20) 18.0±1.5 (17-21)
Lateral field width 5.3±0.7 (5-6) 7±0.5 (6-7) 5.5±0.9 (5-6) 5.7±0.2 (5-6) 6.1±0.6 (5-7) 5.9±0.3 (5-6)
Tail length 22±3.3 (20-26) 27±2.7 (24-32) 20.8±1.7 (19-23) 24.7±3.5 (20-29) 22.2±1.6 (20-25) 24.6±2.1 (22-28)
Anal body diameter 9.1±0.2 (9-10) 11.4±0.4 (11-12) 8.7±0.9 (7-10) 10±0.8 (9-11) 10.6±0.4 (10-11) 10.5±1.2 (9-12)
DNA characterisation. The 28S rRNA gene fragment (about 658 to 712 bp) of the six P. thornei populations studied during this research was amplified by the two primers D2A and D3B. Nblast (https: //blast.ncbi .nlm.nih.gov/Blast.cgi) results indicated that the populations studied (Gorgan, Roodsar and Langerood; KX 258735, KX 258737, KX 258738) are similar to a population from the USA (EU 130880; 100% identity). The P. thornei population from Sari, province of Mazandaran (KX 258740) shows only one nucleotide difference with the population from the USA (EU 130880; 99% identity). Compared to other populations from the USA (EU
130880, 98% identity), the Iranian populations studied (Nowshahr and Ziarat; KX 258739, KX 258736) differed by 15 base pairs.
Pairwise Maximum Composite Likelihood distance among the 28S rDNA region of P. thornei ranges from 0.000 to 0.073. The highest genetic variation was observed for a population from Australia (EU 130872) and one from Iran (JX 261963). Among the Iranian populations identified during this study, the genetic distance ranges from 0.000 to 0.071. The highest variation was observed for P. thornei specimens of the Golestan (present study; KX258736) and Lorestan (JX261963) populations.
Table 2. Nematode species and GenBank accession numbers used for the phylogenetic study.
Species GenBank accession number Reference Origin Sample codes
P. thornei KX258735 Present study Iran (Golestan, Gorgan) IR1
P. thornei KX258736 Present study Iran (Golestan, Ziarat) IR2
P. thornei KX258737 Present study Iran (Guilan, Roodsar) IR3
P. thornei KX258738 Present study Iran (Guilan, Langerood) IR4
P. thornei KX258739 Present study Iran (Mazandaran, Nowshahr) IR5
P. thornei KX258740 Present study Iran (Mazandaran, Sari) IR6
Table 3. Correlation analysis for populations of P. thornei Sher & Allen, 1953.
Character L a b c c' Tail V Pharynx Stylet Secretory excretory pore PVS
L 1
a 0.830(**) 1
b 0.713(**) 0.526(**) 1
c 0.424(**) 0.415(**) 0.283 1
c' 0.000 0.043 -0.146 0.558(**) 1
Tail 0.442(**) 0.309 0.375(*) 0.599(**) 0.483(**) 1
V -0.072 -0.300 0.019 -0.098 -0.022 0.042 1
Pharynx 0.362(*) 0.306 -0.227 0.173 -0.013 0.091 -0.129 1
Stylet 0.293 0.037 0.153 0.119 0.131 0.112 0.109 0.196 1
Secretory-excretory 0.476(**) 0.364(*) 0.002 -0.017 0.356(*) 0.409(*) -0.282 0.404(*) 0.375(*) 1
pore
PVS 0.353(*) 0.013 0.531(**) 0.256 0.515(**) 0.080 0.139 0.139 0.158 0.425(**) 1
* - correlation is significant at the 0.05 level (2-tailed).
** - correlation is significant at the 0.01 level (2-tailed).
Morphometric analysis on the P. thornei. The
results based on two-tailed Pearson correlation within the six P. thornei populations showed that some morphometric data, obtained from 46 females, showed significant correlations with others. Some important morphometric data, such as body length and length of the post vulval sac, need to be considered to understand the correlation with each other. The results indicate that body length has significant correlation with other morphometric data (a, b and c index, tail, pharynx, secretory-excretory pore and post vulval sac) in females (Table 3). On the other hand, body length had no correlation with some important morphometric characters such as c' and V indices in females. Interestingly, c index had a significant correlation (r = -0.599, P < 0.01) with tail length. Stylet length only showed correlation
with the positionof the secretory-excretory pore (r = 0.375, P < 0.01). Post vulval sac length had a significant correlation with the indices b (r = 0.531), c' (r = 0.515), body length (r = 0.353) and secretory-excretory pore (r = 0.425).
Hierarchical clustering. The dendrograms (Fig. 4) show the average linkage method of hierarchical clustering with P values of 14 populations of P. thornei from different localities in the world (Sher & Allen, 1953; Loof, 1960; D'Errico, 1970; Khan & Singh, 1975; Ryss, 1988; Yu, 1997; Pourjam et al., 1999; Mirzaipour et al., 2016). The hierarchical analysis based on important morphometric characters clustered the P. thornei populations studied into two groups. One group consisted of populations from Germany, Iran and Italy and with 99 and 100 AU (approximately unbiased) values. The other group consisted of populations from Canada,
Fig. 2. Pratylenchus thornei Sher & Allen, 1953. A-I: Anterior region. Sampled from three provinces in the north of Iran (Golestan, Guilan and Mazandaran) A: (IR1, Golestan, Gorgan region); B: (IR5, Mazandaran, Nowshahr region) C: (IR4, Guilan, Langerood region); D, E: (IR2, Golestan, Ziarat region); F, G: (IR3, Guilan, Roodsar region); H, I: (IR6, Mazandaran, Sari region).
A BCD E
В 25 цш
F G H I J
Fig. 3. Pratylenchus thornei Sher & Allen, 1953. A-J: Posterior end of females. Sampled from three provinces in the north of Iran (Golestan, Guilan and Mazandaran). A, B: (IR6, Mazandaran, Sari); C: (IR5, Mazandaran, Nowshahr); D, E: (IR4, Guilan, Langerood); F, G: (IR2, Golestan, Ziarat); H, I: (IR3, Guilan, Roodsar); J: (IR1, Golestan, Gorgan).
USA and Russia with 100 AU (approximately unbiased) values (Fig. 4). This analysis demonstrated that the Iranian populations are very closely related to Italian populations, representing a sister group. Moreover, the geographic pattern comprises of three continents with two of them (Asia and America) plotting completely separate from the European populations of P. thornei. On the other hand, the Russian population of P. thornei is placed in a group separate from the European populations.
DISCUSSION
The usefulness of morphometric analyses as a tool to identify Pratylenchus spp., due to significant correlations existing between some morphometric characteristics of females of the six P. thornei populations examined, has been demonstrated as a result of this study. In addition, morphometric cluster analysis is also suggested as a useful tool to characterise Pratylenchus spp. However, use of a third method, namely molecular analysis of the six P.
Fig. 4. Cluster dendrogram for different populations of Pratylenchus thornei using morphometric data. Red values on the left branch represent AU (Approximated Unbiased) values, while green values on the right branch indicate BP (Bootstrap Probability) values in percentage, and cluster labels (bottom).
Fig. 5. The Bayesian tree inferred of Pratylenchus thornei Sher and Allen, 1953 newly sequences from Iran and the other sequences of the Gene Bank, based on sequences of the 28S rDNA region.
thornei populations according to the 28S rDNA gene, is suggested to be more accurate than morphometric analysis to identify Pratylenchus spp., as has also been shown by Subbotin et al. (2008).
Regarding morphometric analysis, the authors could not find reports that demonstrated that correlations of such measurements or indices have been used to aid in identifying Pratylenchus spp. In our study, the indices a, b and c generally correlated with the body length of the P. thornei female specimens studied from the six Iranian populations. Hence, it is suggested to be more useful than other indices accurately to identify the species. By contrast, some characteristics, e.g. stylet with body length; r = 293, showed no or insignificant correlations, which may be an indication that the genes responsible for these characteristics are located at different loci or far apart (high genetic distances). Correlation between morphological characteristics of nematode specimens has been suggested as a useful method to identify species of nematodes. For example, Fortuner (1984) noted that the indices a, c and c' are often useful for accurate identification of species of Helicotylenchus Steiner, 1945. Amirzadi et al. (2013) also showed that b and c are more useful in identifying Acrobeles spp. Fortuner (1990) also revealed that the features related to body size (length and indices of a and b) showed high correlation to each other for Hirschmanniella belli. Subbotin et al. (1999) also stated that morphometric character analysis is suitable for separating populations within the H. avenae group. As a result of this study, cluster analysis according to morphometric characteristics grouped the six Iranian populations of P. thornei close to, but in a separate clade from, the European populations previously reported (Ryss, 1988).
The phylogenetic position of the P. thornei populations was studied by the extensive use of 42 large subunit rDNA sequences of nematodes belonging to the genus Pratylenchus from GenBank. The consensus tree based on LSU, showed that the P. thornei species is represented by a monophyletic group. The phylogenetic analysis grouped Pratylenchus spp. into five clades: I) P. thornei; II) P. neglectus and P. brzeskii; III) P. bhatii, P. zeae, P. parazeae and P. bolivianus; IV) P. vulnus, P. penetrans, P. dunensis, P. brachyurus and P. crenatus; and V) P. scribneri, P. pseudocoffeae, P. coffeae, P. jaehni, P. hippeastri, P. loosi and P. speijeri. Pratylenchus brezeski and P. neglectus grouped as a sister group and close to the P. thornei populations, as indicated by Subbotin et al. (2008). The six P. thornei populations from Iran studied
grouped together with the same species from USA, Australia, Spain, Iran and others countries (Subbotin et al., 2008; Majd Taheri et al., 2013). Although the P. thornei populations grouped close to P. brezeski and P. neglectus phylogenetically, P. thornei specimens are distinguished from P. brezeski by having three lip annuli and indistinct spermatheca, but both of them have a smooth tail terminus. Pratylenchus neglectus differs by having two lip annuli and also due to conoid-round tail shape. The spermatheca of both species is reduced. These three species also placed closely together in the study presented by Subbotin et al. (2008), while according to ITS rDNA P. thornei was placed in a group separated from P. neglectus (De Luca et al., 2011). The latter authors indicated that P. thornei is more closely related to P. mediterraneus Corbett, 1983. These two species are similar in lip annuli, tail shape and tail with smooth tip. However, they differ in lip shape, spermatheca and the presence of sperm. According to morphological characters, these species hence do not group closely together. Phylogenetic analysis based on morphological characters, as reported by Ryss (2002), showed that P. thornei places close to P. pinguicaudatus and P. coffeae (Zimmermann, 1898) Filipjev & Schuurmans Stekhoven, 1941 and P. dasi Fortuner, 1985. It is similar to P. pinguicaudatus in lip annuli, stylet length, and smooth tip tail but different in lip shape. It resembles P. coffeae due to its smooth tip tail but differs in lip annuli (two annuli), shape and spermatheca. Consequently, the phylogenetic position of these species based on morphological characters are not acceptable because they differ substantially in terms of morphological traits. Therefore, use of molecular analyses, especially with data that has been published on the 28S rDNA sequences as presented, is a more accurate way to identify these species.
In addition, more studies on DNA sequence data will improve our knowledge of the phylogenetic position of Pratylenchus spp.
ACKNOWLEDGEMENTS
The author is grateful for supplying types and other specimens from nematode collections or for providing information on morphological details of selected species, to A.M. Golden (f) and Z.A. Handoo, Beltsville; A.Yu. Ryss, St. Petersburg; J. Rowe and D.J. Hooper, formerly Rothamsted; E.A. Krall (f) and M. Rahi, Tartu"; Z. Tanha Maafi, Tehran; W.M. Wouts, formerly Auckland; S.A. Subbotin, Sacramento; R.N. Inserra, Gainesville; P.A.A. Loof and G. Karssen, Wageningen; M.A.
Maqbool, Karachi; S.D. Sidikov, Tashkent; C.
Nguyen and P.Q. Trinh, Hanoi; T. Mekete, Addis
Abeba; J. and M. Sturhan, Münster, and several
others, also for the opportunity to use the nematode
slide collections on visits of the author to Auckland,
Beltsville and St. Petersburg.
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Резюме. При проведении исследований по фитопаразитическим нематодам на севере Ирана были выявлены 6 популяций Pratylenchus thornei, исследованные затем морфологическими и молекулярными методами. Было показано, что длина тела этих нематод четко коррелирует с другими морфометрическими показателями, за исключением показателя c' и положения поры дорсальной железы пищевода. Наиболее выраженная корелляция была отмечена между длиной тела и индексом 'a' (r = 0.805). Также значительная корелляция была отмечена между длиной стилета и длиной тела (r = 0.511), длиной хвостового отдела (r = 0.300) и индексом V (r = 0.324). Среди индексов Де-Мана, значения 'b' и 'a' наиболее надежны для разграничения видов. Молекулярно-филогенетический анализ последовательностей 28S rDNA P. thornei показал значительное сходство иранских популяций с формами этого вида из США, Марокко, Молдавии, Испании и Великобритании. Показана монофилия P. thornei. Представлены таблица измерений, иллюстрации и филогенетическое древо этого вида.
