New data on known species of Hirschmanniella and Pratylenchus (Rhabditida, Pratylenchidae) from Iran and South Africa

The description of female body of Hirschmanniella anchoryzae is as follows: body length is from 1,740 to 1,895 µm, slightly curved to ‘C’ shaped after fixation, some specimens slightly straight; cuticle is finely annulated with 1.2 to 1.5 µm wide at midbody; maximum body diameter is of 26 to 29 µm; cephalic region is continuous with the body; lip region is flat, with a slight depression, bearing four to five annuli; lateral field is with four lines, 8 to 10 µm width, occupying about 31 to 34% of midbody diameter, aereolated along the body especially in the tail region; stylet length is from 19 to 21 µm, basal knobs usually rounded, and stylet conus 46 to 50% of the total length of the stylet; dorsal pharyngeal gland opening (DGO) is at 3 to 4 µm posterior to stylet base; median bulb is spherical to oval with 7 to 11×9 to 10 µm length and width, respectively; nerve ring is located just after isthmus, at 27 to 28% of the neck (from head to end of pharyngeal gland overlapping); excretory pores are at 1 to 8 µm anterior to the pharyngeal-intestinal junction, at 35 to 37% of the neck; hemizonid is 5 to 8 µm anterior to excretory pore; pharyngeal glands are overlapped with 8 to 10 times than the corresponding body diameter; pharynx is 380 to 410 µm long, about 22% of the body length; in reproductive system didelphic amphidelphic, ovary is not reaching the pharyngeal glands; oocytes are in one or two rows; vulva occupies 35 to 40% of the corresponding body diameter; spermatheca is visible, oval shape, with sperm; vulva is with a transverse slit with not protruded lips, V = 53 to 57; tail is 96 to 106 µm long, conical, elongated with 63 to 80 ventral annuli and axial mucro, in some specimens the appendage visible as a notch; and phasmid is located at about middle of the tail, 57 to 61% of the tail length.

The structure of male body is similar to the female body with hypoptygma. Reproductive system is monorchid. Spicules tylenchoidis of 30 to 35 µm length, paired, separate in ventral view, smooth, and ventrally arcuate in lateral view; rounded manubrium; calamus very short, lamina thin, ventral curved end. In lateral view, the gubernaculum is bent, 5 to 8 µm length, 18 to 26% of spicule length. Bursa is leptoderan. Phasmid is 60 to 70% of the tail length. Tail is conical, elongated, 81 to 87 µm with axial mucro at tail tip.

Four females from Royan (Mazandaran Province, Iran) in a good state of preservation were studied. Iranian population of Hirschmanniella is similar to H. anchoryzae based on the original description and the identification key (Ebsary and Anderson, 1982). In comparison with the H. anchoryzae reported previously from Iran (Pourjam et al., 2000), they differ in female body length (1,740–1,895 vs 1,580–1,680 µm) and spicule length (27–30 µm vs 39–40 µm). This population differentiate from H. oryzae (Luc and Goodey, 1964) in having longer spicule (27–30 µm vs 18–26 µm) and shorter gubernaculum (5–8 µm vs 9–14 µm) (Khan and Bala, 2003). This population also resembles H. gracilis (De Man, 1880; Luc and Goodey, 1964). However, the two differed in the upper range of the body length (1.9 and 1.6 mm for female and male, respectively, vs 2.2 and 2.0 mm for female and male, respectively), stylet length (19–21 µm vs 20–24 µm), and gubernaculum length (5–8 µm vs 9–15 µm) (Loof, 1991).

The principal component analysis was performed to study the variation within the populations of H. anchoryzae (Fig. 2). An accumulated variability of 53.90% was detected in the female by the F1 (31.12%) and F2 (22.79%). All characters exhibited positive correlations among the populations and were responsible for the variability of the F1, except for a, b, c′ and tail length (Fig. 2). Some characters such as c, c′, pharynx length, stylet length, and tail length with 22.1, 18.8, 17.1, 13.5, and 12.6% showed the most contribution to the variability and had a high correlation with the F1, while excretory pore to anterior end and body length with 34.3 and 28.2% showed a high correlation with the F2. The result indicated that two population of H. anchoryzae from Iran and Canada place close each other. Two populations of H. anchoryzae place close with the H. halophila from Germany.

Figure 2:

The genes 28S rDNA and COI of mtDNA for H. anchoryzae yielded 687 and 385 bp, respectively. Nblast of the 28S rDNA showed 93% identity with a Dutch population (acc. nr: EU620464; EU620465) of H. halophila (Sturhan and Hallmann, 2010) with 35 nucleotides differences. Compare with a population of H. santarosae, De Ley et al. (2007) showed 90% identity with 59 nucleotide differences. In comparison with H. pomponienis, Abdel-Rahman and Maggenti (1987) showed 89% identity with 70 nucleotide differences. Regarding COI of mtDNA, Nblast showed 95% identity with an unidentified population (acc. nr: KX349428) of Hirschmanniella from China showing 18 nucleotide differences. In comparison with H. mucronata (KY424110), our sequence showed 84% identity with 61 nucleotide differences, whereas another population of H. mucronata (KR819278) from China there was 71% identity with 89 nucleotide differences. Genetic pairwise distance (Table 4) indicated the lowest (0.073) and the highest range (0.166) among H. anchoryzae (MK571451), obtained from H. halophila (EU620464, EU620465) and H. mucronata (KP179327, KP179333, KF201167), respectively. The lowest range (0.000) was observed between an unidentified Hirschmanniella (DQ328686) from Vietnam and H. oryzae from Myanmar and the Philippines.

The Bayesian inference tree of 28S rDNA of Hirschmanniella species (Fig. 3) grouped them into three clades including (i) H. oryzae, H. belli (Sher, 1968), and unidentified Hirschamnniella with 1.00 posterior probability; (ii) H. loofi, H. kwazuna, H. mucronata, and unidentified Hirschamnniella with 1.00 posterior probability, and (iii) H. anchoryzae, H. pomponiensis, H. santarosae, and H. halophila with 1.00 posterior probability.

Figure 3:

The description of female body of Pratylenchus hippeastri: body length is 423 to 614 µm, slightly curved after fixation, some specimens straight; cuticle is finely annulated with 0.9 to 1.2 µm wide at midbody; maximum body diameter is 17 to 27 µm; cephalic region is continuous with the body; lip region is round to flat, with a slight depression, bearing two annuli; lateral field with four lines, started with two lines 7.5 µm from anterior end, ending at tail terminus with three lines, occupies about 25 to 36% of midbody diameter and around 53% at the vulval region; stylet length is 13.3 to 18.0 µm, basal knobs usually rounded and flatted; dorsal esophageal gland opening (DGO) is at 1.7 to 3.6 µm posterior to stylet base; median bulb oval, nerve ring located just after isthmus, is at 48 to 66% of the neck; excretory pore is at 80 to 112 µm from the anterior body, at 60 to 70% of the neck; hemizonid one annuli is anterior to excretory pore; pharyngeal glands overlapped with intestine are about 32 to 43 µm; neck is 116 to 149 µm long, body length about 3.5 to 4.9 times than pharynx length; ovary is not reaching the pharyngeal glands; columnar cells of uterus are distinct and disposed in four rows; oocytes are in one row at growth zone (V = 75-82); spermatheca is visible, circular to oval shape, without sperm; post-vulval uterine sac is 11 to 26 µm long, vulva with a transverse slit and protruded lips; tail is conoid with bluntly pointed, usually with a ventral constriction at the middle or subhemispherical, slightly smooth terminus; tail is with slightly indented terminus observed in some specimens; tail is 26 to 43 µm long, about 2.0 to 2.5 times than anal body diameter; phasmid is located at about middle of the tail, 46 to 55% of the tail length; and the hyaline portion of tail terminus is 2.5 to 3.8 µm long.

Not found.

Seven females from Potchefstroom (South Africa) in a good state of preservation were studied. This species was previously reported from Florida, USA (Inserra et al., 2007; De Luca et al., 2012), Japan (Gu et al., 2014), and China (Wang et al., 2016). In the present study, this species has been studied from South Africa. The morphometric characters resemble those studied previously. However, compared to the specimens studied from Florida (Inserra et al., 2007), the specimens from Potchefstroom have shorter tail lengths (26–33 μm vs 32–42 μm) and shorter post uterine sacs (11–26 μm vs 21–45 μm). The lip region is reported to contain two annuli with the first being deeper than the second one (Inserra et al., 2007; De Luca et al., 2012; Wang et al., 2016). The results indicated the uncompleted third incisure in some specimens of P. hippeastri. SEM observation of the Potchefstroom individuals indicated that two lip annuli are present, in which the first one being deeper. Uncompleted lip annuli and tail variation forms were not observed in Potchefstroom specimens. According to the identification key presented by Inserra et al. (2007), P. hippeastri is similar to P. scribneri Steiner in Sherbakoff and Stanley’s (1943) study reported from Ohio (USA). These authors separated P. hippeastri from P. scribneri by a longer tail (36.6 vs 26.7 μm), slightly longer stylet (15.4 vs 14.7 μm), and shape of tail terminus, but often bluntly pointed and smooth terminus vs the consistently hemispherical and smooth tail terminus. The present results indicated that the stylet is overlapped in the two mentioned species (stylet length 13.3-15.4 µm in South African population in this study). Concerning the tail end morphology, none of the South African specimens studied had smooth ends. Hence, based on the results of this study, the tail morphology of P. hippeastri seems to be different from that of P. scribneri as shown in the study of Inserra et al. (2007). However, more specimens from different localities of South Africa are needed for the detailed description of the test populations.

The results based on two-tailed Pearson correlation within the five P. hippeastri populations studied showed that some morphometric data, obtained from 63 females (except for the South African specimens, the average based on the available data have been used), showed significant correlations with others. Some important morphometric data such as body length seemed to be considered to understand the correlation with each other. Results indicated that body length showed the highest correlation with the b (r = 0.786) and tail (r = 0.768), respectively (Table 5). On the other hand, body length had no significant correlation with some morphometric characters such as stylet length (r = 0.277). Despite the stylet length, body length showed the highest significant correlation (r = 0.931, P = 0.05) with MB (median bulb to anterior end as % of the pharynx). Tail length showed a highly significant correlation with the index b (r = 0.904).

The principal component analysis was performed to study the variation within the populations of P. hippeastri (Fig. 7). An accumulated variability of 72.75% was detected in the female by the F1 (47.63%) and F2 (25.12%). All characters exhibited positive correlations among the populations and were responsible for the variability of the F1, except for a, c, V, DGO, and pharynx (Fig. 7). Some characters such as body length, b, MB, and tail length with 11.7, 13.2, 12.5, and 11 percent showed the most contribution to the variability and had a positive and high correlation with the F1.

Figure 7:

The dendrogram (Fig. 8) showed the average linkage method of hierarchical clustering with p-values of five populations of P. hippeastri from different localities in the world (Inserra et al., 2007; De Luca et al., 2010; Gu et al., 2014; Wang et al., 2016; present study). The hierarchical analysis based on important morphometric characters clustered the P. hippeastri populations studied into two groups. One group consisted of populations from the USA (Florida) and South Africa with 100 (AU: approximately unbiased) values and the second group consisted of populations from China and Japan with 100AU values (Fig. 8). This analysis demonstrated that the South African populations are very closely related to American populations. Moreover, the geographic pattern comprises of three continents with two of them (Africa and America) plotting completely separate from the Asian populations of P. hippeastri.

Figure 8:

The 18S rDNA (859 bp), ITS rDNA (910 bp), 28S rRNA (707 and 713 bp), and COI (mtDNA) (382 bp) gene fragments of the P. hippeastri studied during this research were amplified and compared using Nblast. The 18S rDNA of our population (MH324470) is similar to those of populations from the Israel and China (KJ001716 and KY424116; 99% identity) with three and five nucleotide difference, respectively. In addition, it is similar to a Dutch population of P. scribneri (EU669958; 99% identity) with two nucleotide differences. The ITS rDNA of the population studied (MH324471) is similar to those of populations from the USA (FN554886 and FN554888; 99% identity with one nucleotide difference only). Compared to the Chinese population of the same species, the Potchefstroom population showed two nucleotide differences (KY424236; KY424237; 99% identity). The 28S rDNA of the population studied (MH324472; MH324473) are similar with populations from the USA (FN554879 and FM994117; 99% identity) and China (KP161608 and KC796707; 99% identity), with one and two nucleotide difference, respectively. The COI gene of mtDNA of the population studied (MH324474) is similar to those of populations from China (KY424098 and KY424099; 97% identity), with 11 and 10 nucleotide differences, respectively.

Regarding P. hippeastri, the Bayesian inference trees constructed on the basis of the 18S rDNA, ITS rDNA, D2 to D3 segment of 28S and COI of mtDNA sequences are shown in Figures 9 to 12, respectively. All populations of P. hippeastri are placed in one group with highly supported bootstrap values of 100%. Based on the phylogenetic analysis using ITS (Fig. 10) and 28S rDNA, P. hippeastri placed close to P. floridensis (De Luca et al., 2010) and P. parafloridensis (De Luca et al., 2010).

Figure 9:

Figure 10:

Figure 12:

According to the 28S rDNA phylogenetic tree results (Fig. 11), the South African populations are more closely related to the Floridian (USA) (FM994114; FN554879) populations than to the Chinese populations (KC796706; KC796707). However, based on 18S rDNA (Fig. 9), the South African population of P. hippeastri placed close to the same species from China (KY424166; KJ001716) as well as P. scribneri Steiner in Sherbakoff and Stanley (1943) from the Netherlands (EU669958), and P. araucensis (Múnera et al., 2009) from Germany (FJ154950). The COI phylogenetic results (Fig. 12) indicated that the South African population grouped with the Chinese population of P. hippeastri (KY424098; KY424099).

Figure 11: