First report of root-lesion nematode, Pratylenchus oleae from pistachio in Iran

Morphometric of P. oleae, found in roots and around soil from Pistachio tree in Iran are presented in Table 1. The morphological and molecular analyses confirmed that the species was P. oleae as well. P. oleae is characterized by morphological features in females: Vermiform body, lip region with three annuli, slightly offset. Stylet well developed with distinct rounded knobs slightly directed anteriorly. Lateral field with four incisures, metacorpus oval to rounded, isthmus rather short, surrounded by nerve ring, pharyngeal glands well developed, with rather long ventral overlap. Excretory pore either slightly anterior pharyngo-intestinal junction or opposite. Genital branch with one row of oocyte, spermathecal rounded, vagina a straight tube. Vulva transverse slit. Post-vulval uterine sac short, not differentiated. Tail sub-cylindrical, terminus rounded to conical, smooth, male unknown (Fig. 1). P. oleae is morphologically closely related to P. cruciferus, P. delattrei and P. kumamotoensis.

Figure 1:

Morphometric mean, standard deviation and range values of P. oleae females were (n  =  10): L  =  522.5 ± 34.9 (463.0–565.0) μm; a  =  28.3 ± 1.6 (25.7–31.0)  μm; b  =  5.9 ± 0.4 (5.2–6.7)  μm; c  =  20.2 ± 0.8 (19.1–21.0) μm; Stylet length  =  17.3 ± 0.7 (16.0–18.0) μm; Median bulb 55.6 ± 2.3 (51.0–59.0) μm; Anterior to Excretory Pore 88.9 ± 2.7 (85.0–93.0) μm; Anterior end to pharyngeal junction 89.0 ± 4.6 (78.0–94.0) μm; Pharynx Length 123.5 ± 3.4 (118.0–129.0) μm; Max. Body diam. 18.5 ± 1.0 (17.0–20.0) μm; Body diam. at anus 12.6 ± 0.5 (12.0–13.0)  μm and tail length  =  25.9 ± 1.7  (22.0–27.0)  μm (Table 1).

The D2–D3 region of the 28S-rDNA amplified with the primer sets D2A/D3B and yielded single fragments of 800 bp (Fig. 2), based on direct fragment sequencing. The blastn test of 28S rDNA showed that only seven accession numbers of P. oleae belong to Spain (three isolates of Agua Amarga-Nijar, Almeria, wild olive and one isolates of Albaricoques-Nijar, Almeria, cultivated olive Picnal) and Tunisia (three isolates of Ouled Chamekh, cultivated olive Koroneiki). Pratylenchus capsici, 9 isolates are most similar to P. oleae, but differs in presence of males, a functional spermatheca, a larger body and shorter stylet (Qing et al., 2019). BLAST searches indicated 99–100% identity (similarity) with sequences of P. oleae and 100% similarity with sequences of P. capsici (Table 2). Sequences from other species of Pratylenchus obtained from NCBI were used for further phylogenetic studies. D2–D3 expansion segments of 28S rRNA of P. oleae were deposited in GenBank under the accession numbers MW338666 from cultivated pistachio trees cv. Badami from Ardakan city, Yazd province matched well with the Pratylenchus spp. deposited in GenBank. This sequence had intra-specific variation 100% similarity (2 nucleotide differences) with P. oleae Spain isolate (KJ510861) and 99% (2–7 nucleotide differences) similar to P. oleae Spain isolates (KJ510855, KJ510856, KJ510857) and P. oleae Tunisia isolates (KJ510858, KJ510859, KJ510860), while P. dunensis and P. penetrans are 89% and 87% similar (61 and 82 nucleotide differences with 6 and 13 Gaps). The gap variations and nucleotide differences between 9 isolates of P. capsici and P. oleae (MW338666) are between 3–9 and 30–37 nucleotides respectively (Table 2).

Figure 2:

In total, 46 sequences were included in the phylogenetic analysis of D2-D3 expansion segments of the 28S rDNA gene. The phylogenetic tree based on 28S rDNA, placed the Iran P. oleae population in a clade together with other P. oleae populations and revealed the strong relationship of P. oleae with P. penetrans, P. dunensis and P. capsici. (Fig. 3). The molecular characterization of several isolates of P. oleae suggested that they formed a monophyletic group. To the best of our knowledge, this is the first record of P. oleae from pistachio rhizosphere and root in the world. The species identification of P. oleae was confirmed using PCR by species-specific primers Poleae_fw1_4_36/Poleae_rv1_ _525_551 and produced a 547-bp fragment (Fig. 2), which was the same as reported by Palomares-Rius et al. (2014).

Figure 3:

Their morphology and morphometric of P. oleae population in Iran followed the original species descriptions, which were reported previously from two locations of Spain (Agua Amarga-Níjar and Albaricoques-Níjar) and one location of Tunisia (Ouled Chamekh) (Palomares-Rius et al., 2014), But some morphometric discrepancies were found. Body, stylet length, anterior to excretory pore, pharynx length, tail length of Iranian specimen was slightly 68 μm, 0.8 μm, 6.9 μm, 21.5 μm and 3.2 μm longer, respectively. The Iranian isolate of P. oleae is characterized by lip region consisting of three annuli and four incisures along with oblique lines in middle of body, tail sub-cylindrical to mild conoid like to other P. oleae isolates.

The sequence of Iranian isolate showed high nucleotide similarity with D2-D3 region of P. oleae deposited in GenBank database. The test population had no gap (base pair), which was different to those of P. oleae from Spain (KJ510855 and KJ510861) with 100% similarity, one gap with Spain (KJ510856 and KJ510857) and Tunisia (KJ510858) with 99% similarity respectively and two gaps with Tunisia (KJ510859 and KJ510860) with 99% similarity. In summary, Iranian P. oleae was isolated from pistachio trees in Ardakan, Yazd province. It is reported for the first time from Iran and pistachio tree in the world. This nematode can be considered as a risk to the economy in pistachio orchards.