Distribution and characterization of the root-knot nematode, Meloidogyne javanica (Treub, 1885) Chitwood, 1949 (Meloidogynidae) isolates, in some regions of two main pistachio growing provinces of Iran

Eighty-four different localities of pistachio orchards in Kerman and Khorasan Razavi provinces, respectively in southeastern and northeastern Iran were selected for sampling during 2018–2024. Samples were kept in plastic bags (each soil + infected root inside a nylon bag was regarded as one sample) and the necessary data including codes, locality, and GPS information were recorded (Table 1) and then transferred to the laboratory for further processing. Due to difficulties in separating intact female specimens from woody roots of pistachio, all RKN isolates were reared on tomato (Solanum lycopersicum cv. Early Urbana) in the greenhouse using one egg mass, separated from infected roots (one egg mass from one infected root sample was inoculated into one pot including four seedlings). After 45 days, the infected tomato roots were cut and mature females were isolated.

The females, males, and juveniles were heat-killed by adding boiling 4% formaldehyde solution, transferred to anhydrous glycerin, and mounted on permanent slides (De Grisse, 1969). The specimens were observed using a stereomicroscope and a light Olympus BH2 microscope. Perineal patterns were cut from female specimens preserved in lactic acid (Taylor and Netscher, 1974). The recovered populations of Meloidogyne species were primarily identified morphologically based on the characteristics of perineal pattern of females (Jepson, 1987). In second step, species-specific primers Fjav and Rjav (Zijlstra et al., 2000) were used. Finally, the infestation percent of the sampling points to M. javanica was calculated. Eight isolates of M. javanica (KS1-1, KS1-10, KN7-1, KR3-1, KR8-1, KSh1-1, KhF1-4, and KhF2-1) showing morphological/morphometric variations in comparison with the other isolates were selected for detailed studies. Interpretation of morphological features was based on Jepson (1987) and Subbotin et al. (2021).

A single female nematode specimen of each isolate was picked out and transferred to a small drop of tris-ethylenediaminetetraacetic acid (TE) buffer (10 mM Tris-Cl; 0.5 mM ethylenediaminetetraacetic acid; pH 9.0) on a clean slide and squashed using a clean coverslip. The suspension was collected by adding 50 μl of TE buffer. The prepared raw DNA samples were stored at −20°C until used as a polymerase chain reaction (PCR) template and no further purification was performed.

The primers used for PCR and DNA sequencing are given in Table 2. The 25 μl PCR mixture contained 12.5 μl 2X Taq red master mix DNA polymerase (Amplicon, Stenhuggervej 22 DK-5230 Odense M Denmark), 7.5 μl distilled water, 1 μl each of 10 μM forward and reverse primers, and 3 μl of raw DNA template. The PCR was performed in a Biometra^® thermocycler (Analytik Jena AG, Jena, Germany). All amplifications were carried out under the following cycling conditions: 94°C for 5 min, then 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, and final extension at 72°C for 10 min.

Amplified PCR products were loaded into 1% agarose gel and were electrophoresed at 80 V for 40 min with Tris, borate, and ethylenediaminetetraacetic (TBE) buffer and Green-Viewer^® for gel staining. The successfully amplified PCR products were sequenced using the same primers used in PCR by Macrogen Co., South Korea. The accession numbers of newly obtained sequences are given in Table 3. Molecular identification using species-specific primers (Fjav and Rjav) was exploited to identify 37 isolates of M. javanica (Zijlstra et al., 2000) (no sequencing was performed for this purpose).

The basic local alignment search tool for nucleotide (BLASTn) at the National Center for Biotechnology Information (NCBI) was used to unravel the identity of the newly generated sequences compared with previously deposited ones. The relevant sequences were downloaded for the three loci to infer phylogenetic relationships of the newly generated sequences. The SSU rDNA sequence of M. artiellia Franklin, 1961 and Pratylenchus thornei Sher & Allen, 1953, COII-16S sequence of M. enterolobii Yang & Eisenback, 1983 and Radopholus similis (Cobb, 1893) Thorne, 1949 and Nad5 sequences of two isolates of M. enterolobii were used as outgroups for phylogenies of corresponding loci (Kiewnick et al., 2014; Trinh et al., 2022).

The Q-INS-i algorithm of the online version of MAFFT (version 0.91b) was used to align the SSU rDNA, COII-16S, and Nad5 datasets, and the resultant alignments were manually edited using MEGA7 (Kumar et al., 2016). The model of base substitution was selected using MrModeltest 2. The Akaike-supported model, a general time reversible model, including among-site rate heterogeneity and estimates of invariant sites (GTR + G + I), was used in phylogenetic analyses of three loci. Bayesian analyses were performed using MrBayes v3.1.2 running the chains for two million generations. After discarding burn-in samples (the burn-in was set to 25%), the remaining samples were retained for further analysis. The Markov chain Monte Carlo method within a Bayesian framework was used to estimate the Bayesian posterior probabilities (BPP) of the phylogenetic trees using the 50% majority rule. Convergence of model parameters and topologies were assessed based on an average standard deviation of split frequencies and potential scale reduction factor values. Adequacy of the posterior sample size was evaluated using autocorrelation statistics as implemented in Tracer v.1.7 (Rambaut et al., 2018). The output files of the trees were visualized using Dendroscope v3.2.8 and trees were digitally drawn using CorelDRAW software, version 2020.

Based on the dataset summarized in Table 1, QGIS (https://qgis.org/) was employed to visualize the spatial distribution of infested and non-infested localities associated with RKNs, including Meloidogyne javanica, M. incognita, M. arenaria, and M. cruciani Garcia-Martinez et al., (1982), across studied regions in Kerman and Khorasan Razavi provinces (Fig. 1). A Digital Elevation Model (DEM) was integrated as the base layer to improve interpretation of the geographical and topographical characteristics of the highlighted regions. All spatial datasets were projected using the WGS 1984 geographic coordinate system.

Figure 1

Geographical distribution of root-knot nematode species (RKNs) in the infested and non-infested regions in Khorasan Razavi and Kerman (A and B), two major pistachio-producing provinces of Iran. (a) Khorasan Razavi province and (b) Kerman Province. (c and d) Enlarged maps displayed with a DEM background; c: Mahvelat county, d: Cities of Shahre-e Babak, Anar, Rafsanjan. Legends and scale bars are shown.

Thirty-seven isolates of M. javanica were identified from pistachio orchards in Kerman (17 isolates) and Khorasan Razavi (20 isolates) provinces. Eight isolates of M. javanica showing morphological/morphometric variations in comparison with other presently and formerly recovered isolates were studied in detail.

The morphology and morphometrics of the eight studied isolates were similar to those given by Whitehead (1968) and Orton Williams (1972). Compared to other isolates, the presently studied isolates showed morphological and morphometric variations. For instance, the female body in isolate KN7-1 was longer (not pear shaped). Body length of females ranged from 590 to 1,050 μm. The KSh1-1 isolate had the shortest body while KN7-1 isolate had the longest body. Their body width ranged from 315 to 700 μm. The KN7-1 isolate had the lowest and KhF1-4 isolate had the highest range. Their stylet length ranged from 14.0 to 19.5 µm, the KS1-1 isolate had the lowest and KhF1-4 isolate had the highest range. The excretory pore (EP) distance from anterior end showed remarkable variation, ranging from 20.0 to 57.5 µm. The lower range belonged to the KR8-1 isolate and the greatest range belonged to the KS1-10 isolate. The perineal patterns of the isolates were variable, but the general pattern was conserved. The vulval slit ranged from 20.0 to 29.5 µm wide. The shortest in the KS1-10 isolate and the longest in the KhF2-1 and KS1-1 isolates. The distance between anus to vulval slit ranged from 13 to 26 µm. The shortest in KS1-10 and the longest in KR3-1. The inter-phasmid distance ranged from 14 to 39 µm. The shortest belonging to KR8-1 and the longest to KR3-1. Males were not common in all studied isolates and were only seen in KS1-1, KS1-10, KN7-1, and KSh1-1 isolates. Their body length ranged from 900 to 1,350 μm. The KSh1-1 isolate had the shortest and KS1-1 and KS1-10 had the longest body. Their stylet ranged from 20.5 to 22.3 µm. The KSh1-1 isolate had the lowest and KS1-10 had the highest range. Mean male DGO ranged from 2.7 to 3.8 µm. The shortest in KS1-10 isolate and the longest in KS1-1 isolate. Spicules length ranged from 27 to 37 µm. The KS1-10 isolate had the lowest and KN7-1 isolate had the longest ones. The J2s body length showed no remarkable variation between isolates. Their stylet ranged from 9.5 to 14.5 µm. The Khorasan Razavi isolates (KhF1-4 and KhF2-1) had the shortest stylet. Their tail length ranged from 35 to 57 µm. The KS1-10 isolate showed both values. The hyaline region of tail ranged from 10 to 17 µm. The KhF1-4 isolate had the shortest and KS1-10 isolate had the longest hyaline region.