Genetic intraspecific diversity of Meloidogyne javanica parasitizing vegetables in southern Iran

In total, 37 populations of M. javanica were collected from vegetables (tomato, cucumber, okra, and eggplant) and weeds of vegetable fields in several regions of southern Iran, during 2018 and 2019 (Table 1). Nearly 700 fields and greenhouses were sampled, randomly or sometimes from infested patches of affected plants showing symptoms, in several times during 2017 and 2018 years. In total, 57 populations were recovered on roots of crops such as tomato, cucumber, eggplant, and okra in different regions of Fars, Boushehr, Kerman, and Kohgiloyeh and Boyer-Ahmad provinces. Pure cultures of all populations were produced by rearing a single egg mass on tomato (cv. Early Urbana) grown in sterilized river sand in clay pot, at a temperature ranging 20 to 30°C. After 70 days, mature females were extracted from stained roots for further morphometric analysis of the females and preparing perineal patterns. Second-stage juveniles (J2s) and males, if present, were killed and fixed by hot FPG (4:1:1, formaldehyde: propionic acid: glycerin), and processed to anhydrous glycerin (de Grisse, 1969). The J2s, males, and perineal patterns were then mounted on permanent slides and examined for morphological and morphometric purposes using an Olympus BX41 light microscope, equipped with Dino-eye microscope eye-piece camera and its Dino Capture version 2.0 software.

One full egg mass was put in 16 µl ddH₂O in a 0.2 ml tube. DNA was extracted according to Tanha Maafi et al. (2003) with some modifications: tubes were frozen at –80°C for at least 15 min and crushed by vortexing, and 20 µl worm lysis buffer (500 mM KCl, 100 mM Tris-Cl pH 8, 15 mM MgCl₂, 0.05% Mercaptoethanol, and 4.5% Tween 20) and 4 µl proteinase K (600 µg/ml) were added, respectively. The samples were incubated at 65°C for 1 hr and at 95°C for 10 min. After incubation, the tubes were centrifuged for 2 min at 38,000 g and kept at –20°C for next uses.

PCR was performed containing 1 µl of DNA template, 8 µl mastermix (Amplicon Red), 1 µl of each primer (10 pmol) and 4 µl ddH2O to a final volume of 15 µl. Four set species-specific primers (Table 2) were used for PCR reactions (Zijlstra, 2000; Zijlstra et al., 2000). A negative control containing the PCR mixture without DNA template was also included.

The PCR amplification profile consisted of 5 min at 94°C, 35 cycles of 30 sec at 94°C, 30 sec at annealing temperature (64°C for M. javanica, 54°C for M. incognita, 61°C for M. arenaria, and 58°C for M. hapla) and 45 sec at 72°C, followed by a final step of 5 min at 72°C. All steps were repeated twice to insure the results. PCR success was visualized loading 4 µl of the PCR product into a 1.7% TBE buffered agarose gel (75 V, 60 min) and visualized with ethidium bromide.

The ISSR-PCR reactions were performed in a 20 μl volume containing 2 µl of genomic DNA, 2 µl of primer, 8 µl mastermix (Amplicon Red), and 8 µl ddH₂O. Ten primers including (CCA)₅, (GTG)₆, (ATG)₆, (GAG)₄GC, (GACA)₄, (GA)₈C, (AC)₈T, (CA)₈G, (CTC)₄GC, and (GAGA)₄GG (Table 2) were implemented (Lax et al., 2007).

PCR amplification reactions were programmed for an initial denaturation at 94°C for 3 min, followed by 35 cycles of 30 sec at 93°C, 90 sec at 48°C, 1 min at 72°C, and a final extension of 10 min at 72°C. PCR success was visualized by loading 7 µl of the PCR product into a 1.7% TBE buffered agarose gel (75 V, 120 min).

Amplification reactions were carried out in reactions of 20 µl total volume containing 2 µl of genomic DNA, 2 µl of primer, 8 µl mastermix (Amplicon Red), and 8 µl ddH₂O. Ten primers (Table 2) were used in RAPD-PCR (Mokaram Hesar et al., 2011).

For RAPD amplifications, the thermos-cycle profile was 5 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 35°C and 2 min at 72°C, followed by a final step of 5 min at 72°C. PCR success was visualized loading 7 µl of the PCR product into a 1.7% TBE buffered agarose gel (75 V, 120 min).

For the ISSR and RAPD fragment analysis, data were scored for all primers: “1” for the presence of band and “0” for the absence of band. To be sure, all steps were repeated twice and smeared or weak bands were not counted. Distance matrixes were calculated using the Dice and Jaccard coefficients. Cluster analysis was performed with the Numerical Taxonomy Multivariate Analysis System (NTSYSPC V-2.02) software package (Rohlf, 2000). The genetic dissimilarity matrix and ultrametric distance matrix produced from UPGMA-based dendrogram with COPH module nested in the same software was compared using Mantel’s matrix correspondence test (Mantel, 1967). Also, we try to use Mantel test in linear shape for searching correlation between different matrix produced by RAPD and ISSR. Bootstrapping was done using the Winboot with 1000 replicates (Yap and Nelson, 1996) and measurements under 60 were excluded. Frequency analyses were performed to estimate the variance components and their significance levels of genetic variation within and among populations using GenALEx version 6.5 (Peakall and Smouse, 2006). We used P (percentage of polymorphic loci), H (the expected heterozygosity) and I (Shannon’s information index) to calculate genetic diversity. Also, F _ST (Fixation index) and Nm (Number of migrants) used to calculate the differences of populations and amount of gene flow between populations. These two indices are inversely correlated, and by increasing fixation index, Nm is decreased (Milgroom, 2015).

In total, 41 populations of RKNs were recovered including 34 populations from vegetable fields and nine populations from greenhouses in southern Iran. Two nematodes populations were obtained from weeds growing in vegetable fields. All the recovered populations in this study were tested with the species-specific molecular marker type Sequence Characterized Amplified Region (SCAR) for the four major species (Zijlstra, 2000; Zijlstra et al., 2000). Expected band for M. javanica, M. incognita, M. arenaria, and M. hapla are 670 bp, 1200 bp, 420 bp, and 960 bp, respectively. The species-specific primers of M. javanica (Fjav and Rjav) yielded one unique fragment of approximately 670 bp in 37 populations, and no PCR product was obtained in the control template (due to the limitation of the number of wells in electrophoresis, the PCR products were run separately, and the results of 25 populations were presented in Fig. 1). Also, no bands were detected in the gels of the 37 isolates of other three species (data not presented). The primers of M. incognita yielded a 1,200 bp band for four isolates of greenhouse cucumber in Fasa region (Fig. 2). Morphometric characters for the females, J2s, and males of some populations of M. javanica are given in Table 3.

Figure 1:

Figure 2:

With the 10 random primers used in the RAPD analysis, a total of 180 bands were produced. All primers resulted in amplification and different patterns were tested for population differences. The resulting gel electrophoresis for 19 populations for (CCA)₅ primer of ISSR and OPAD10 primer of RAPD is presented in Figure 3. The number of reproducible amplified fragments varied from 14 to 23 per primer (average 18), their size ranging from 200 to 2500 bp. The percentage of polymorphic bands (P%) within each population ranged from 16.1 to 36.1%, (in populations of Khafr and Abdan, respectively) and I index ranged from 0.093 to 0.218 (same as P%). The highest value of H index was in populations of Abdan (0.150). The mean gene variations for the 27 populations were 0.103 and 0.148 by H and I, respectively (Table 4).

Figure 3:

In ISSR, a total of 164 loci, with average of 16.4 loci per primer, were scored. P, H, and I were used to estimate genetic diversity within the M. javanica populations. The populations of Abdan and Bardkhon exhibited the highest level of genetic diversity (P% = 25.6, H = 0.110, I = 0.158; P% = 24.39, H = 0.097, I = 0.141, respectively), whereas the lowest value was shown in the populations of Jiroft (P% = 10.37, H = 0.039, I = 0.058). In 164 loci, 19.08% were polymorphic in all individuals of studied populations. The mean genetic variations of total indicated by H and I, were 0.080 and 0.115, respectively.

The Nei’s (1978) genetic distances (D) between pairs of M. javanica populations with RAPD analysis were 0.043 to 0.162 (Table 5). The lowest value was between the populations of Abdan and Beiza, and the highest between the populations of Kazeron and Khafr. While for ISSR analysis, pairwise D values were ranged from 0.032 (populations of Abdan and Deir) to 0.091 (populations of Kazeron and Deir, also Abdan and Beiza). Mantel test showed a positive correlation between RAPD and ISSR (Fig. 6) and an increasing on each marker values was observed with an increasing on another marker values.

The UPGMA trees obtained with RAPD and ISSR data showed some differences in tree topologies between calculations based on Dice and Jaccard. The positions of some populations are different by ISSR and RAPD, although the number of clades was consistent as both methods revealed four clades. None of these clades grouped M. javanica populations according to their geographical region and host plants. The UPGMA trees generated using Dice coefficient for RAPD and ISSR are presented in Figures 4 and 5, respectively. We use bootstrap values upper 60 for separating main clades, and in other clades or subclades, bootstrap is under 60. Bootstrap values written only for main clades.

Figure 4:

Figure 5:

In analyses of molecular variance analysis, percentages of molecular variance within and among populations were 11.36% and 2.62% for RAPD, and 8.26% and 1.33% for ISSR, respectively (Table 6). F _ST and Nm for RAPD are 0.188 (P = 0.010) 1.079 however for ISSR are 0.140 (P = 0.010) and 1.535.