Genetic diversity of the recovered populations of Mesocriconema xenoplax (Nematoda: Criconematidae) from orchards in Fars province, Southern Iran

A total of 14 populations of Mesocriconema xenoplax were collected in 2018–2019 from the rhizosphere of fruit and nut trees including walnut (five populations), grapevine (two populations), almond (two populations), apricot, apple, peach, sour cherry and greengage from several localities in Fars province, Southern Iran (Table 1). Soil composite samples were prepared by taking soil from three cores around the trunk of 5–10 trees in each orchard. Nematode specimens were extracted from the soil using the centrifugal flotation method (Jenkins, 1964), killed and fixed with hot FPG (4:1:1, formaldehyde: propionic acid: glycerol), and processed to anhydrous glycerol (de Grisse, 1969). They were then mounted in glycerol on permanent slides using paraffin wax and were studied using an Olympus BX41 light microscope equipped with a Dino-eye microscope eye-piece camera and its Dino Capture version 2.0 software. For each population, the nematodes were examined and photographed, and 10 females were measured using the same microscope. All populations were characterized based on morphological and morphometric characters using identification keys (e.g., Geraert, 2010). Using Minitab ver.16 software, a cluster analysis was performed with an agglomerative hierarchical method to assess the relative variations of the different populations. The Euclidean method was used as a distance measure to create a dendrogram for clustering the populations.

ISSR-PCR reactions were performed in a 20 μl volume containing 2 μl of genomic DNA, 2 μl of each primer, 8 μl of Master Mix RED (Ampliqon A/S, Denmark) and 8 μl of ddH₂O. Seven primers, including (CCA)₅, (GTG)₆, (GAG)₄GC, (GACA)₄, (AC)₈T, (CA)₈G and (GAGA)₄GG were used (Lax et al., 2007). PCR amplification reactions were programmed for initial denaturation at 94°C for 3 min, followed by 35 cycles of 30 s at 93°C, 90 s at 48°C, 1 min at 72°C, and a final extension of 10 min at 72°C. PCR success was visualized by running 7 μl of the PCR product on a 1.7% TBE buffered agarose gel (75 V, 120 min).

For ISSR fragment analysis, data were scored for all primers: “1” for the presence of the band and “0” for the absence of the band. Distance matrixes (D) were calculated using the Dice and Jaccard coefficients (Huff et al., 1993), performed by a Principal Coordinate Analysis (PCA) to obtain a graphical representation of the relationship between nematode populations. The basic population parameters (Nei, 1973; McDermott and McDonald, 1993) including polymorphism (P), heterozygosity (H), Shannon diversity (I), and Nei’s gene diversity (uHe) were estimated based on the banding pattern observed. The fixation index (F_st) and indirect gene flow (Nm) were calculated based on formulae developed by Slatkin and Barton (1989).

A cluster analysis was performed using the Numerical Taxonomy Multivariate Analysis System (NTSYSPC V-2.02) software package (Rolf, 2000). We used the ISSR marker to create UPGMA and NJ trees. The genetic dissimilarity matrix and ultrametric distance matrix generated from a UPGMA-based dendrogram with a COPH module in the same software were compared with the Mantel matrix correspondence test (Mantel, 1967). The result of this test is a cophenetic correlation coefficient, r, indicating how well the dendrogram represents similar data. For the allelic frequency analyses, we defined the populations in two procedures: the first for each associated plant without considering the geographic regions and the second for the geographic regions at the level of a city without considering the associated plant. The allele frequency and analysis of molecular variance (AMOVA) were carried out using GenALEx version 6.5 (Peakall and Smouse, 2006).

The recovered populations were characterized as M. xenoplax based on morphological and morphometric characteristics. The measurements of all recovered M. xenoplax populations are listed in Table S1 (see supplementary data). Cluster analysis identified four distinct groups labeled as A to D where A and B were closely related to each other and both were associated with group C, while a population recovered from the rhizosphere of walnut (Ghasrodasht) formed the last group D (Fig. 1).

Figure 1:

Similarity dendrogram of 14 populations of Mesocriconema xenoplax as compared by UPGMA analysis of morphological characters.

The D2-D3 alignment was 541 bp long and consisted of 56 sequences as ingroups and two sequences, including Aglenchus agricola (de Man, 1884) Andrassy, 1954 and Eutylenchus excretorius Ebsary and Eveleigh, 1981 as outgroups. Fourteen new sequences of the D2-D3 expansion fragments of the 28S rRNA gene were obtained in the present study. The phylogenetic relationships of the Iranian populations within other populations of M. xenoplax and certain closely related species, inferred from the analysis of these partial 28S rRNA gene sequences with collapsed branches, with a PP of less than 70%, are shown in Fig. 3.

Figure 3:

Bayesian Inference (BI) tree of D2-D3 expansion segments of 28S rRNA nucleotide sequences. New sequences are in bold.

Nucleotide diversity within the Iranian populations varied between zero and 61 nucleotides (0–11.46%). It seems that two distinct lineages could be considered for populations of M. xenoplax. A large proportion of the populations of M. xenoplax with 13 Iranian populations of this species formed one lineage (M. nebraskense showed high affinity with this lineage) and the grapevine-Khan Zeniyan population formed another lineage with two populations from Portugal, one population from Japan, and one population from China (one population of M. ornatum with close affinity to this lineage).

A total of 89 loci with an average of 12.7 loci per primer were scored with the ISSR marker. ISSR patterns for 14 populations of M. xenoplax generated with primer (CA)₈G are shown in Fig. 4 (data not shown for other primers).

Figure 4:

ISSR patterns for 14 populations of Mesocriconema xenoplax generated with the primer (CA)₈G. DNA Ladder = 200 bp.

Considering the populations by associated plants, the mean genetic variations of the total indicated by P, I, H and uHe were 6.18%, 0.039, 0.027 and 0.033, respectively, indicating a very low genetic diversity. Since only one population each from sour cherry, apricot, apple, greengage and peach was collected, values of P, I, H and uHe could not be calculated for these populations. However, after excluding these populations, five walnut populations exhibited the highest genetic diversity (P% = 23.60, I = 0.154, H = 0.108 and uHe = 0.121). It seems that there is no correlation between population size and genetic diversity, as two populations were evaluated for both grapevine and almond, but the genetic diversity of the two almond populations (P% = 19.10, I = 0.116, H = 0.079 and uHe = 0.105) was much higher than that of grapevine (P% = 6.74, I = 0.041, H = 0.028 and uHe = 0.037), almost close to the walnut populations.

For the second comparison based on regions, the populations of Ghasrodasht, Maharloo and Bajghah were excluded as they had only one population. The populations from Dokohak and Khan Zeniyan showed the highest genetic diversity (P% = 34.83, I = 0.231, H = 0.164, uHe = 0.182 and P% = 21.35, I = 0.129, H = 0.088, uHe = 0.118, respectively), while the lowest value was found in the populations from Sepidan (P% = 4.49, I = 0.027, H = 0.019 and uHe = 0.025). The mean total genetic variation indicated by P, I, H and uHe, were 10.91%, 0.069, 0.048 and 0.059, respectively, indicating low genetic diversity.

The genetic distances (D) between pairs of M. xenoplax populations by associated plants determined by Nei (1978) varied from 0.034 to 0.269. The lowest value was between populations of sour cherry and apricot, and the highest between populations of grapevine and apricot. In the ISSR analysis by geographic region, pairwise D values varied from 0.082 (for the populations of Dokohak and Khan Zeniyan) to 0.315 (for the populations of Sepidan and Ghasrodasht) (Table 2).

The UPGMA and NJ trees obtained from the ISSR data were presented in Fig. 5 and Fig. 6, respectively. For each tree, five different groups of A-E were obtained. The trees were generally very similar and only some minor differences were found in the placement of certain groups and populations. The main differences were the separation of the grapevine population (Khan Zeniyan) and the placement of this population in a separate line in the NJ tree (Fig. 6). However, this population formed a well-separated clade in the UPGMA tree with the grapevine (Dokohak) and almond (Arjan) (Fig. 5). In addition, groups A and B were closely related to group C in the UPGMA tree, but these two groups were closer to group D than to group C in the NJ tree. PCA plots for PC1 vs. PC2 and PC1 vs. PC3 based on plants and geographic regions were shown in Fig 7. In the PCA plots, similar five groups of UPGMA and NJ trees were highlighted. The two principal coordinates (PCs) were accumulated at 64% and 68%, and the three PCs were accumulated at 78% and 81% for plants and geographic regions, respectively.

Figure 5:

UPGMA cluster diagram based on Dice coefficient estimated from 89 ISSR markers for 14 populations of the Mesocriconema xenoplax (host plants and geographic regions are written in the front of branches).

Figure 6:

NJ cluster diagram from 89 ISSR markers for 14 populations of the Mesocriconema xenoplax (host plants and geographic regions are written in the front of branches).

Figure 7:

The bi-dimensional dispersion according to the principal coordinates analysis (PC1 vs PC2 and PC1 vs PC3) obtained using the GenAlex software for Mesocriconema xenoplax populations from Fars province, Southern Iran by ISSR analyses (above: populations defined by plants, below: populations defined by geographic regions).

The percentage of molecular variance (AMOVA) among and within populations was 61% and 39% for the populations grouped by plant, and 50% when grouped by geographic region. The values of F_ST and Nm by plant were 0.611 (P = 0.010) and 0.159, respectively, and by geographic region were 0.504 (P = 0.010) and 0.246, respectively (Table 3). The values of F_ST and Nm among populations of the different plants are shown in Table 4. For a large proportion of the plant relationships, a good separation was obtained among the populations of the individual plants and the other plants (F_ST > 0.800 and Nm < 0.100). The highest F_ST value (and consequently the lowest value of Nm) was obtained between apricot and apple (F_ST = 0.911 and Nm = 0.024), while the F_ST value between walnut and almond, and grapevine and almond was very low (0.103 and 0.120, respectively), indicating a very high gene flow between nematode populations associated with these plants (Nm was 2.177 and 1.833, respectively).

The values of F_ST and Nm among populations of different geographical regions were also determined (Table 4). The F_ST values among populations from different geographic regions were lower than those for among plants (F_ST < 0.400), and a moderate to strong gene flow occurred among populations of the different geographic regions (Nm > 0.400). The highest F_ST value and consequently the lowest Nm value was found for Arjan-Bajgah (F_ST = 0.382 and Nm = 0.404) and minimum F_ST value (0.111) for Khan Zeniyan-Arjan (Nm = 2.002).