Genetic intraspecific diversity of Meloidogyne javanica parasitizing vegetables in southern Iran

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Genetic intraspecific diversity of Meloidogyne javanica parasitizing vegetables in southern Iran

Reza Ghaderi * / Ali Asghar Dehghan / Abbas Mokaram Hesar / Akbar Karegar

Keywords : Diversity, ISSR, RAPD, root-knot nematodes, species-specific primer

Citation Information : Journal of Nematology. Volume 52, Pages 1-13, DOI: https://doi.org/10.21307/jofnem-2020-035

License : (CC-BY-4.0)

Received Date : 30-October-2019 / Published Online: 24-April-2020

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ABSTRACT

In order to investigate different species of root-knot nematodes associated with vegetable production in southern regions of Iran, 37 populations of the most predominant species, Meloidogyne javanica, were recovered. Morphological and morphometric studies were carried on the characters of females, males, J2s and perineal patterns. Species-specific Sequence Characterized Amplified Region (SCAR) primers confirmed morphological studies, and all these populations produced specific band in 670 bp using Fjav and Rjav primers. Genetic diversity of different populations was studied by Inter Simple Sequence Repeats (ISSR) and Random Amplified Polymorphic DNA (RAPD) markers implementing 10 primers for each approach. Results revealed a relatively low genetic diversity (the percentage of polymorphic bands were 19.08 and 24.60 for ISSR and RAPD, respectively). The analyses of molecular variance indicated that the variation resulted from genotypic variations within region and variances among regions are 81% and 19% for RAPD, and 86% and 14% for ISSR, respectively. On the other hand, FST and Nm values are 0.140 and 1.535 for ISSR while these values are 0.188 and 1.079 for RAPD. So it can be concluded that there is a great deal of gene flow between populations due to the movement of plant material contaminated with nematodes, which results in high mixing between populations. ISSR and RAPD datasets failed to group populations according to their geographic region. There were no pathotypes or other intraspecific biological entities observed in the species.

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Plant-parasitic nematodes are an important limiting factor in vegetable production, and in many areas a major factor requiring extensive use of pesticides. Root-knot nematodes (RKNs) of the genus Meloidogyne (Göldi, 1887), which their population increase to damaging levels within a few seasons under susceptible crops, are so common in subtropical and tropical vegetable production that frequently they are taken to represent “nematodes” in general (Sikora and Fernandez, 2005). The populations of M. incognita (Kofoid and White, 1919) Chitwood, 1949 and M. javanica (Treub, 1885) Chitwood, 1949 were the most recovered populations (more than 80%) from vegetable fields worldwide (Taylor and Sasser, 1978). Five species including M. arenaria (Neal, 1889) Chitwood, 1949, M. cruciani Garcia-Martinez, 1982, M. hapla Chitwood, 1949, M. incognita and M. javanica are the recovered RKN in vegetable production in Iran (Mahdikhani Moghadam, 2018).

Considering that morphological and morphometrical methods for the identification species of RKNs are considerably time consuming and there is a lot of ambiguity, using species-specific primers could be a rapid method for detection (these primers listed in Cunha et al., 2018).

The genetic diversity among M. javanica populations has been inferred by different markers (reviewed in Blok and Powers, 2009; Mokaram Hesar et al., 2011). Statistical analysis of the morphological characters and RAPD markers demonstrated 86 and 93% similarity among 21 populations of M. javanica recovered from north east of Iran. Intraspecific variation was less influenced by geographical origin of nematodes and/or their host plants (Mokaram Hesar et al., 2011). RAPD-PCR technique was utilized successfully to differentiate among three important RKN species, M. arenaria, M. incognita, and M.  avanica, and also, to evaluate genetic variations among different populations of M. javanica (Naz et al., 2013). RAPD markers revealed a low intraspecific genetic diversity (14%) among 17 isolates of this species parasitizing potato in Brazil (Medina et al., 2017). Principal component and phylogenetic analyses revealed distant relationship between M. javanica and M. enterolobii Yang and Eisenback, 1983 populations from South Africa (Rashidifard et al., 2018).

Molecular markers that have proved to be useful in these studies are mitochondrial gene cytochrome C oxidase subunit I (COI) (Ye et al., 2015; Powers et al., 2018), the small subunit 18 S rRNA gene (Tigano et al., 2005; Ye et al., 2015), D2-D3 expansion segment of the large subunit 28 S rRNA (Palomares-Rius et al., 2007; Ye et al., 2015) and ribosomal internal transcribed spacer (ITS1 and ITS2) (Powers et al., 1997; Ye et al., 2015). These markers were useful for separating species of RKNs; however, they could not separate main species including M. javanica, M. incognita, M. hapla, and M. arenaria, and there is overlapping between these species (Kiewnick et al., 2014).

Although ISSR technique has been used to resolve genetic diversity in certain species of RKNs such as M. enterolobii (Tigano et al., 2010) and M. arenaria (Carneiro et al., 2008), it has not been implemented for M. javanica so far.

However, ISSR and RAPD markers are similar in some aspects, but there are a number of differences between them that make the two markers act as complementing of each other. RAPD primers are short (10 nucleotides) with a random sequence and markers are considered to be uniformly distributed along the genome, whereas ISSR primers is based on the amplification of regions flanked by repeating sequences which found only between microsatellite loci. In the other hands, annealing temperature in RAPD is lower than ISSR (Lax et al., 2007).

The aim of the present study is to infer the genetic intraspecific diversity among populations of M. javanica collected from major vegetable growing areas in southern regions of Iran, using RAPD and ISSR markers.

Material and methods

Nematode populations and light microscopy

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.

Table 1.

Populations of Meloidogyne species collected in this study.

10.21307_jofnem-2020-035-t001.jpg

DNA extraction

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 using species-specific primers

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.

Table 2.

All primers used for ISSR, RAPD and species-specific identification of Meloidogyne species during the present study.

10.21307_jofnem-2020-035-t002.jpg

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.

ISSR-PCR

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).

RAPD-PCR

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).

ISSR and RAPD data analysis

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, FST (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).

Results

Characterization of the recovered populations

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.

Table 3.

Average of the morphometrics of females, J2s and males of five populations of Meloidogyne javanica parasitizing vegetables in southern Iran.

10.21307_jofnem-2020-035-t003.jpg
Figure 1:

Species-specific 670 bp band in the populations of Meloidogyne javanica recovered from southern Iran. (For population codes, see Table 1).

10.21307_jofnem-2020-035-f001.jpg
Figure 2:

Species-specific 1200 bp band in the populations of Meloidogyne incognita recovered from southern Iran. (For population codes, see Table 1).

10.21307_jofnem-2020-035-f002.jpg

Inferring genetic diversity by RAPD and ISSR markers

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).

Table 4.

Genetic diversity estimates of Meloidogyne javanica populations recovered from southern Iran by RAPD and ISSR analyses.

10.21307_jofnem-2020-035-t004.jpg
Figure 3:

RAPD (left) and ISSR (right) patterns for 19 populations of Meloidogyne javanica generated with the primer (CCA)5 and OPAD10. DNA Ladder = 200 bp. (For population codes, see Table 1).

10.21307_jofnem-2020-035-f003.jpg

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.

Table 5.

Nei’s (1978) unbiased genetic distance between pairs of Meloidogyne javanica populations detected by RAPD and ISSR analyses.

10.21307_jofnem-2020-035-t005.jpg

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:

UPGMA cluster diagram based on Dice coefficient estimated from 180 RAPD markers for 37 populations of Meloidogyne javanica (For population codes, see Table 1).

10.21307_jofnem-2020-035-f004.jpg
Figure 5:

UPGMA cluster diagram based on Dice coefficient estimated from 164 ISSR markers for 37 populations of the Meloidogyne javanica (For population codes, see Table 1).

10.21307_jofnem-2020-035-f005.jpg

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). FST and Nm for RAPD are 0.188 (P = 0.010) 1.079 however for ISSR are 0.140 (P = 0.010) and 1.535.

Table 6.

Analysis of molecular variance (AMOVA) of Meloidogyne javanica populations based on RAPD and ISSR data.

10.21307_jofnem-2020-035-t006.jpg

Discussion

This is the first comprehensive study of RKN species in southern regions of Iran which demonstrated that M. javanica was the most predominant species of RKN infesting vegetable fields and greenhouses in southern Iran. Although there was a slight genetic variation at the intraspecific level, populations of M. javanica generally showed a low level of genetic diversity. In the present study, surveyed Meloidogyne species were characterized using morphological and morphometric characters and confirmed with species-specific SCAR-PCR. The genetic diversity among populations of M. javanica was further explored by ISSR and RAPD markers.

Accurate distinction of M. enterolobii, M. javanica, and M. incognita was still challenging using various molecular techniques, except for SCAR-PCR (Zijlstra et al., 2000) and genotyping by sequencing method for identifying diagnostic single nucleotide polymorphisms (Rashidifard et al., 2018). In our study, SCAR markers were used successfully for distinguishing populations of M. javanica from M. incognita, so 37 populations produced specific band of M. javanica and four populations produced the band typical for M. incognita.

On the whole, P, H, and I revealed a low level of genetic diversity within populations by incorporation of RAPD and ISSR data (Table 4). To our knowledge, this is the first study for the genetic structure of M. javanica populations revealed by ISSR marker. The RAPD and ISSR markers could not reveal any population-specific band and they failed to discriminate different populations of M. javanica by their geographical origin or plant hosts; which in agreement with those obtained by Adam et al. (2005) and Mokaram Hesar et al. (2011).

The mean FST and Nm values for the studied populations were 0.188 and 1.079 by RAPD while 0.140 and 1.535 by ISSR, respectively (Table 6), indicating that most of the total genetic variances (81 or 86%) were at the intra-population level. The greater the FST values are, the greater the differences between populations. FST and Nm are inversely related and when values of FST are low, values of gene flow are high, it means that a lot of gene exchange has occurred between populations. In nematodes and especially RKNs, the most important method of their distribution is through agricultural inputs; thus, populations could be distributed by infected transplants and other plant material. In this study, low values of FST and average values of gene flow may indicate some integration of populations with together. So higher values of genotypic variations within regions, rather than among regions, confirms this fact and that is the amount of genetic variation related to this species. The low genetic diversity observed for M. javanica isolates during the present study was similar to results reported for other populations of M. javanica from different crops (Carneiro et al., 1998; Randig et al., 2002; Cofcewicz et al., 2004; Medina et al., 2017). This finding might be attributed to the parthenogenetic mode of reproduction in M. javanica, which results in clonal progenies (Triantaphyllou, 1985). Measured individuals of the selected five populations of M. javanica in the present study could not be sorted in regard of none of the diagnostic characters (Table 3).

The amount of genetic diversity of RAPD marker in this study is higher than that of the northern regions of Iran, which has previously reported by Mokaram hesar et al. (2011), although the host plants are almost identical. Moreover, it seems that genetic diversity revealed by ISSR marker, is slightly higher; however, there is not any other reference that can be compared with the present results. One possible source might be related to the presence of males in the studied populations. Genetic changes in nematode population are partially driven by the environment including the use of resistant varieties and pesticides. Exploring such genetic changes at the population level is crucial for understanding and predicting the behavior of nematodes in the field (Golden and Birchfield, 1978). When we compare warmer conditions in southern regions with those in northern regions of Iran, it may be concluded that the crop cultivation is carried out consecutively throughout the year, and it seems that activity of RKNs continuing during the year, which may be a cause of increased genetic diversity among the present populations.

It should also be pointed that the results of Mantel test revealed that there is a low correlation between the two genetic diversity sets of RAPD and ISSR (r = 0.266, P ≤ 0.01), and it seems that these two markers may work independently (Fig. 6).

Figure 6:

A Mantel test showing correlation between RAPD and ISSR datasets of the populations of Meloidogyne javanica recovered from southern Iran.

10.21307_jofnem-2020-035-f006.jpg

In this paper, the species-specific SCAR primer technique proved to be a powerful tool to distinguish the populations of the two widely distributed RKNs (M. javanica and M. incognita) from each other. The PCR-based techniques using RAPD and ISSR markers were shown to be helpful for obtaining detailed information about the diversity that exists among M. javanica populations. Perhaps including more populations from distant geographic regions, and even different countries, provide better understanding of the genetic structure and gene flow of these nematodes. Further intra- and inter-specific genetic diversity of M. javanica may be explored by implementing next-generation sequencing (NGS) techniques.

Acknowledgements

The authors wish to thank the Iranian National Science Foundation (INSF; project No. 96008747) and Shiraz University (Iran) for financial support.

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  25. Tanha Maafi, Z. T. , Subbotin, S. A. and Moens, M. 2003. Molecular identification of cyst-forming nematodes (Heteroderidae) from Iran and a phylogeny based on ITS-rDNA sequences. Nematology 5:99–111.
  26. Taylor, A. L. and Sasser, J. N. 1978. Biology, Identification and Control of Root-knot Nematodes (Meloidogyne species). A Cooperative Publication of the Department of Plant Pathology, North Carolina State University and the US Agency for International Development, Raleigh, North Carolina.
  27. Tigano, M. S. , Carneiro, R. M. D. G. , Jeyaprakash, A. , Dickson, D. W. and Adams, B. J. 2005. Phylogeny of Meloidogyne spp. based on 18S rDNA and the intergenic region of mitochondrial DNA sequences. Nematology 7:851–62, doi: 10.1163/156854105776186325.
  28. Tigano, M. , De Siqueira, K. , Castagnone Sereno, P. , Mulet, K. , Queiroz, P. , Dos Santos, M. , Teixeira, C. , Almeida, M. , Silva, J. and Carneiro, R. M. D. G. 2010. Genetic diversity of the root-knot nematode Meloidogyne enterolobii and development of a SCAR marker for this guava damaging species. Plant Pathology 59:1054–1061.
  29. Triantaphyllou, A. C. 1985. “Cytogenetics, cytotaxonomy and phylogeny of root-knot nematodes”, in Sasser, J. N. and Carter, C. C. (Eds), An advanced Treatise on Meloidogyne 1 North Carolina State University Graphics, Raleigh, NC, pp. 113–126.
  30. Yap, I. V. and Nelson, R. J. 1996. WINBOOT: a program for performing bootstrap analysis for binary data to determine the confidence limits of UPGMA-based dendrograms. Discussion Paper Series No. 14, International Rice Research Institute, Manila, Philippines.
  31. Ye, W. , Zeng, Y. and Kerns, J. 2015. Molecular characterisation and diagnosis of root-knot nematodes (Meloidogyne spp.) from turfgrasses in North Carolina, USA. PLoS One 10, pp. 1–16, doi: 10.1371/journal.pone.0143556.
  32. Zijlstra, C. 2000. Identification of Meloidogyne chitwoodi, M. fallax and M. hapla based on SCAR-PCR: a powerful way of enabling reliable identification of populations or individuals that share common traits. European Journal of Plant Pathology 106:283–290.
  33. Zijlstra, C. , Donkers-Venne, D. T. H. M. and Fargette, M. 2000. Identification of Meloidogyne incognita, M. javanica and M. arenaria using sequence characterised amplified region (SCAR) based PCR assays. Nematology 2:847–853.
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FIGURES & TABLES

Figure 1:

Species-specific 670 bp band in the populations of Meloidogyne javanica recovered from southern Iran. (For population codes, see Table 1).

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Figure 2:

Species-specific 1200 bp band in the populations of Meloidogyne incognita recovered from southern Iran. (For population codes, see Table 1).

Full Size   |   Slide (.pptx)

Figure 3:

RAPD (left) and ISSR (right) patterns for 19 populations of Meloidogyne javanica generated with the primer (CCA)5 and OPAD10. DNA Ladder = 200 bp. (For population codes, see Table 1).

Full Size   |   Slide (.pptx)

Figure 4:

UPGMA cluster diagram based on Dice coefficient estimated from 180 RAPD markers for 37 populations of Meloidogyne javanica (For population codes, see Table 1).

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Figure 5:

UPGMA cluster diagram based on Dice coefficient estimated from 164 ISSR markers for 37 populations of the Meloidogyne javanica (For population codes, see Table 1).

Full Size   |   Slide (.pptx)

Figure 6:

A Mantel test showing correlation between RAPD and ISSR datasets of the populations of Meloidogyne javanica recovered from southern Iran.

Full Size   |   Slide (.pptx)

Full Size   |   Slide (.pptx)

Full Size   |   Slide (.pptx)

Full Size   |   Slide (.pptx)

Full Size   |   Slide (.pptx)

Full Size   |   Slide (.pptx)

Full Size   |   Slide (.pptx)

REFERENCES

  1. Adam, M. A. M. , Phillips, M. S. and Blok, V. C. 2005. Identification of Meloidogyne spp. from north east Libya and comparison of their inter- and intra-specific genetic variation using RAPDs. Nematology 7:599–609.
  2. Blok, V. C. and Powers, T. O. 2009. “Biochemical and molecular identification”, in Perry, R. N. , Moens, M. and Starr, J. L. (Eds), Root-Knot Nematodes CAB International, Wallingford, pp. 98–118.
  3. Carneiro, R. M. D. G. , Castagnone-Sereno, P. and Dickson, D. W. 1998. Variability among four populations of Meloidogyne javanica from Brazil. Fundamental and Applied Nematology 21:319–326.
  4. Carneiro, R. M. D. G. , dos Santos, M. F. A. , Almeida, M. R. A. , Mota, F. C. , Gomes, A. C. M. M. and Tigano, M. S. 2008. Diversity of Meloidogyne arenaria using morphological, cytological and molecular approaches. Nematology 10:819–834.
  5. Cofcewicz, E. T. , Carneiro, R. M. D. G. , Castagnone-Sereno, P. and Quénéhervé, P. 2004. Enzyme phenotypes and genetic diversity of root-knot nematodes parasitising Musa in Brazil. Nematology 6:85–95.
  6. Cunha, T. G. D. , Visôtto, L. E. , Lopes, E. A. , Oliveira, C. M. G. and God, P. I. V. G. 2018. Diagnostic methods for identification of root-knot nematodes species from Brazil. Ciência Rural 48:1–12.
  7. de Grisse, A. T. 1969. Redescription ou modifications de quelques techniques utilisées dans l'étude des nématodes phytoparasitaires. Mededlingen van den Rijksfaculteit Landbouwwetenschappen Gent 34:351–369.
  8. Golden, A. M. and Birchfield, W. 1978. Meloidogyne incognita wartellei n. subsp. (Meloidogynidae), a root-knot nematode on resistant soybeans in Louisiana. Journal of Nematology 10:269–277.
  9. Kiewnick, S. , Holterman, M. , van den Elsen, S. , van Megen, H. , Frey, J. E. and Helder, J. 2014. Comparison of two short DNA barcoding loci (COI and COII) and two longer ribosomal DNA genes (SSU & LSU rRNA) for specimen identification among quarantine root-knot nematodes (Meloidogyne spp.) and their close relatives. European Journal of Plant Pathology 140:97–110, doi: 10.1007/s10658-014-0446-1.
  10. Lax, P. , Dueñas, J. R. , Gardenal, C. and Doucet, M. 2007. Assessment of genetic variability in populations of Nacobbus aberrans (Thorne, 1935) Thorne & Allen, 1944 (Nematoda: Pratylenchidae) from Argentina. Nematology 9:261–270.
  11. Mahdikhani Moghadam, E. 2018. “Nematodes of the family Meloidogynidae”, in Ghaderi, R. , Kashi, L. and Karegar, A. (Eds), Plant-Parasitic Nematodes in Iran Marjaeelm & Iranian Society of Nematology, Shiraz, pp. 397–418.
  12. Mantel, N. 1967. The detection of disease clustering and a generalized regression approach. Cancer Research 27:209–220.
  13. Medina, I. L. , Gomes, C. B. , Correa, V. R. , Mattos, V. S. , Castagnone-Sereno, P. and Carneiro, R. M. D. G. 2017. Genetic diversity of Meloidogyne spp. Parasitising potato in Brazil and aggressiveness of M. javanica populations on susceptible cultivars. Nematology 19:69–80.
  14. Milgroom, M. G. 2015. Population Biology of Plant Pathogens: Genetics, Ecology, and Evolution (No. 581.2 M644p) APS Press, Minnesota, MN.
  15. Mokaram Hesar, A. , Mehdikhani Moghadam, E. and Tanha Maafi, Z. 2011. Morphometrical and genetic diversity of Meloidogyne javanica isolates from the north east of Iran. Journal of Nematode Morphology and Systematics 14:1–11.
  16. Naz, I. , Palomares-Rius, J. E. , Blok, V. , Ahmad, M. and Ali, S. 2013. Species identification of root knot nematodes in Pakistan by random amplified polymorphic DNA (RAPD-PCR). Sarhad Journal of Agriculture 29:71–78.
  17. Palomares-Rius, J. E. , Vovlas, N. , Troccoli, A. , LieBanas, G. , Landa, B. B. and Castillo, P. 2007. A new root-knot nematode parasitizing sea rocket from Spanish Mediterranean coastal sand dunes: Meloidogyne dunensis n. sp. (Nematoda: Meloidogynidae). Journal of Nematology 39:190–202.
  18. Peakall, R. O. D. and Smouse, P. E. 2006. GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes 6:288–295.
  19. Powers, T. , Harris, T. , Higgins, R. , Mullin, P. and Powers, K. 2018. Discovery and identification of Meloidogyne species using COI DNA barcoding. Journal of Nematology 50:399–412.
  20. Powers, T. O. , Todd, T. C. , Burnell, A. M. , Murray, P. C. B. , Fleming, C. C. , Szalanski, A. L. , Adams, B. J. and Harris, T. S. 1997. The rDNA internal transcribed spacer region as a taxonomic marker for nematodes. Journal of Nematology 29:441–50.
  21. Randig, O. , Bongiovanni, M. , Carneiro, R. M. D. G. and Castagnone-Sereno, P. 2002. Genetic diversity of root-knot nematodes from Brazil and development of SCAR markers specific for the coffee-damaging species. Genome 45:862–870.
  22. Rashidifard, M. , Fourie, H. , Véronneau, P. , Marais, M. , Daneel, M. S. and Mimee, B. 2018. Genetic diversity and phylogeny of South African Meloidogyne populations using genotyping by sequencing. Scientific Reports 8:13816.
  23. Rohlf, F. J. 2000. Numerical taxonomy and multivariate analysis system. Version 2.11 T Exeter Software, Setauket, NY.
  24. Sikora, R. A. and Fernandez, E. 2005. Nematode parasites of vegetables, in Luc, M. , Sikora, R. A. and Bridge, J. (Eds), Plant parasitic nematodes in subtropical and tropical agriculture. CAB International, Wallingford UK, pp. 319–392.
  25. Tanha Maafi, Z. T. , Subbotin, S. A. and Moens, M. 2003. Molecular identification of cyst-forming nematodes (Heteroderidae) from Iran and a phylogeny based on ITS-rDNA sequences. Nematology 5:99–111.
  26. Taylor, A. L. and Sasser, J. N. 1978. Biology, Identification and Control of Root-knot Nematodes (Meloidogyne species). A Cooperative Publication of the Department of Plant Pathology, North Carolina State University and the US Agency for International Development, Raleigh, North Carolina.
  27. Tigano, M. S. , Carneiro, R. M. D. G. , Jeyaprakash, A. , Dickson, D. W. and Adams, B. J. 2005. Phylogeny of Meloidogyne spp. based on 18S rDNA and the intergenic region of mitochondrial DNA sequences. Nematology 7:851–62, doi: 10.1163/156854105776186325.
  28. Tigano, M. , De Siqueira, K. , Castagnone Sereno, P. , Mulet, K. , Queiroz, P. , Dos Santos, M. , Teixeira, C. , Almeida, M. , Silva, J. and Carneiro, R. M. D. G. 2010. Genetic diversity of the root-knot nematode Meloidogyne enterolobii and development of a SCAR marker for this guava damaging species. Plant Pathology 59:1054–1061.
  29. Triantaphyllou, A. C. 1985. “Cytogenetics, cytotaxonomy and phylogeny of root-knot nematodes”, in Sasser, J. N. and Carter, C. C. (Eds), An advanced Treatise on Meloidogyne 1 North Carolina State University Graphics, Raleigh, NC, pp. 113–126.
  30. Yap, I. V. and Nelson, R. J. 1996. WINBOOT: a program for performing bootstrap analysis for binary data to determine the confidence limits of UPGMA-based dendrograms. Discussion Paper Series No. 14, International Rice Research Institute, Manila, Philippines.
  31. Ye, W. , Zeng, Y. and Kerns, J. 2015. Molecular characterisation and diagnosis of root-knot nematodes (Meloidogyne spp.) from turfgrasses in North Carolina, USA. PLoS One 10, pp. 1–16, doi: 10.1371/journal.pone.0143556.
  32. Zijlstra, C. 2000. Identification of Meloidogyne chitwoodi, M. fallax and M. hapla based on SCAR-PCR: a powerful way of enabling reliable identification of populations or individuals that share common traits. European Journal of Plant Pathology 106:283–290.
  33. Zijlstra, C. , Donkers-Venne, D. T. H. M. and Fargette, M. 2000. Identification of Meloidogyne incognita, M. javanica and M. arenaria using sequence characterised amplified region (SCAR) based PCR assays. Nematology 2:847–853.

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