Molecular-genetic analysis of Meloidogyne (Heteroderidae) species from Surxondaryo Region, Uzbekistan based on ITS markers

S. Choriyev; A. Khurramov; O. Amirov; D. Mardonayeva; S. Bobokeldiyeva; B. Asliddin; G. Narzullaeva; N. Khusanov

doi:10.15421/0225217

S. Choriyev Termez State University
A. Khurramov Termez State University
O. Amirov Institute of Zoology
D. Mardonayeva Termez State University
S. Bobokeldiyeva Termez State University
B. Asliddin Denov Institute of Entrepreneurship and Pedagogy
G. Narzullaeva Termez Branch of Tashkent Medical Academy
N. Khusanov National University of Uzbekistan named after Mirzo Ulugbek

Keywords: Meloidogyne, ITS, phylogeny, genetic diversity, K2P, haplotype, Uzbekistan.

Abstract

This study investigated the molecular-genetic characteristics of Meloidogyne species from Surxondaryo region, Uzbekistan, using ITS (Internal Transcribed Spacer) nucleotide sequences. A total of 765 root samples from peanut, carrot, sugar beet, cucumber, and tomato were collected and analyzed. Nematodes were morphologically identified, and genomic DNA was extracted from mature females. The 5.8S–ITS2 region was amplified using TW81 and AB28 primers, sequenced, and analyzed phylogenetically using Maximum Likelihood methods with IQ-TREE 2 and 1000 bootstrap replicates. Multiple sequence alignments were performed with MAFFT, and genetic diversity indices, inclu d ing haplotype diversity (Hd), nucleotide diversity (π), Tajima’s D, and Kimura 2-parameter (K2P) distances, were calculated in R using ape and pegas packages. Phylogenetic analyses revealed four main clusters corresponding to M. arenaria , M. incognita , M. javanica , and M. konaensis , with high bootstrap support (≥70%), confirming their mon o phyletic nature and clear inter-species differentiation. K2P distance analyses showed very low intra-species divergence (0.0015–0.0095) and high inter-species differentiation (0.040–0.055), indicating strong molecular conservation within species. Haplotype analysis revealed high diversity (Hd = 0.937), while nucleotide diversity was low (π = 0.052), su g gesting recent population expansion, close evolutionary relationships, or selective pressures shaping the genetic stru c ture. Overall, the results demonstrate that the ITS marker is a reliable molecular tool for species identification and pop u lation-genetic studies in Meloidogyne . The inclusion of Uzbek isolates provides molecular confirmation of their ta x onomic status and establishes a foundation for future ecological and genetic research.

References

Blok, V. C., & Powers, T. O. (2009). Biochemical and molecular identification. In: Perry, R. N., Moens, M., & Starr, J. L. (Eds.). Root-knot nematodes. CABI Publishing, Wallingford. Pp. 98–118.

Blok, V. C., Phillips, M. S., & Fargette, M. (2008). Genetic diversity and phylogeny of Meloidogyne javanica populations. Nematology, 10(3), 345–354.

Cabrera, J., Barcala, M., Fenoll, C., & Escobar, C. (2016). The power of omics to identify plant susceptibility factors and to study resistance to root-knot nematodes. Current Issues in Molecular Biology, 19, 53–72.

Carneiro, R., Dickson, D., Jeyaprakash, A., Adams, B., & Tigano, M. (2005). Phylogeny of Meloidogyne spp. based on 18S rDNA and the intergenic region of mitochondrial DNA sequences. Nematology, 7(6), 851–862.

Castagnone-Sereno, P., Bongiovanni, M., Wajnberg, E., & Dalmasso, A. (2013). Genetic structure and evolutionary relationships among populations of Meloidogyne arenaria. Journal of Nematology, 45(2), 123–133.

Choriyev, S., Khurramov, A., Khurramov, S., & Mardonayeva, D. (2024). Ecological analysis of peanut nematodes in Surkhondaryo Region. In: BIO Web of Conferences, 100, 04006.

Curran, J. A., Argyle, D. J., Cox, P., Onions, D. E., & Nicolson, L. (1994). Nucleotide sequence of the equine interferon gamma cDNA. DNA Sequence, 4(6), 405–407.

Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39(4), 783–791.

Forghani, F., & Hajihassani, A. (2020). Recent advances in the development of environmentally benign treatments to control root-knot nematodes. Frontiers in Plant Science, 11, 1125.

Hunt, D. J., & Handoo, Z. A. (2009). Taxonomy, identification and principal species. In: Perry, R. N., Moens, M., & Starr, J. L. (Eds.). Root-knot nematodes. CABI Publishing, Wallingford. Pp. 55–97.

Hussey, R. S. (1979). Biochemical systematics of nematodes – a review. Helminthological Abstracts, Series B, Plant Nematology, 48, 141–148.

Hyman, B. C. (1990). Molecular diagnosis of Meloidogyne species. Journal of Nematology, 22(1), 24–30.

Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution, 30(4), 772–780.

Kavuluko, J., Gichuki, C., Wanjohi, W., & Runo, S. (2016). Molecular characterization and genetic variation of root-knot nematode (Meloidogyne spp.) in selected legume production areas of Eastern Kenya. American Scientific Research Journal for Engineering, Technology, and Sciences, 18(1), 67–83.

Khanal, C., Robbins, R. T., Faske, T. R., Szalanski, A. L., McGawley, E. C., & Overstreet, C. (2016). Identification and haplotype designation of Meloidogyne spp. of Arkansas using molecular diagnostics. Nematropica, 46(2), 261–270.

Khuramov, A., Bobokeldieva, L., Choriyev, S., Rakhmatullaev, B., Khimmatov, N., Raimov, S., Narzullaeva, G., Bobokeldiyeva, S., Mukhiddinova, M., & Karshieva, M. (2024). A comprehensive study of phytonematodes of grape plants in the conditions of the Surkhandarya Valley, Uzbekistan. Biodiversitas Journal of Biological Diversity, 25(11), 3.

Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16(2), 111–120.

Mendy, B., Wang’ombe, M. W., Radakovic, Z. S., Holbein, J., Ilyas, M., Chopra, D., Holton, N., Zipfel, C., Grundler, F. M. W., & Siddique, S. (2017). Arabidopsis leucine-rich repeat receptor–like kinase NILR1 is required for induction of innate immunity to parasitic nematodes. PLoS Pathogens, 13(4), e1006284.

Minh, B. Q., Schmidt, H. A., Chernomor, O., Schrempf, D., Woodhams, M. D., von Haeseler, A., & Lanfear, R. (2020). IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Molecular Biology and Evolution, 37(5), 1530–1534.

Paradis, E. (2010). pegas: An R package for population genetics with an integrated-modular approach. Bioinformatics, 26(3), 419–420.

Paradis, E., & Schliep, K. (2018). ape 5.0: An environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics, 35(3), 526–528.

Sasser, J. N. (1980). Root-knot nematodes: A global menace to crop production. Plant Disease, 64(1), 36–47.

Somasekhar, N., & Prasad, J. S. (2011). Plant – nematode interactions: Consequences of climate change. In: Venkateswarlu, B., Shanker, A. K., Shanker, C., & Maheswari, M. (Eds.). Crop stress and its management: Perspectives and strategies. Springer, Dordrecht. Pp. 547–564.

Tajima, F. (1989). Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics, 123(3), 585–595.

Whitehead, A. G. (1968). Taxonomy of Meloidogyne (Nematodea: Heteroderidae) with descriptions of four new species. The Transactions of the Zoological Society of London, 31(3), 263–401.

Ye, W., Robbins, R. T., & Kirkpatrick, T. (2019). Molecular characterization of root-knot nematodes (Meloidogyne spp.) from Arkansas, USA. Scientific Reports, 9, 15680.

Ye, W., Zeng, Y., & Kerns, J. (2015). Molecular characterisation and diagnosis of root-knot nematodes (Meloidogyne spp.) from turfgrasses in North Carolina, USA. PLoS One, 10(11), e0143556.

Zeng, Y., Ye, W., Tredway, L., Martin, S., & Martin, M. (2012). Taxonomy and morphology of plant-parasitic nematodes associated with turfgrasses in North and South Carolina, USA. Zootaxa, 3452(1), 1–46.