Morphological Redescription and Molecular Phylogeny of Tylenchulus semipenetrans Populations on Citrus in Mediterranean Agroecosystems

Abstract

Citrus slow decline caused by Tylenchulus semipenetrans is a major constraint in Mediterranean agroecosystems, where long-term perennial cultivation favors nematode persistence and spread. The present study focuses on morphological redescription and molecular phylogeny of T. semipenetrans populations associated with citrus orchards. Nematode populations were extracted from rhizosphere soil and infected roots collected from multiple citrus-growing sites. Detailed morphological examination was conducted on key diagnostic features, including female body shape, stylet structure, esophageal gland morphology, and tail characteristics of juvenile stages. Morphometric measurements revealed variability among populations, while retaining core species-defining traits consistent with T. semipenetrans. For molecular characterization, genomic DNA was extracted and amplified using rDNA-ITS and D2–D3 expansion regions of the 28S rRNA gene. Sequencing and phylogenetic analyses confirmed the identity of all populations as T. semipenetrans, while revealing intra-specific genetic diversity among geographically separated populations. Phylogenetic clustering indicated partial grouping according to location, suggesting limited gene flow and possible local adaptation within Mediterranean citrus systems. Combined morphological and molecular data provided robust species delimitation and clarified subtle morphological variations previously reported within the species complex. Environmental conditions such as sandy-loam soils, warm temperatures, and continuous citrus cultivation were associated with higher nematode abundance and infestation severity. The study highlights the importance of integrative taxonomy for accurate identification of plant-parasitic nematodes. Overall, the results enhance understanding of morphological variability and genetic structure of T. semipenetrans populations and provide a strong basis for developing region-specific nematode management strategies in Mediterranean citrus agroecosystems.