Document Type : Research Paper
Authors
1 Department of Plant Protection, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
2 Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran
Abstract
Keywords
Extended Abstract
Introduction
The genus Crassolabium Yeates, 1967 is a cosmopolitan taxon with an intricate taxonomical history. It consists of 38 valid species (Nguyen & Peña-Santiago, 2021). Till now molecular study on the species of this genus is rare and only four sequences are available for two species Crassolabium circuliferum (Loof, 1961) Peña-Santiago & Ciobanu, 2008 and C. costaricense Varela-Benavides & Peña-Santiago, 2018 in the GenBank. Nine species of this genus have been recorded from Iran so far. In this research, the molecular and morphological characteristics of four species namely C. angulosum, C. pumilum and C. rhopalocercum, and C. circuliferum were studied and presented.
Material and Methods
Soil samples were collected from the rhizosphere of forest trees in the southeast of Khorramabad County during 2022 and 2023. The nematodes were extracted using the modified Brown & Boag (1988) method, killed, fixed, and transferred to anhydrous glycerol by De Grisse (1969) and mounted on permanent glass slides. The species were identified using a light microscope equipped with Dino-Eye Eyepiece Camera based on morphological and morphometric characteristics. Photographs were taken with the same microscope equipped with a DP50 digital camera. Raw photographs were edited using Adobe® Photoshop® CS. Drawings were made using Corel DRAW® software version 12. For molecular study, the DNA was extracted from a single fresh individual (Archidona-Yuste et al., 2016), and the D2-D3 expansion segments of 28S rDNA were amplified using the D2A and D3B primers. PCR products were purified and sent for sequencing to Bioneer Corporation in South Korea. The obtained sequences were deposited into the GenBank database under accession numbers PP971540, PP971543, PP971541, and PP971544 for C. angulosum, C. pumilum, C. rhopalocercum and C. circuliferum respectively. The Basic Local Alignment Search Tool (BLAST) homology search program was used to compare the newly generated sequences with other available sequences in the GenBank database. The sequences were aligned using MEGA6 software and phylogenetic analysis of the sequences was performed using MrBayes 3.1.2. under the general time-reversible model with invariable sites and a gamma-shaped distribution (GTR + I + G). The tree was visualized using Fig Tree v.1.4.2
Results and Discussion
Based on morphological and morphometric characteristics, four species C. angulosum, C. pumilum, C. rhopalocercum, and C. circuliferum of the genus Crassolabium were identified. C. angulosum is reported for the first time for nematode fauna of Iran. The species is distinguished by cylindrical body, 1.63-1.77 mm length, open C-shape when heat relaxed and slightly curved ventrad, lip region angular and offset by marked depression, 13.5-14.0 µm wide or 2.0 to 2.5 times its height, lips slightly amalgamated with protruding papillae, amphidial fovea cup-like, amphidial aperture situated at the level of lip region base, 5.5-6.5 µm wide, odontostyle and odontophore 13-15 and 20-28 µm long, respectively, guiding ring simple and delicate, anterior part of pharynx muscular and cylindrical, pharyngeal expansion 157.5-180.0 µm long, cardia conical, genital system didelphic-amphidelphic, opposed, vulva longitudinal with two glandular cells present near the vagina, pretectum 2.5-3.0 anal body width long, rectum 35.5-38.5 µm long, tail rounded to conoid and dorsally convex. In this study for the first time, LSU D2-D3 sequences of C. angulosum, C. pumilum, and C. rhopalocercum were obtained and deposited into the GenBank database. The inferred phylogenetic tree showed that C. angulosum is clustered together with Nevadanema nevadense (JN242245) in a well-supported clade (BPP = 1) C. circuliferum along with some other species that already reported, have placed in the same clade together with Epidorylaimus lugdunensis (AY593036, AY593035) and E. procerus (MT612088) the two species that belonging to the family Qudsianematidae. C. rhopalocerum and Amblydorylaimus isokaryon (KM092520) from Aporcelaimidae have grouped in the same clade and C. pumilum is separated from the other species mentioned above and appeared as an independent lineage.
Conclusion
Four species of the genus Crassolabium namely C. angulosum, C. circuliferum, C. pumilum, and C. rhopalocercum belonging to Qudsianematidae were recovered and identified from the rhizosphere of oak trees in southeast of Khorramabad County. C. angulosum is reported for the first time from Iran. Nematodes of this genus are found in natural habitats and agricultural ecosystems and are considered a part of the bioindicators of ecosystem health evaluation studies. In the molecular phylogenetic study, the D2-D3 28S rDNA region of all four populations was sequenced and regarding the position of the four species in the phylogenetic tree, it seems that the genus Crassolabium could be currently considered as a polyphyletic genus because none of the sequences that have been registered so far, do form a separate clade altogether and are placed in different positions in the evolutionary tree derived from this study. Other researchers have already addressed this issue and the results of the present study confirm their findings.
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