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29Russian Journal of Nematology, 2015, 23 (1), 21 - 28
Characterisation of Longidorus iranicus Sturhan & Barooti, 1983 (Nematoda: Longidoridae) from Iran and synonymisation of L. moesicus Lamberti, Choleva
& Agostinelli, 1983
1 2, 3 4 1
Zahra Tanha Maafi , Sergei A. Subbotin , Dieter Sturhan , Shapour Barooti and
1
Zahra Majd Taheri
1 Iranian Research Institute of Plant Protection, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran 2 Plant Pest Diagnostic Center, California Department of Food and Agriculture, 3294 Meadowview Road,
95832, Sacramento, CA, USA 3 Center of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences,
Leninskii Prospect 33, 117071, Moscow, Russia 4 Arnethstrasse 13D, 48159, Münster, Germany e-mail: [email protected]
Accepted for publication 16 March 2015
Summary. The needle nematode, Longidorus iranicus Sturhan & Barooti, 1983 has been described from Iran and not reported from any other country so far. Based on the studies of a population from the rhizosphere of raspberry in the north of Iran a detailed description is presented and males are described for the first time. Among the total of more than 150 Longidorus species, L. iranicus is morphologically most close to L. moesicus. Comparative morphological and morphometric characters are given for three populations of L. iranicus and populations of L. moesicus, including the original descriptions of both species and other references. Morphological and molecular studies indicate species identity and synonymy of L. moesicus with L. iranicus is proposed in the present paper. Key words: morphology, morphometrics, needle nematode, synonymy, taxonomy.
The needle nematodes of the genus Longidorus Micoletzky, 1922 comprise more than 150 valid species (Decraemer & Robbins, 2007). Presently, fifteen species have been reported from Iran and three of them were only found in Iran: L. iranicus Sturhan & Barooti, 1983, L. kheirii Pedram, Niknam, Robbins, Ye & Karegar, 2008 and L. tabrizicus Niknam, Pedram, Nejad, Ye, Robbins & Maafi, 2010.
The species L. africanus Merny, 1966, L. pisi Edward, Misra & Singh, 1964 and L. vineacola Sturhan & Weischer, 1964 were reported from the rhizosphere of pistachio, pomegranate, citrus and watermelon in southeast and north of Iran (Kheiri & Barooti, 1984). Longidorus crassus Thorne, 1974, L. elongatus (de Man, 1876) Micoletzky, 1922, L. protae Lamberti & Bleve-Zacheo, 1977 and L. proximus Sturhan & Argo, 1983 were found in the northwest from crops and grapevine (Ghaderi et al., 2012). Four species of Longidorus: L. leptocephalus
Hooper, 1961, L. profundorum Hooper, 1966, L. euonymus Mali & Hooper, 1974 and L. sturhani Rubtsova, Subbotin, Brown & Moens, 2001 were isolated from woody plants in the northwest (Pedram et al., 2008). Longidorus orientalis Loof, 1983 was mainly recorded from date palm in the southeast and south of Iran (Noruzi & Barooti, 2005; Gharakhani et al., 2007; Palomares-Rius et al, 2010).
Longidorus iranicus is one of the three species described only from Iran. This species was first reported from the rhizosphere of several crops, i.e., grapevine, olive, apple, pear, rice, cabbage and tomato, in the provinces of Markazi, Ghazvin, Mazandaran, Gilan, East Azerbaijan and West Azerbaijan (Sturhan & Barooti, 1983) located in the central, north and northwest of Iran. Longidorus iranicus was also recorded in the southern part of the country from the rhizosphere of tomato, citrus and date palm (Ghaderi et al., 2012). It was also
reported from soil around fruit trees: apple, pear, plum and apricot in the northwest of Iran (Fadaei Tehrani & Kheiri, 2004) and peach, sweet cherry, hazelnut from Ardabil (Ghaderi et al, 2012). Based on the studies of a population from the rhizosphere of raspberry in Mazandaran province a detailed description is presented and males are described for the first time in this study.
Longidorus moesicus was firstly described by Lamberti et al. (1983) from the rhizosphere of black currant (Ribes nigrum) at Kostinbrod near Sofia, Bulgaria. Afterwards it was reported from different regions of Serbia from the rhizosphere of many plants, such as Populus sp., Trifolium sp., pear and grapevine, Urtica sp. and Rubus sp. (Barsi & Lamberti, 2004). More recently, L. moesicus was reported from Slovenia in a vineyard soil sample and characterised morphologically and molecularly along with five other Longidorus species (Sirca & Urek, 2009). The Crete Island in Greece was the latest locality where L. moesicus was found in the rhizosphere of grapevine and morphologically and molecularly characterised (Tzortzakakis et al., 2014).
Morphological and molecular studies of the populations of L. iranicus and comparison of the data with the available records for L. moesicus were the main purposes of this study.
MATERIALS AND METHODS
Soil samples were collected from the rhizosphere of raspberry, citrus (Mazandaran province), walnut, raspberry, alder (Alnus sp.) (Golestan province) and oak (Ilam province). Nematodes were extracted from soil and fixed using the tray method (Whitehead & Hemming, 1965), fixed in formalin, transferred in dehydrated glycerin and processed onto permanent slides (De Grisse, 1969). Females and males were subsequently used for morphological studies. Morphometric features were measured by using a camera lucida installed on a light microscope (Olympus BH-2). The measurements are expressed in micrometers (mm for body length and ^m for other characters) and presented as a mean and the standard deviation of the mean with the range in parentheses. Photomicrographs were taken by digital camera (Dino Capture 2.0) via a light microscope Olympus BH-2 with Nomarski differential interference contrast.
DNA was extracted from single females. Protocols for DNA extraction, PCR and sequencing were described by Tanha Maafi et al. (2003). The following primers were used for amplification of two rRNA gene fragments. The ITS1 region using
forward primer 18S (5' -TTGATTACGTCCCTGCC CTTT-3') and reverse primer rDNA1 (5'-ACGAGC CGAGTGATCCACCG-3') as described in Wang et al. (2003), the D2-D3 expansion segments of 28S rRNA gene with the forward D2A (5'-ACAAGTAC CGTGAGGGAAAGTTG-3') and the reverse D3B (5' -TCGGAAGGAACCAGCTACTA-3') primers (Rubtsova et al., 2001). The PCR products were sequenced at MWG Biotech (Ebersberg, Munich, Germany) and the sequences were submitted to the GenBank database under the accession numbers KP222294 and KP222295.
DESCRIPTION
Longidorus iranicus Sturhan & Barooti, 1983 (Figs 1 & 2)
Measurements. Table 1.
Female. Body long, variously shaped after fixation: ventrally curved to open C, open coiled to irregular (Figs 1O & P). Lip region rounded and narrow, continuous to slightly offset from the rest of the body. Amphidial pouches long, more than half to 70% of the distance from oral opening to guiding ring, slightly bilobed at base, with lobes of almost equal length (Fig. 2). Guiding ring typical of the genus. Odontostyle long and slender, odontophore less than half its length. Nerve ring located about equivalent to the corresponding body diameter posterior to odontophore. Pharynx typical dorylaimoid, Pharyngeal bulb muscular, dorsal pharyngeal gland nucleus location is variable from one-third of pharyngeal bulb length to mid-bulb, nuclei of the two ventro-sublateral glands located at the posterior third part of the bulb. Pharyngeal intestinal valve rounded. Rectum almost equal to body width at anus. Genital tract didelphic, amphidelphic with reflexed ovaries, occupying 78% of the total body length, oviduct as a narrow tube separated from the uterus by a sphincter, uterus long, without sperm. Vulva a transverse slit, vagina occupying more than half the corresponding body width with conspicuous sphincter muscles. Tail bluntly convex conoid, short, slightly less than one anal body width long, one pair of caudal pores on each side visible.
Male. Body long, curved ventrally after fixation, the posterior part of body being complete spiral specially near tail (Figs 1M & N). Lip region rounded, slightly offset from the rest of the body. Amphidial pouches similar to female with small lobes, guiding ring conspicuous and located less than three corresponding lip region widths from the oral aperture, odontostyle long and slender, odontophore less than half of odontostyle length,
Characterisation of Longidorus iranicus
nerve ring located posterior to odontophore. Dorsal pharyngeal gland nucleus located almost in the anterior part of pharyngeal bulb and at one-third of its length, nuclei of the two ventro-sublateral glands located at 62-64% of the bulb length. Testis diorchic, opposed, with conspicuous fusiform to oval sperm, posterior testis reflexed, T = 38% and
53%, anterior testis longer, T = 45% and 61%. Spicules curved ventrally, heavily sclerotised, lateral guiding pieces (crura) sclerotised, 15 and 17 pm long. An adanal pair plus a ventral series of about 8 to 10 supplementary papillae present. Tail conoid, dorsally convex, terminus rounded, with two pairs of caudal pores.
Fig. 1. Longidorus iranicus. A, B, F-K, O, P: female; C-E, L-N: male. A-D: anterior end; F: pharyngeal bulb; G-I: tail of three juvenile stages; J-K: female tail; O, P: shape of fixed-relaxed female; C, D: anterior end, E-F: pharyngeal bulb, L: male posterior part; M, N: shape of fixed-relaxed male.
Fig. 2. Longidorus iranicus photomicrographs. A, B, E: female; C, D, F-J: male. A-D: anterior end; E: tail; F: sperm in testis, H, I: pharyngeal bulb showing pharyngeal ventro-sublateral glands nuclei; G, J: tail showing spicules and preanal supplementary papillae.
Table 1. Morphometric characters of Longidous ircmicus and L. moesicus (measurements in mm for body length and in |im for other characters except ratios)
Characters L. ircmicus L. moesicus
Sturhan & Barooti (1983) Fadaei Tehrani & Kheiri (2004) The present study Lamberti et al. (1983) Barsi & Lamberti (2004) Sirca & Urek (2009) Tzortzakakis etal. (2014)
2 females
n 8 females 10 females 11 females 2 males 10 females 15 females 17 females (P. Elias population)
L 5.09(5.22-7.1) 5.7(5-6.5) 6.28±0.6 (5.1-7.14) 4.92, 7.5 7.2 (6.4-8) 6.5±0.63 (5.7-7.56) 6.9i0.7 (5.9-8.4) 5.06, 5.61
a 103 (92-115) 104.6(100-112) 90.3±9.8 (78.4-109.8) 82.1, 124.4 120(96-147) 124.4±6.46 (115.4-139.5) 128.6i7.4 (117-145.5) 97.5, 105.9
b 12.7(9-14.9) 12.6(10-15.2) 13.5il.8 (11.5-17.2) 10.9, 18.7 15.8(13.8-18.8) 13.8Ü.3 (11.5-16.4) 15.8il.4 (13.1-18.3) 13.5, 16.3
c 180(151-220) 162.5 (150-182) 183.4±30.3 (137.6-237.8) 111.9, 196.4 170(146-186) 166.8±15.28 (141-197) 164.3Ü8 (129-200.8) 137.0, 160.4
c" 0.84 (0.78-1) 0.92 (0.81-1.2) 0.8±0.1 (0.7-0.9) 0.9, 1 1 (0.8-1.2) 0.94±0.09 (0.79-1.06) 1.05i0.08 (0.90-1.23) 0.9, 1.0
V% 50.6 (48.8-52) 50.7(49-53) 49.4±2.3 (44.5-52.9) - 53 (50-54) 48.9±1.73 (44.8-51.5) 52.7il.4 (49.6-54.6) 48, 49
Stylet length - - 143.7±6.2 (131-150) 136, 144 - 174.3±5.05 (163.8-183.8) 155.8i5.9 (143.1-161) -
Odontostyle length 112(106-118) 104.5 (100-110) 104.5±4.1 (97-111) 96, 103 119(115-124) 114±5.60 (102.5-125) 107.Ü4.4 (96.3-114.6) 97, 104
Odontophore length 43 (40-48) 65 (58-72) 41.7±3.1 (34-49) 40,41 63 (59-66) 60.3±3.70 (55-67.5) 48.7i4 (42-56.4) 51, 55
Oral aperture to guide ring 35 (33.5-36.5) 35.3 (30-40) 35.1±1 (33-37) 38 34(32-38) 37.6±1.80 (34.4-41.9) 35.6il.6 (32.8-39.1) 30, 34.5
Body diameter at lip region - - 14.3±1.1 (13-16) 14, 16 12(11-13) 13.1±0.36 (12.8-13.8) 12.4il (10.5-13.9) 10.5, 11.5
Body diameter at guide ring - - 27.7±3.2 (23-34) 25,33 25 (24-26) 25.1±0.81 (23.4-26.3) 26.6il.l (24.2-28.4) -
Body diameter at base of pharynx - - 55.5±3.9 (50-62) 50, 64 50 (45-61) 44.4±2.50 (40-49.7) 46i2.9 (41.9-50.8) -
Distance from oral aperture to the base 459±26.3 387, 425 437.6i27.5
of pharynx (410-495) (381-466)
Body diameter at vulva - - 68.2±3.9 (62-75) - 61 (52-71) 52.1±3.04 (48.1-58.4) 53.6i3.7 (48.4-59.9) -
Body diameter at anus - 43±2 (41-46) - 42 (39-49) 40.5il.67 (37.8-43.1) 40.2i3 (36.3-46.1) -
J (hyaline portion of tail) - - 12.7il.8 (10-15) - 13 (12-13) ll.3il.31 (9.4-13.8) - -
Body diameter at beginning of J - - 28.5±2.2 (24-32) - 27 (24-30) 24.2il.60 (21.3-28.3) - -
Tail length 34 (29-41) - 33.4±3.2 (28-38) - 43 (40-49) 39i2.45 (34.3-42.5) 42.2i3.8 (36.2-49.4) 35,37
Spicule length 55. 58
Molecular characterisation. A comparison of the results of the obtained sequences of the D2-D3 expansion segments of the 28S rRNA gene from L. iranicus with those from L. moesicus from Slovenia and Crete, Greece, showed 99% similarity and differed by only two nucleotides, with one of the differences being in the poly T and A regions, which are rather common regions for mutations. The ITS1 of rRNA gene sequence of L. iranicus contained several ambiguous nucleotide positions and it was different from that of the Crete L. moesicus at least by one nucleotide.
DISCUSSION
Chen et al. (1997) indicated morphological similarities of four species of Longidorus including L. iranicus, L. iuglandis Roca, Lamberti & Agostinelli, 1984, L. moesicus and L. olegi Kankina & Metlitskaya, 1983 and pointed out the difficulties of their separation due to sharing morphological and morphometrics characters, i.e., similar body and odontostyle length, rounded lip region, symmetrical bilobed amphidial pouches and width of the lip region.
The morphometric characters of three populations of L. iranicus and of three populations of L. moesicus, including the original descriptions of both species and of the recently reported Cretan population are presented and compared in Table 1. The morphometric characters of both species are similar, with the same codes of the polytomous key of Chen et al. (1997): A345, B12, C3, D1, E23, F-3, G23, H12, which is also in agreement with Cretan population (Tzortzakakis et al., 2014) except for the key code H. The only noticeable difference between the two species appears to be the presence of males in L. moesicus, while no male was available for the original description of L. iranicus and no sperm was observed in the genital tracts of the females. However, in our present study two males were recovered from the soil sample collected from the rhizosphere of raspberry and alder at Minoodasht in Golestan province. Although, in surveys from Serbia and Crete Island males were not found in L. moesicus (Barsi & Lamberti, 2004; Tzortzakakis et al, 2014).
In addition to morphological, morphometric and molecular similarities, the type habitats of both species are also similar; both species were found mainly in vineyard soil and the rhizosphere of fruit trees as well as the bush plants raspberry and black currant.
Based on the morphological and molecular similarities of L. iranicus and L. moesicus, we
suggest synonymy of these two species. Because of the journal issue with the description of L. iranicus appeared 15/03/1983, and the journal issue containing the description of L. moesicus was published 15/06/1983, the name L. iranicus has the priority over L moesicus; thus, L moesicus is suggested as its junior synonym.
REFERENCES
Barsi, L. & Lamberti, F. 2004. Morphometrics of Longidorus juvenilis and first record of L. aetnaeus and L. moesicus from Serbia. Nematologia Mediterranea 32: 5-14. Chen, Q., Hooper, D.J., Loof, P.A.A. & Xu, J. 1997. A revised polytomous key for the identification of the genus Longidorus Micoletzky, 1922 (Nematoda: Dorylaimoidea). Fundamental and Applied Nematology 20: 15-28. De Grisse, A. 1969. Redescription ou modifications de quelques techniques utilisées dans l'étude des nematodes phytoparasitaires. Mededelingen Faculteit der Landbouwwetenschappen Rijksuniversiteit Gent 34: 351-369.
Decraemer, W. & Robbins, R.T. 2007. The who, what and where of Longidoridae and Trichodoridae. Journal of Nematology 39: 295-297. Fadaei Tehrani, A. & Kheiri, A. 2004. [Some species of Longidorus from Iran]. Sciences and Agricultural Technology 9: 239-248 (in Persian). Ghaderi, R., Kashi Nahanji, L. & Karegar Bideh, A. 2012. [The nematodes of Iran based on the published reports until 2011]. Iran, Agricultural Education and Extension. 371 pp (in Persian). Gharakhani, A., Pourjam, E. & Karegar, A. 2007. [Some plant parasitic nematodes (Criconematoidea and Longidoridae) in Kerman province orchards]. Iranian Journal of Plant Pathology 43: 372-397 (in Persian).
Kheiri, A. & Barooti, Sh. 1984. [Species of Dorylaimoidea (Nematoda: Dorylaimida) from iran]. Iranian Journal of Plant Pathology 20: 8-20 (in Persian).
Lamberti, F., Choleva, B. & Agostinelli, A. 1983. Longidoridae from Bulgaria (Nematoda, Dorylaimida) with description of three new species of Longidorus and two new species of Xiphinema. Nematologia Mediterranea 11: 49-72. Niknam, G., Pedram, M., Nejad, E.G., Ye, W., Robbins, R.T. & Maafi, Z.T. 2010. Morphological and molecular characterization of Longidorus tabrizicus sp. n. and L. sturhani Rubtsova, Subbotin, Brown & Moens, 2001 (Nematoda: Longidoridae) from north-western Iran. Russian Journal of Nematology 18: 127-140.
Characterisation of Longidorus iranicus
Noruzi, R. & Barooti, Sh. 2005. Presence of Longidorus orientalis Loof, 1983 in the South of Iran. Iranian Journal of Plant Pathology 41: 141-142.
Palomares-Rius, J.E., Landa, B.B., Tanha Maafi, Z., Hunt, D.J. & Castillo, P. 2010. Comparative morphometrics and ribosomal DNA sequence analysis of Longidorus orientalis Loof, 1983 (Nematoda: Longidoridae) from Spain and Iran. Nematology 12: 631-640.
Pedram, M., Niknam, G., Robbins, R.T., Ye, W. & Karegar, A. 2008. Longidorus kheirii n. sp. (Nematoda: Longidoridae) from Iran. Systematic Parasitology 71: 199-211.
Rubtsova, T.V., Subbotin, S.A., Brown, D.J.F. & Moens, M. 2001. Description of Longidorus sturhani sp. n. (Nematoda: Longidoridae) and molecular characterization of several longidorid species from Western Europe. Russian Journal of Nematology 9: 127-136.
Sirca, S. & Urek, G. 2009. Morphological and molecular characterization of six Longidorus species (Nematoda: Longidoridae) from Slovenia. Russian Journal of Nematology 17: 95-105.
Sturhan, D. & Barooti, S.H. 1983. Longidorus iranicus n. sp. (Nematoda: Dorylaimida). Systematic Parasitology 5: 21-24.
Tanha Maafi, Z., Subbotin, S.A. & Moens, M. 2003. Molecular identification of cyst-forming nematodes (Heteroderidae) from Iran and a phylogeny based on ITS-rDNA sequences. Nematology 5: 99-111.
Tzortzakakis, E.A., Archidona-Vuste, A., Cantalapiedra-Navarrete, C., Nasiou, E., Lazanaki, M.S., Kabourakis, E.M., Palomares-Rius, J.E. & Castillo, P. 2014. Integrative diagnosis and molecular phylogeny of dagger and needle nematodes of olives and grapevines in the island of Crete, Greece, with description of Xiphinema cretense n. sp. (Nematoda, Longidoridae). European Journal of Plant Pathology 140: 563-590.
Wang, X., Bosselut, N., Castagnone, C., Voisin, R., Abad, P. & Esmenjaud, D. 2003. Multiplex polymerase chain reaction identification of single individuals of the Longidorid nematodes Xiphinema index, X diversicaudatum, X vuittenezi and X. italiae using specific primers from ribosomal genes. Phytopathology 93: 160-166.
Whitehead, A.G. & Hemming, J.R. 1965. Comparison of some quantitative methods of extracting small vermiform nematodes from soil. Annals of Applied Biology 55: 25-38.
Z. Tanha Maafi, S.A. Subbotin, D. Sturhan, Sh. Barooti and Z. Majd Taheri. Данные к описанию Longidorus iranicus Sturhan & Barooti, 1983 (Nematoda: Longidoridae) из Ирана и синонимизация L. moesicus Lamberti, Choleva & Agostinelli, 1983.
Резюме. Нематода Longidorus iranicus Sturhan & Barooti, 1983 до настоящего времени не была отмечена нигде кроме Ирана. Представлено подробное описание этого вида по особям из популяции, выделенной из ризосферы малины в Сев. Иране. Впервые приводится описание самцов. Среди более чем 150 видов Longidorus, L. iranicus морфологически ближе всего к L. moesicus. Морфологические и морфометрические параметры трех популяций L. iranicus и одной популяции L. moesicus приводятся в сравнительном аспекте, включая данные первоописаний и других находок. На основе сравнения морфологических и молекулярных данных предлагается синонимизация L. moesicus и L. iranicus.
