Morphometrical and molecular data on plant parasitic nematodes Longidorus attenuatus Hooper, 1961 and L. danuvii Barsi et al., 2007 (Nematoda: Longidoridae) reported from Ukraine for the first time

Nematode genus Longidorus Micoletzky, 1927 consists of about 150 species of obligatory plant root ectoparasites. Some of these species are of economic importance as pests of agricultural plants (Singh et al., 2013 gives general review). This importance is further augmented by the fact that several of genus species are known to be virus vectors. An example of such important species is Longidorus attenuatus Hooper, 1961, which is associated with ‘docking’ disease of sugarbeet (Whitehead & Hooper, 1970). This species is also a vector of Tomato black ring virus (Harrison, 1964), which makes it important to viticulture industry (Hübschen et al., 2004). This nematode is rather widely distributed, as it is known from several European countries (e.g. Brown & Taylor, 1987). However, up to date it has not been recorded from Ukraine (Peneva et al., 2009). In comparison with L. attenunatus, less is known on L. danuviiBarsi, Lamberti and De Luca, 2007, as data on this species include original description based on populations from Serbia (Barsi et al., 2007) and subsequent description of population from Poland (Kornobis, 2012). Up to date, this nematode was found in association with Amorpha fruticosa L. and Populus sp. (Barsi et al., 2007) and Salix alba L. (Kornobis, 2012). The aim of this Research Note is to present first records of both of the above mentioned nematode species from Ukraine, their morphometrics, D2-D3 rDNA region sequence and data on association of L. danuvii with plants of agricultural importance. Additionally, D2-D3 rDNA sequence was obtained from population described previously from Poland (Kornobis, 2012).

Soil samples containing L. attenuatus and L. danuvii were taken from localities and plants given in Table 1. Nematodes were extracted from soil by sieving and decanting method (Brown & Boag, 1988). From each sample a few specimens were hand-picked under Olympus SZX 10 stereomicroscope and fixed in DESS (Yoder et al., 2006). Remaining nematodes were heat killed, fixed in TAF, processed to glycerol by a slow evaporation method and mounted on permanent slides. Identification and measurements were made using Olympus BX 51 microscope with Nomarski differential interference contrast, equipped with a digital camera Olympus DP 72 and computer program Quick PHOTO MICRO 2.3. DNA was isolated using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instruction. Both PCR and sequencing were done using primers D2A and D3B (Nunn 1992). PCR conditions were as described by Kornobis et al. (2015). Quality of obtained chromatograms was checked in ChromasLite 2.1.1 (Chromas Lite, Technelysium, Queensland, Australia). Sequences were edited in BioEdit (Hall, 1999). Obtained sequences were deposited in Gen-Bank under KT755457 accession number for L. attenuatus and KU507542 and KT755458 for L. danuvii from Poland and Ukraine, respectively. Subsequently three alignments were made: in first L. attenuatus sequence obtained here was aligned with D2-D3 28S rDNA sequence (GenBank accession: AY601572) provided by He et al. (2005). In second L. danuvii sequences obtained in this study were compared, in third alignment these sequences were aligned with D3 28S rDNA sequences AM412364-66 provided by Barsi et al. (2007) Sequences were aligned using MUSCLE (Edgar, 2004) implemented in MEGA 6 (Tamura et al., 2013).

Three populations of Longidorus danuvii were collected in the city of Lviv. This is the first record of this species from Ukraine. Lviv populations were found in relatively dry conditions, with two populations on the tops of the hills (300 – 350 m.a.s.l.) in the central part of the city, in biotopes distant from watercourses, unlike Serbian and Polish populations, which were found on the banks of Danube and Strwiaz rivers (Barsi et al., 2007; Kornobis, 2012). Additionally, L. danuvii was found in the rhizosphere of plants of agricultural importance (Malus domestica Borkh., Pyrus sp., Rubus idaeus L.) for the first time. It is possible that this species has a negative impact on these plant species, further studies are however required to evaluate that. Longidorus attenuatus population (first record for Ukraine) was collected in the garden in rhizosphere of apple tree, forming mixed population with L. danuvii.

Morphometrics of both species are given in Tables 1 and 2. Morphology of L. danuvii is illustrated on Fig. 1 A-H. Three Lviv populations of L. danuvii are morphologically and morphometrically similar to each other. However, some minor morphometric differences are present between them. These populations differ the most clearly in a (mean 101.5 vs 113.5) and c (mean 99.6 vs 120.4) values. Morphometrical characters of Lviv populations are close to the range of variability of type Serbian population (Barsi et al., 2007). Ukrainian populations differ from Serbian ones mainly in thicker body (mean diameter at vulva 37.4 and 37.8 vs 40.5; 42.4 and 43.2 μm) which results in lower a index values (means 100.1; 102.6 and 113.5 vs 119.8 and 123.5). Other worth mentioning differences among these populations include dimensions and shape of tail, which is shorter in Ukrainian populations (mean 40.1; 42.1 and 41.8 vs 45.2 and 46.5 μm), has lower c’ index (1.45; 1.52 and 1.59 vs 1.7 and 1.85) and somewhat more rounded terminus in females (Fig. 1 E-H). In comparison with Polish population (Kornobis, 2012) juveniles from Lviv (Citadel Park) are characterized by shorter odontostylet in J4 (76.1 vs 82.6) and shorter replacement odontostylet in all juvenile stages (J1 – 61.0 vs 64.8; J2 – 68.8 vs 74.3; J3 – 77.9 vs 86.0; J4 – 84.8 vs 92.0). No males were present among collected specimens of L. danuvii.

Fig. 1

Alignment of D2-D3 rDNA marker in L. attenuatus contained 712 positions. In comparison with sequence AY601572 obtained by He et al. (2005), sequence from Ukraine differed in one nucleotide. Alignment of D2-D3 L. danuvii sequences obtained here contained 698 positions, sequences were identical. Finally, alignment of D3 sequences contained 287 positions. In comparison with sequences provided by Barsi et al. (2007) sequences from Ukraine and Poland differed in one nucleotide.