First record of the ectoparasitic nematode Amplimerlinius macrurus (Nematoda: Tylenchida) on the perennial grass Miscanthus × giganteus (Angiosperms: Poaceae) in Ukraine

Miscanthus × giganteus J. M. Greef, Deuter ex Hodk., Renvoize (Angiospermae: Poaceae) (M × g) is a rhizomatous, lignocellulose-rich perennial grass grown worldwide as a biofuel crop and a source of different bio-based products (Clifton-Brown, 2017; Cosentino et al., 2018; Lewandowski et al., 2018). This plant is the most common second-generation biofuel crop for commercial production in Ukraine due to its rapid growth and high yields in agricultural and marginal/contaminated soils of various anthropogenic origins (Kharytonov et al., 2019; Pidlisnyuk et al., 2020). Stefanovska et al. (2017) found several herbivorous insects associated with the cultivation of M × g being grown in the plantation as a source of bioenergy. Plant-parasitic nematodes may also affect M × g yields (Mekete et al., 2009, 2011). Different agronomic practices affect M × g yield and nematode communities, the latest can be used as a bioindication of the phytoremediation process (Almasary et al., 2020). Earlier, a soil survey on identifying plant-parasitic nematodes associated with the cultivation of M × g was conducted in eight locations in Ukraine (Stefanovska et al., 2020) in order to assess the potential for nematodes to reduce yield. During the survey Amplimerlinius macrurus (Goodey, 1932) was detected in the soil under M × g at two of eight locations: those locations were Dolyna (site was located within city) and Grytzyv (village is located in Khmelnitska region).

The history of the species knowledge may serve as an example of confuse state of nomenclature. It started in 1914 with a first note on Aphelenchus dubius spez. nov. by Steiner from Switzerland (Steiner, 1914) and was gone through several name changes and genus transfers. In 1932, Goodey redescribed the species and named it as Anguillulina macrura, which was a feeder in the cortical cell layer of the roots of the grass Lolium perenne L. (Goodey, 1943). The next name change was made in 1936 in the new genus Tylenchorhynchus macrurus (Filipjev, 1936) and redescribed by Wallace and Greet (1964). Finally, the last replacement of the species was done by Siddiqii (1976), while it was renamed Amplimerlinius macrurus (Filipjev, 1936; Goodey, 1932). The problem with taxonomic uncertainty was shown by study of Siddiqii and Klingler (1980) which collected specimens of A. dubius. Steiner in the type locality of A. dubius near Thalwil (Switzerland) proposed Amplimerlinius dubius comb. n. for A. dubius (Steiner, 1914).

A. macrurus was found in the soil under different land use and cover types, i.e.: crops, meadows, pastures, grasslands, forest, fruit, nut, and olive orchards. The species was reported by researchers from the United States (Norton et al., 1984), as A. dubius (Steiner, 1914) and different parts of Europe (Háněl and Čerevková, 2006; Lamberti and Vovlas, 1993; Renčo, 2013), Caucasus, i.e.: Armenia (Poghossian, 1979) and Azerbaijan (Niknam et al., 2008), Asia, i.e.: Tajikistan (Ivanova, 1982); countries in the Middle East (Bahmani et al., 2013; Ghaderi et al., 2017; Hashim, 1982, 1983; Panahandeh and Pourjam, 2014; Stefan et al., 1985). Specifically, Ghaderi and Karegar (2014) and Ghaderi et al. (2014) provided lists of different field crops and A. macrurus was associated with Iran. It is presented in the latest updated parasitic annotated checklist of parasitic nematodes in Germany (Sturhan, 2014) and Belgium (Bert et al., 2003).

A. macrurus is associated with many cultivated crops and grasses (Bert et al., 2003; Kheiri et al., 2002; Navas and Talavera, 2002; Winiszewska et al., 2012). However, it was not previously recorded at the energy crop like M × g. The objective of this study was to describe two populations of A. macrurus associated with M × g via morphological, morphometric, and molecular approaches.

The soil samples were collected in 2017 from the rhizosphere of two-year M × g stands located in Dolyna city, Ivano-Frankivsk region, Ukraine (49.15°N, 24.37°E) on Glayic Cambisols: and in Grytzyv village in the vicinity of Shepetivka, Khmelnytskyi region (49 98°N, 21 36°E) from the rhizosphere of five-year M × g stands on Chernozem. A sampling of nematodes was done randomly through late August-early October.

The rapid centrifugation-flotation method (Szczygieł, 1971) was used for extracting nematodes from the soil samples. Isolated nematodes were killed and fixed, then passed through a graded series of glycerol-ethanol solutions, followed by storage in anhydrous glycerol on permanent slides (Seinhorst, 1959). In total, 47 individuals from the sample were identified to the species level morphologically via a Leica biological microscope using the keys of Andrássy (2007), Brzeski (1998), and Ghaderi et al. (2014).

In order to confirm a morphological identification, three putative A. macrurus specimens were fixed in a DESS solution (Yoder et al., 2006) for genetic analysis. After washing with sterilized milli-Q water, selected individual nematodes were transferred to separate 0.2 ml polymerase chain reaction (PCR) tubes containing 25 μl sterile water, then lysed for DNA extraction according to the procedure described by Holterman et al. (2006). The obtained single nematode lysate (crude DNA extract) was used as a DNA template for a PCR reaction or stored at −20°C. 18S rDNA was amplified in two overlapping fragments using the following primer combinations: 988F with 1912R and 1813F combined with 2646R (Holterman et al., 2006). Amplification of the partial 28S rDNA sequence was obtained using the D2A and D3B primers (Nunn, 1992). The 18S and 28S rDNA regions were sequenced by the Sanger method on the ABI 3500L genetic analyzer (Applied Biosystems, Foster City, CA, USA). The sequences reported in this study were deposited in GenBank under the following accession numbers: MK952146 for 18S rDNA and MK951999 for 28S rDNA.

A. macrurus was collected in two locations representing population densities 12 to 110 individuals in 100 g of samples of two soil types: M × g stands in Dolyna (measurements for 20 females and 10 males), and M × g stands in Grytzyv (measurements for 15 females and two males). The original photomicrographs of this species are presented in Figure 1 and morphometrics are given in Table 1. The photomicrographs of the collected specimens showed that the continuous head region and distinct clavate tail shape provided an evidence that it was A. macrurus.

Figure 1: