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

Abstract The growing interest in biomass production of Miscanthus × giganteus (M × g) (Poaceae) on agricultural and marginal lands has prompted researches to identify plant pathogens and diseases affecting this crop which has a great potential for production of biofuels and different bioproducts. A soil survey of nematodes in the M × g rhizosphere and a report on the collection of the plant-parasitic nematode Amplimerlinius macrurus (Belonolaimidae) were accomplished in two locations in Ukraine. It is known that this family of nematodes can reduce the root system and biomass of Poaceae family plants. Both molecular and morphological characters were used to identify the nematodes; measurements and photomicrographs of the species were presented. This is the first documentation and description of A. macrurus in Ukraine to the best of our knowledge. Further investigation is underway to confirm the pathogenicity of this species on perennial grasses plantations.

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(Mekete et al., , 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  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).  (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 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.

Materials and methods
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 μ l 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.

Results and discussion
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.
The amplification of the almost full-length 18S rDNA fragment was successful (1,670 bp, MK952146). The Basic Local Alignment Search Tool (Blast) showed a 99.52% similarity to the sequence of A. macrurus deposited in GenBank from the Netherlands (FJ969114) (Carta et al., 2010). The 28S rDNA sequence alignment from A. macrurus (712 bp, MK951999) showed a 99.30% similarity to the sequence of A. macrurus deposited in GenBank from Iran (KX789694). Our investigation showed that the results of A. macrurus molecular identification corresponded to its morphological diagnosis.
A. marcurus was first isolated in Poland from peat soils in grasslands by Skwiercz (1989) and Brzeski (1998). Brzeski (1998) considered A. macrurus as a sedentary ectoparasitic grass nematode. However, the data regarding the feeding type on new perennial grasses, including M × g, is still lacking. The growing needs for such data are attributable to the rapid upscaling of M × g cultivation in Ukraine in agricultural, marginal, and deteriorated lands and increasing risk of nematode damage and adverse impact to the biomass yield. Generally, the results of the study of morphological and morphometric characters of two Ukrainian A. macrurus populations appeared consistent description after Wallace and Greet (1964) and Saltukoglu et al. (1976). Differential analyze of several populations described from England, after Siddiqii (1976) [S], Spain after Bello et al. (1987) [B], and Iran after Ghaderi and Karegar (2014) [G] showed wider range of the morphometric characters. The most of its indices fits well to the body length, head annuli, a, b, c, V, tail, and stylet length and gubernaculum of Ukrainian To the best of our knowledge, this is the first record of A. macrurus associated with M × g. The received data confirmed the occurrence of this nematode in Ukraine and extended the range of host crops for these species. Our findings provided some basic arguments for considering A. macrurus as a potential pest of perennial crops in Ukraine. However, for demonstration its pathogenicity and host range on M × g it is advisable to validate this study hypotheses in the future research.