Characterisation of plant-parasitic nematodes Helicotylenchus digonicus and H. vulgaris from the rhizosphere of grapevines in Crimea, Russia

During 2016-2020, about 120 soil samples were collected from the rhizosphere of grapes in different vineyards of the Central and Southern regions of Crimea. Two morphologically close nematode species, Helicotylenchus digonicus and Helicotylenchus vulgaris, were found at all sampling sites in the Yalta, Sevastopol, Simferopol and Bakhchisarai districts. Morphologically, these species could be differentiated by the stylet length and the shape of tails. In addition, these species have significant differences in sequences of the D2-D3 expansion segments of the 28S rRNA.


Introduction
One of the main branches of agriculture in the Republic of Crimea is viticulture. In 2020, the total area of vineyards in the republic was about 19 thousand hectares, of which 16 thousand were fruit-bearing. However, the yield from these vineyards remains very low. In 2020, the average yield in Crimea was about 56 centners per hectare, while the normal yield from the specialized plantations of industrial vineyards is considered to be 120-150 centners per hectare. It is known that plant-parasitic nematodes can seriously reduce the yield of grapes and even cause the death of individual plants. Despite this, data on the species composition of plant-parasitic nematodes and their distribution in vineyards of the Republic of Crimea are very limited.
During routine nematological surveys conducted in 2016-2020 in the Crimean Peninsula, Helicotylenchus digonicus Perry, 1959 and Helicotylenchus vulgaris Yuen, 1964 were the most frequently encountered spiral nematodes in the rhizosphere of grapevines.

Materials and methods
The soil samples for this study were collected near the village of Nekrasovka in the Bakhchisarai district (44.754265 N, 33.687089 E). Nematodes were extracted using a modification of the funnel method [1]. For morphological studies, the nematodes were killed with hot water, fixed in a 5-% formalin solution, and mounted in glycerine on slides using the Seinhorst technique [2]. Molecular studies were performed using the scientific equipment of the Core Research Facility of the "Bioengineering" Centre (Moscow, Russia). For this work, nematodes frozen in distilled water were used. Their total DNA was extracted from several specimens using the Wizard kit (Promega, USA) according to the manufacturer's instructions. The forward Nem_18S_F (5′-CGC GAA TRG CTC ATT ACA ACA GC-3′) and the reverse Nem_18S_R (5′-GGG CGG TAT CTG ATC GCC-3′) primers were used for the amplification of the fragment of the 18S rRNA gene. The D2-D3 expansion segments of the 28S rRNA gene were amplified using the forward D2A (5′-CAA GTA CCG TGA GGG AAA GTT G-3′) and the reverse D3B (5′-TCG GAA GGA ACC AGC TAC TA-3′) primers. The partial cytochrome c oxidase subunit 1 gene was amplified with the forward primer JB3 (5′-TTT TTT GGG CAT CCT GAG GTT-3′) and the reverse primer JB5 (5′-AGC ACC TAA ACT TAA AAC ATA ATG AAA ATG-3′). The amplifications were performed in a Tetrad thermal cycler (Bio-Rad, USA). PCR products were purified using the WizardPCRPreps kit (Promega, USA). The sequencing of the PCR products was carried out with the same primers using the genetic analyser "ABI 3730" (Applied Biosystems, USA). Low-quality segments of sequences at the 5′ and 3′ ends were removed. The newly obtained sequences of H. digonicus were submitted to the GenBank database under accession numbers MW881576 (18S rRNA), MW881577 (28S rRNA) and MW881605 (cox1). The newly obtained sequences of H. vulgaris were submitted to the GenBank database under accession numbers MW881606 (18S rRNA), MW881607 (28S rRNA) and MW881650 (cox1). The dendrogram was constructed using the neighbourjoining method [3]. The 28S rRNA sequence of Criconemoides informis Micoletzky, 1922, which was also found in the rhizosphere of grapevines in Crimea [4], was used as the outgroup.
from the morphologically close species Helicotylenchus minzi by the tip of the tail and the absence of males in the population.
Molecular characterisation. The sequences of the 18S rRNA gene, the D2-D3 expansion segments of the 28S rRNA gene, and the COI gene obtained from different individuals in this study were identical within each marker.
Remarks. The morphological characteristics of the Bakhchisarai population of H. vulgaris resemble the original description [6] and some other descriptions of this species [7,8].  (Fig. 2). Despite the wide geographical distribution of H. vulgaris, as it is known from the literature, there are no cox1 sequences of this species in the GenBank. The sequences of the COI gene were most similar to Helicotylenchus varicaudatus from Belgium (MN782380), with 88.48% similarity, and was less similar with other hoplolaimid sequences deposited in the GenBank.

Conclusion
As a result of the conducted work, it was found that the Crimean populations of morphologically similar species of H. digonicus and H. vulgaris can be distinguished by 1) the length of the stylet, 2) the shape of the tail, and 3) the width of the cuticle annules. The molecular diagnostics of these species should be carried out using the D2-D3 expansion segments of the 28S gene. The sequences of the 18S gene are not suitable for this work due to the presence of large fragments identical for many species of this genus. The sequences of the COI gene are variable, so they are also not suitable for the accurate identification of these species.