Diagnosis of potato rot nematode Ditylenchus destructor using PCR-RFLP

During an investigation of nematodes in the Moscow region of Russia in 2019, a known species Ditylenchus destructor was recovered from tubers of potato plants. The genus Destructor is one of the most problematic genera of plant-parasitic nematodes. The numerous species reported for this genus have been cited from various sources. Due to the morphological similarity of many species and the lack of separation characteristics, the identification of D. destructor is difficult. Molecular taxonomy and phylogeny were used to confirm the identification. In the current study, PCR-RFLP illustrative models for the amplification of the ITS-rRNA gene were provided with two enzymes that could recognize D. destructor in potato tubers. Analysis of the rDNA sequences spanning both ITS1-ITS2 regions was carried out on the collected populations. The digestion of the PCR product of the ITS1-5.8S-ITS2 region with the enzyme TaqI produced three fragments; 100, 190, 550, and with Tru1I, two fragments were produced; 300 and 480 bp. The obtained DNA sequences were compared with those DNA sequences deposited in GenBank of populations isolated in other countries. The results showed no distinction between populations isolated from different host plant species, including populations found in the Russian Federation. New sequences from ITS-rRNA were deposited in the GenBank under accession number MN122076, MN658597, MN658599, MN658637, MN658638.


Introduction
Nematodes are an important group of organisms having evident biological progress, characterized by the high amount of density growth, the broad spectrum of spreading, and having numerous intraspecific variable symptoms. They are listed among the quarantine pests in some European countries [1][2][3] and they were also evaluated as a serious pest in the globe [4]. Ditylenchus destructor could be a significant parasite in different host plants. Polyphagous nematode infects large diversity of plants in cropping systems, when there are no host plants, it is capable of feeding and reproducing on mycelia of many fungi. All nematode parasites undergo a process of host invasion, in which they reproduce, disperse, and pursue new hosts [5][6][7][8]. Recently, morphological identification has been investigated to recognize D. destructor, but morphology cannot be used when eggs, juveniles, and males are the only resources available. Moreover, the genus Ditylenchus includes several species with small interspecific morphological differences, and D. destructor usually occurs simultaneously with other nematode species. Without taxonomic expertise, the distinguishing of D. destructor from other species is difficult. There are some methods using PCR with specific primers designed based on nucleotides combination in the ITS rRNA gene which were also developed for the detection of D. destructor [2,5,[9][10][11]. Molecular identification of D. destructor is based on the specific sequences of rDNA region by the conventional PCR methods [8,12] and the most customarily used regions are the large subunits [13], small subunits and the internal transcribed spacer (ITS) of ribosomal RNA [14,15]. Recently, several molecular practices have been evaluated and suggested by the European and Mediterranean Plant Protection Organization (EPPO) for identifying D. destructor, i. e. a PCR-RFLP-based assay of ITS fragment [16]. This research attempts to use EPPO protocol for the characterization and identification of D. destructor nematode. The objective of this study was to identify D. destructor that was recently found in potato tubers in the Russian Federation, by the means of the PCR-RFLP technique.

Materials and methods
Nematode population. Potato rot nematodes (D. destructor) were extracted from potato tubers (Solanum tuberosum L.) in the Russian Federation. Species identification was confirmed through morphological and molecular methods.
For the preparation of nematodes, a tipped pipette was used for picking individual nematodes in a suspension, and a sucking tipped pipette was prepared by burning the tips of two Pasteur pipettes pressed against each other. The pipettes were then pulled apart after melting began, resulting in a tiny syringe-like opening, which served the purpose of sucking nematodes from the suspension with the capillary action.

PLANT PROTECTION
For morphometric analysis, nematodes (males and females) were picked for each population by sucking with the tipped pipette and placing them onto a glass slide (Menzel GmbH, Braunschweig, Germany), thus creating temporary slide mounts. Two drops of distilled water were added into each glass slide. The slides were then heated at 55 °C for 3…5 seconds on a hot plate. Thereafter, covered slides were placed on the water droplets, and samples were mounted on a ZEISS Axioskop50 microscope equipped with a camera. D. destructor remained straight when killed by heat (approximately 60 °C), a typical feature of Ditylenchus spp. Morphometric data and light microscopic images were obtained from digital images on a computer screen with the aid of AxioVision software version 4.8.2 (Carl Zeiss MicroImaging GmbH, Jena, Germany) ( Fig. 1). DNA extraction was carried out by treating the specimens with Proteinase K, which was performed by removing proteins without using organic solvents in the extraction process. A DNA-Ekstran-2 set No EX-511-100 (Synthol, Moscow) was applied for this purpose. Nematodes were extracted from the infected potato tubers. About 6…10 nematodes were collected from the potatoes and put in 20 μl lysis buffer and crashed, then 280 μl of lysis solution and 1 μl mercaptoethanol were added. Thereafter, 10 μl of Proteinase K was added to the tubers.
The mixture was then vortexed and incubated at 55 °C for 24 hours. By the next day, 100 μl of the precipitated solution was put into tubes; vortexed for 20 seconds, and centrifuged at 13000 rpm for 5 minutes. Subsequently, 2 μl of glycogen was put into new tubes and the supernatant derived from the last stage was also added to them. The tubes were manually (by hand) shaken 10 to 14 times, then 300 μl of precipitating solution was added and shaken manually for 10 to 14 times before being centrifuged at 13000 rpm for 5 minutes. At this point, DNA became visible at the bottom of the tubes. In the next step, 400 μl of wash solution was added and the contents were shaken, and then centrifuged at 13000 rpm for 2 minutes. One more time, the extra solution was discarded. Afterward, the tubes only had the DNA in them and were opened, then put in the oven at 37 °C for 12…15 minutes to evaporate the alcohol. Finally, 50 μl DNA dissolution was added, then shaken, and also put in the oven at 60 °C for 5 minutes.
PCR. The PCR reaction was performed with samples (final volume 25 μl), containing 5 μl of 10X reaction buffer, 1 μl of each primer; universal primers 18S (5´-TTG ATT ACG TCC CTG CCC TTT-3´) and 26S (5´-TTT CAC TCG CCG TTA CTA AGG-3´) ( PCR-RFLP. In carrying out RFLP analysis, 30 μl of the amplification product was digested with one of the restriction enzymes (Tru1I and TaqI) in a buffer stipulated by the supplier. Each digestion reaction involved of 2 μl of 10X reaction buffer, 10 μl of the direct PCR product, and 1 μl of restriction enzyme (10 μg -1 μl) thus increasing to a total volume of 17 μl with double distilled water. The digestion mixture was incubated for 5 minutes at 65 °C. The digested DNA fragments were separated on buffered (0.5 % TAE) 1 % agarose gel which contained 15 μl of 10,000X GelRed.
Sequence analysis was performed by the commonly accepted protocol of utilizing the Genetic Analyser AB-3500. Primitive comparison of sequencing results with the GeneBank genetic sequence database was done by the NCBI BLAST website (http://www. ncbi.nlm.nih.gov/BLAST) ( Table 2). BioEdit v.7.0.5.3, sequence alignment editor was used for checking the sequence, editing, and alignment. The tree diagrams were created by using the maximum likelihood technique (ML method) available in Mega 10 software. Bootstrap Test via building 1000 alternative trees was used to analyze and confirm the validity of the tree diagrams. The results are presented in percentage values, the DNA sequences available in the GeneBank homologous to those examined were analyzed along with the newly sequenced. These sequences were deposited in the NCBI database with access numbers: MN122076, MN658597, MN658599, MN658637, MN658638. Results and discussion Using the RFLP-PCR and nematode DNA extracted from potato tubers, PCR fragments were successfully amplified at the expected product size for primer set in D. destructor samples. PCR-RFLP showed the distinction of D. destructor with similar species such as D. dipsaci and D. myceliophagus [17]. In some populations of sweet potato, the RFLP method indicated different band lengths [18]. The amplification of ITS1-5.8S-ITS2 of the ribosomal DNA produced a fragment of almost 1000 bp. Nowadays, the molecular diagnostic of D. destructor is primarily based on the specific sequences of some conserved regions by the conventional PCR, and the most commonly used regions are small subunits and internal transcribed spacer (ITS) of ribosomal RNA [2].

ЗАЩИТА РАСТЕНИЙ
The digestion of the PCR product of the ITS1-5.8S-ITS2 region with the enzyme TaqI produced three fragments; 100, 190, 550, and with Tru1I, two fragments were produced; 300 and 480 bp (Fig. 2). ITS1 differed in length from 315 to 473 bp, 5.8S measured ca 154bp and ITS2 was 207 bp. The identified distinction in length in the ITS1 was affected by the insertion of 57 to 188 bp in the entire length in some D. destructor [2]. A mentioned primer set can be used for specific identification of D. destructor. All populations from potatoes were identified as D. destructor by DNA sequencing. DNA fragments produced bands with the primer set tested.
Diagnostic of D. destructor is difficult by the morphological identification only. Hence, molecular techniques help scientists to identify this nematode accurately. It allows to the suggestion that D. destructor may present a species or subspecies complexity]18[.
Recently, methods and knowledge of molecular biology have been investigated as a powerful practice to identify various nematode species [13,17]. It was also revealed that rDNA ITS regions can be desirably used for phylogenetic analyzes [5,16].
Similar research by BLAST of the sequences of D. destructor was found in potato tubers with sequences of the GenBank from both markers of the rDNA previously deposited in the GenBank displayed three clades of D. destructor, D. dipsaci, and Meloidogyne javanica as an outgroup species. Our deposited sequences formed a highly supported (PP = 100 %) with whole populations that were involved from China, Czech Republic, and South Korea.
Phylogenetic relationships within potato rot nematode as inferred from the ITS1-5.8S-ITS2 rRNA gene sequences using Maximum Likelihood Method inference is given in Fig. 3. In the GenBank database, there are many sequences of D. destructor retrieved from various plants and geographical regions [5,7].

Conclusions
The accurate identification of nematode is a significant step for effective control of the host plant. It must be recognized that species determinations have an essential practical application in nematology. Regulatory decisions depend on quick and also accurate identification. Overall, economic management options require a greater resolution in nematode identification. Based on the PCR-RFLP restriction pattern obtained with the Tru1I, TaqI, it is possible to indicate that the rot nematode D. destructor was found in potato tubers in the Russian Federation territory. It is necessary to carry out studies of differential hosts in the populations and use new sequencing to obtain useful molecular markers for the differentiation of the D. destructor in the Russian Federation.