Identi cation and establishment of a culture method for the spiral nematode Helicotylenchus microlobus from tomato in China

Yanhui Xia College of Plant Protection, Henan Agricultural University, Zhengzhou, China; Jin Li Institute of Nuclear Technology and Biotechnology, Xinjiang Academy of Agricultural Sciences; Feifei Xu College of Plant Protection, Henan Agricultural University, Zhengzhou, China; Bin Lei Institute of Nuclear Technology and Biotechnology, Xinjiang Academy of Agricultural Sciences; Honglian Li National Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou, China; Ke Wang College of Plant Protection, Henan Agricultural University, Zhengzhou, China; Yu Li (  liyuzhibao@henau.edu.cn ) College of Plant Protection, Henan Agricultural University, Zhengzhou, China;


Background
Tomato (Solanum lycopersicum L.) is the most economically important member of the family Solanaceae and is cultivated worldwide. It is one of the most important vegetable crops in China and is rich in minerals, vitamins, antioxidants and other micronutrients [1]. China has the largest area of tomato cultivation in the world, and Henan Province is the dominant production area for tomato cultivation.
Tomatoes are susceptible to damage from pests (insects, mites, nematodes, and so on) as well as fungi, viruses, or bacteria [2]. Nematode diseases are the major factor limiting tomato production worldwide and cause severe economic losses.
The genus Helicotylenchus Steiner 1945 is one of the ten most important plant parasitic nematodes in the world, and these nematodes are commonly referred to as spiral nematodes because of their coiled habitus mortis [3]. Within the genus Helicotylenchus, more than 230 nominal species have been described worldwide [4,5]. Spiral nematodes are ectoparasites or semi-endoparasites that may occur in large numbers and feed on the roots of a variety of plants, causing plant growth reduction [6]. Many of the described species have not received widespread attention as serious plant pathogens, because there are no available data showing that they damage and inhibit plant growth [4]. However, plant growth inhibition has been consistently associated with several cosmopolitan species that have also increased secondary infections of fungal pathogens [7], such as, H. dihystera, H. digonicus, H. multicinctus, H. pseudorobustus and H. varicaudatus [8][9][10]. At present, 50 valid species are listed as spiral nematodes in China, and these species are parasitic on a large variety of plants including ornamental plants, fruit trees, cucumber, rice, grape and megranate [11][12][13][14]. However, only Qi [19][20][21]. At present, carrot disks have been successfully applied in the culture of plant-parasitic nematodes, but carrot disks are not suitable for rearing all migratory plant-parasitic nematodes, and only a small number of plant-parasitic nematodes can be cultured [21]. However, the establishment of a culture method for Helicotylenchus has not been reported. In 2019, soil samples were collected from the rhizosphere of plants in a tomato eld near Zhuma village in Tongxu County of Kaifeng city, Henan Province, China. Spiral nematodes were separated from the samples and one individual female nematode was selected for propagation on carrot disks at 25 °C by parthenogenesis. In this study, the puri ed spiral nematode LQ-1 populations were identi ed based on morphological and molecular biological technology, a culture method for spiral nematodes was established, and the reproduction of H. microlobus was detected on carrot disks. To our knowledge, this is the rst report of H. microlobus from tomato in China using morphological and molecular biological technology. It also represents the establishment of an arti cial culture method for H. microlobus. The purpose of this study was to identify a species of spiral nematode from tomato in China and to establish an arti cial culture method for H. microlobus, which would be very useful for other related biological studies of H. microlobus.

Nematode collection
In August 2019, ve samples of roots and corresponding rhizosphere soils were collected from a tomato (cv. Maohong 801) elds near Zhuma village in Tongxu County of Kaifeng city, Henan Province, China. Each sample consisted of at least ve sampling points from patches of poor growth. Samples were placed in plastic bags and stored at 16 to 18 °C [21]. Nematodes were extracted using the modi ed Baermann funnel method for 2 days [38], by which time 100 g of tomato soil and root samples were extracted from each sample.

Nematode culturing and experiments
Carrot disks were prepared in the following way. The surface of a carrot was sterilized with 95% ethanol.
The external tissues were removed with a sterile knife, and then the carrot was cut into disks (approximately 6 mm thick). Each carrot disk was placed in a petri dish, in an incubator maintained at 25°C approximately 15 days prior to use. One individual female nematode was selected and sterilized with 0.3% streptomycin sulfate, and then transferred to carrot disks at 25 ℃ in the dark for propagation. After that, the puri ed spiral nematode LQ-1 population cultured on carrot disks were used for morphological and molecular analysis.

Morphological identi cation
Nematodes were heat-killed, xed in FG solution (formalin: glycerin: water=10:1:89) [39], and processed to glycerin by the formalin glycerin method [40]. Photomicrographs and morphometric data of the specimen LQ-1 population were obtained using a Nikon Eclipse Ti-S microscope (Japan). Images of key morphological features were processed using Photoshop CS5. The De Man formula was used for measurements. All measurements are expressed in micrometers (μm), unless otherwise stated.
The obtained ITS region and D2-D3 region of the 28S sequences were subjected to multiple alignment using Clustal W in MEGA 5.0 [45] with other spiral nematode species sequences published in the NCBI GenBank database. Outgroup taxa were selected based on a previous study [20]. Sequence datasets were analysed with Bayesian inference (BI) using MrBayes 3.2.6 [46] under the best-t model of GTR+G+I, according to Akaike Information Criteria [47]. BI analysis for each gene was run with a random starting tree and four Markov chains for 1×10 6 generations. The Markov chains were sampled at intervals of 100 generations. After discarding burns in samples, the remaining samples were used to generate a 50% majority rule consensus tree. Posterior probabilities (pp) are given on appropriate clades.

Reproduction tests
Using the carrot disk method, experiments were conducted to determine the reproductive potential of H. microlobus females and the optimum temperature for culturing H. microlobus on carrot disks. In the experiment, 30 females were surface sterilized with 0.3% streptomycin sulfate and then transferred to one carrot disk in a petri dish. These petri dishes were sealed with para lm and incubated at 25, 27.5, and 30°C in the dark. The number of nematodes and reproduction rate (Rr = nal number of nematodes/initial number of nematodes) on each carrot disk were determined at 90 days after inoculation.
A maceration method was used to collect nematodes [21]: the carrot tissues were placed in sterile water and macerated in a blender, and to isolate nematodes from carrot tissue, the suspension was then passed through 0.250-mm and 0.150-mm-pore sieves. The nematodes were collected through the sieves into a beaker, and carrot tissues were discarded. Then, the nematode suspension in the beaker was left to settle for at least 4 h, and the supernatant was removed. The total nematode number was observed under a stereomicroscope to assess whether H. microlobus reproduction had occurred, and the Rr ( nal number of nematodes/initial number of nematodes) was determined. There were ve replicates for each experiment, and each experiment was conducted twice.

Morphology of the spiral nematodes
The morphology of the spiral nematode LQ-1 population collected from tomato was photographed (Fig.  1). The morphometric data from the LQ-1 population closely resembled H. microlobus as described previously [22] ( Table 1).
Female spiral nematodes had a spiral body shape after being heat-killed ( Fig. 1 A). The lip region was hemispherical, composed of 4-5 annuli ( Fig. 1 B, C, D). The stylet was robust, with hemispherical knobs that varied little in shape, and was approximately 2-3 µm high and 4-6 µm wide ( Fig. 1 B, C, D). An excretory pore was immediately posterior to the hemizonid (Fig. 1 E). Lateral elds with four longitudinal lines occurred, not areolated in the tail region ( Fig. 1 K). Pharyngeal glands overlapped the intestine ventrally ( Fig. 1 F). Inner lateral eld incisures in the tail region were mostly fused distally into a Y-shaped con guration (Fig. 1 H, I, J). Mean anal body ped con guration. Mean of anal body diameter ranged from 12.7 to 14.4 ( Fig. 1 H, I, J). The tail was generally longer than the anal body diameter, bearing 6-13 ventral annuli and ending in a pronounced ventral projection, usually rounded terminally, without a mucro (Fig. 1   J). The distal half of the tail was either without annulation or indistinctly annulated with marked dorsally convex-conoids ( Fig. 1) (Table. 1).
Male spiral nematodes were not found.

Molecular characterization and phylogenetic relationships of H. microlobus
The primers TW81/AB28 and D2A/D3B were used to amplify the ITS and D2-D3 regions, respectively, of the 28S rRNA gene sequences of the spiral nematode LQ-1 population. The ampli ed PCR products were 28S rRNA sequences in this study were deposited in the NCBI GenBank database for a BLAST search. Phylogenetic analysis within the genus Helicotylenchus based on ITS rRNA gene sequences was performed and indicated 28 ingroups and one outgroup taxon (Fig. 3) [15,16]. However, these studies on the parasitism of tomato by spiral nematodes include only census data, or morphological data and lack detailed molecular data.
In this study, morphological identi cation of the spiral nematode LQ-1 population collected from tomato rhizosphere soils and roots in Henan province, China, was carried out. We found that the main morphological characteristics of the LQ-1 population were basically consistent with H. microlobus as described previously, with only slight differences in some measurements. Compared with the data of Mwamula et al. (2020), the L, b, c and max body diameter values for female nematodes were relatively smaller [22]; the pharynx length and m value of measurements were relatively larger. The reason for differences may be related to underlying genetic variation and geographical position. In addition, spiral nematodes are a genera of plant parasitic nematodes known to contain various species complexes that normally exhibit similar diagnostic characteristics [27]. However, species delineation within the genus is not always reliable due to enormous intraspeci c variability in characters apparently in uenced by environmental conditions [28]. This adds to taxonomic problems and leads to some misidenti cations within the genus, due to lack of consensus among different taxonomists on the validity of some species [29,4]. Therefore, to facilitate species identi cation, a molecular approach is needed. In recent years, sequences of nuclear ribosomal RNA genes have been used for reconstruction of phylogenetic relationships and molecular characterization within Helicotylenchus [30]. This study revealed that the species of the spiral nematode extracted from the rhizosphere of tomato in Kaifeng city, Henan Province, was H. microlobus. The Bayesian tree showed that the results of the phylogenetic analysis of the ITS rRNA gene and 28S rRNA gene D2-D3 region were consistent, and clearly separated H. microlobus from its sister Helicotylenchus species. This is the rst report of H. microlobus from tomato in China using morphological and molecular biological technology.
Obtaining a large number of plant-parasitic nematodes is very important because many types of studies can be performed with these nematodes, such as pathogenicity tests and biological and genetic studies [21]. For a long time, researchers have been looking for methods to culture plant-parasitic nematodes using plant tissues [19][20][21]. Some culture methods for plant-parasitic nematodes have been successfully employed, but there have been great differences in culture techniques. For example, Aphelenchoides besseyi, Ditylenchus destructor and Bursaphelenchus xylophilus can be cultured on certain fungi [31][32][33]. A. ritzemabosi and D. dipsaci can reproduce rapidly on alfalfa tissue [19]. The carrot callus method is suitable for culturing A. ritzemabosi, A. besseyi, R. similis, and most species of Pratylenchus [21,34,35]. Until now, successful mass culture of Helicotylenchus has not been reported. Sterile carrot disks are usually regarded as a relatively low-cost, straightforward and effective method for culturing migratory endoparasitic nematodes that results in greater nematode multiplication compared with other methods [37]. However, our study is the rst to establish an arti cial culture method for H. microlobus. The carrot disk method produces large numbers of nematodes quickly and requires less time and labour. Once the culture technique is established, growth can be subdivided into many carrot disks, which can be kept for at least two months and contain many live nematodes.

Ethics approval and consent to participate
We collected samples of roots and corresponding rhizosphere soils in areas where no speci c permits were required. The land used as the collection area is neither privately owned nor protected in any way, and the eld studies did not involve endangered or protected species. Procedures for eld sample collection were explained to village authorities and their verbal agreement was obtained before collected samples.

Consent for publication
Not applicable

Con ict of interests
The authors of this work declare that there is no con ict of interest.
Note: All measurements are in µm and in the form of mean ± SD (range). n: Number of specimens measured; L: Body length; a: Body length/greatest body width; b: Body length/length from the lips to the junction of esophageal gland and intestine; b': Body length/ length from the lips to esophageal gland end; c: Body length/ tail length; c': Tail length/tail diameter at anus; V: Distance of vulva from the lips×100/body length; o: DGO from stylet base×100/Stylet length; m: Conus length×100/Stylet length; DGO: Distance between dorsal esophageal gland opening and stylet knobs.    Bayesian tree of Helicotylenchus as inferred from ITS rRNA gene sequences under GTR+I+G model. Posterior probabilities more than 50% are given for appropriate clades. Newly obtained sequence is indicated in bold font.

Figure 4
Bayesian tree of Helicotylenchus as inferred from the D2-D3 region of 28S rRNA gene sequences under GTR+I+G model. Posterior probabilities more than 50% are given for appropriate clades. Newly obtained sequence is indicated in bold font.