Background

Tomato (Solanum lycopersicum L.) is one of the most economically important members of the family Solanaceae and is cultivated worldwide [1]. It is one of the most important vegetable crops in China and is rich in minerals, vitamins, antioxidants and other micronutrients [2]. China has the largest area of tomato cultivation in the world, and Henan Province is the dominant production area for tomato cultivation [3]. Many plant pathogens can infect tomato, and nematodes perhaps are one of the important pathogens limiting tomato production worldwide and can cause severe economic losses [4].

The genus Helicotylenchus Steiner 1945 is considered one of the ten most important plant parasitic nematodes in the world [5]. Within the genus Helicotylenchus, more than 200 nominal species have been described worldwide [6]. The genus Helicotylenchus is classified as semi-endoparasitic that may occur in large numbers, causing plant growth reduction [7, 8]. After infection by Helicotylenchus, plants of outer layer of cortical tissue and the root system were damaged, further resulting in reduced ability of plants to absorb water and nutrients. For example, Helicotylenchus species associated with Musaceae damaged the cortical tissue of roots, and reduced the ability of roots for the uptake of water and nutrients [9]. H. microlobus infected Paspalum vaginatum causing brown lesions on their roots [10]; H. multicinctus, H. dihystera and H. erythrinae have been found to be harmful to banana and plantain crops around the world [9]. Inoculating H. dihystera on Olive seedlings caused 78% reduction in weight, and the development of the lateral roots occurred retardation [11]. Some studies suggests that H. pseudorobustus feeding in the cortical parenchyma of corn and soybean roots and causing serious damage [8, 12]. At present, 50 Helicotylenchus species are reported from China, and these species are reported from a variety of plants including ornamental plants, fruit trees, cucumber, rice, grape and pomegranate [13,14,15]. However, only H. dihystera is currently reported associated with tomatoes in China [16, 17]. At present, H. microlobus are not reported from tomato in China. Subbotin et al. recorded that H. microlobus was collected in California, Illinois, and Iowa, in the United States of America and in several European countries, including Spain, Italy and Russia [18]; Yan et al. reported H. microlobus infecting soybean in North Dakota [19]. Abraham and Dong reported H. microlobus in turfgrass in Korea [20].

In addition, a mass pure culture of plant-parasitic nematodes is key to pathogenicity studies and many other biological studies [21]. For most species of plant- parasitic nematodes, establishing a rapid and efficient culture method is a challenge. At present, only a small percentage of plant-parasitic nematodes can be successful cultured. The successfully establishment of an efficient culture method for Helicotylenchus has not been reported, and this increased the difficulty to study the pathogenicity and some biology of Helicotylenchus.

In 2019, during a survey of the plant-parasitic nematode associated with tomato, a population of Helicotylenchus was collected from tomato roots and soil samples in Zhuma village, Tongxu County of Kaifeng city, Henan Province, China. The objectives of this study were to confirm the specie of Helicotylenchus obtained from the tomato samples based on morphological and molecular characteristics and establish a method of culturing Helicotylenchus on carrot disks.

Results

Helicotylenchus microlobus Perry in Perry, Darling and Thorne, 1959

The Helicotylenchus individual population collected from tomato was photographed (Fig. 1). The morphometric data from the population closely resembled H. microlobus as described previously [18, 19, 22] (Table 1). Distance between dorsal esophageal gland opening and stylet knobs.

Fig. 1
figure 1

Light micrographs of Helicotylenchus microlobus female. A Entire body, B-D Lip region, e Anterior region, F-G Two genital branches, H Lateral lines, I The junction of genital gland and intestine, J Vulval region, K-N Tail region, O-P Lateral field at tail region. Scale bars: 50 µm (A) and 10 µm (B-P); an = annuli; s = stylet; sk = stylet knob; oc = ovary cells; lf = lateral field; mb = median bulb; eg = esophageal glands; vu = vulval; a = anal

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Table 1 Morphometrics of females of Helicotylenchus microlobus
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Female; Habitus spiral. (Fig. 1 A). Lip region hemispherical, with 4–5 annules (Fig. 1 B, C, D). Stylet robust, with rounded knobs that varied little in shape, 2–3 µm high and 4–6 µm wide (Fig. 1 B, C, D). Median pharyngeal bulb oval to rounded (Fig. 1 E, I). An excretory pore was immediately posterior to the hemizonid (Fig. 1 E). Two genital branches, both functional, outstretched (Fig. 1 F, G). Lateral field with four longitudinal lines (Fig. 1 H), absence of areolation in the tail region (Fig. 1 O, P). Inner lateral field incisures in tail region mostly fused distally into a Y-shaped configuration (Fig. 1 O, P). Pharyngeal glands overlapped the intestine ventrally (Fig. 1 E, I). Measurement of anal body diameter ranged from 12.7 to 14.4 µm (Fig. 1 K, L, M, N). Tail longer than the anal body diameter, with 6–13 ventral annuli, ending in a pronounced ventral projection, usually rounded terminally, without a mucro (Fig. 1 K, L, M, N). Tail tip without annulation or indistinctly annulated.

Male; Not observed.

Remarks: The specimens were identified as H. microlobus because of the presence of the lateral field not areolated on the tail, inner incisures of the lateral field fused distally for about two annuli in a Y-shaped pattern and tail projection not annulated. The several characteristics were consistent with the description of Siddiqi (1972) [23]. So, taking into consideration the results of our morphological and morphometric data, we consider the population as representatives of H. microlobus rather than H. pseudorobustus or another taxon. In this study, the morphological characteristics of the population of H. microlobus collected in this study were compared with data of Mwamula et al. (2020), Yan et al. (2017) and Subbotin et al. (2015) [18, 19, 22]. In comparison with the materials studied by Mwamula et al. (2020), the L (679.7 µm vs 732.5 µm), c (33.1 vs 37.7) and max body diameter values (25.4 µm vs 28.6 µm) for female nematodes were relatively smaller; the pharynx length (144.3 µm vs 123.6 µm) of measurements and m value (48.8 vs 43.7) were relatively larger [23]. In comparison with the North Dakota specimens studied by Yan et al. (2017), they differed in tail annules (10.0–14.0 vs 9.0–12.0) and a value (21.4–27.1 vs 25.6–28.1) [19]. Compared with the California specimens studied by Subbotin et al. (2015), the body length (679.7 µm vs 739.0 µm), anterior end to excretory pore (110.0 µm vs 128.0 µm) and stylet length (25.6 µm vs 28.9 µm) were relatively smaller; and Compared with the China specimens studied by Subbotin et al. (2015), the body length (679.7 µm vs 622.0 µm), b value (5.3 vs 4.7), Anterior to median bulb length (78.0 µm vs 58.0 µm), and tail length (20.7 µm vs 17.0 µm) were relatively larger [20].

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 H. microlobus. The amplified PCR products were 1105 bp and 787 bp in length, respectively (Fig. 2). The obtained ITS sequences and the D2-D3 region of 28S rRNA sequences in this study were submitted to GenBank database. The ITS rRNA gene sequences obtained in this study (GenBank accession No. MZ2708013) showed 99.17%-99.89% similarity with H. microlobus sequences from the California populations (KM506859 and KM506860) and the Korea populations (MN764342 and MN764343). The obtained D2-D3 region of the 28S rRNA gene sequences of H. microlobus (MZ2707554) showed 99.2%-100% similarity with H. microlobus sequences from the Korea populations (MN764328, MN764324, MN764323 and MN7643253) and the Island populations (MG770481).

Fig. 2
figure 2

PCR amplification of the D2-D3 region of 28S rRNA and the ITS rRNA gene of Helicotylenchus microlobus. M: DL2000 Marker. 1: 28S. 2: ITS

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Phylogenetic analysis within the genus Helicotylenchus based on ITS rRNA gene sequences was performed and contained 41 sequences with 954 positions in length. The 50% majority rule consensus tree inferred from the ITS data set by Bayesian analysis is shown in Fig. 3. The phylogenetic tree showed that the newly obtained sequence for H. microlobus (MZ2708013) formed a 100% supported clade with other H. microlobus population. Phylogenetic analysis within the genus Helicotylenchus based on the D2-D3 region of the 28S rRNA gene contained 57 sequences with 557 positions in length. The 50% majority rule consensus tree inferred from the 28S data set by Bayesian analysis is shown in Fig. 4. This phylogenetic tree indicated that the newly obtained sequence for H. microlobus (MZ2707554) formed a highly supported clade with H. microlobus and H. pseudorobustus type B (pp = 100%).

Fig. 3
figure 3

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

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Fig. 4
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

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Effects of temperature on the reproduction rate (Rr) of H. microlobus

At 90 days after inoculation with 30 females, reproduction occurred on each carrot disk, and the Rr reached 232, 406, and 53 at 25, 27.5 and 30 °C, respectively (Table 2). The nematode Rr at 27.5 °C was significantly higher than that at 25 and 30 °C (P < 0.05). In addition, when incubated at 25 and 27.5 °C for 90 days, H. microlobus gathered on the surface of the Petri dish (Fig. 5), and the number of nematodes reached 6947(35.9% females, 34.4% juveniles, 29.7% eggs and no males) and 12,190(34.0% females, 29.9% juveniles, 36.1% eggs and no males), respectively (Fig. 5) (Table. 2). At 90 days after inoculation with 30 females, the carrot disks presented obvious infection symptoms and turned brown or dark-brown. These results demonstrate that the carrot disk method is suitable for the culture of H. microlobus.

Table 2 Effect of temperature (Tm) on the reproduction of Helicotylenchus microlobus on carrot callus 90 days after inoculation with 30 females x
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Fig. 5
figure 5

The carrot disks with disease symptoms and nematode populations at 25, 27.5 and 30 °C after 90 days. A: control without nematodes; B-D: the carrot disks with disease symptoms at 25 ℃, 27.5 ℃ and 30 ℃, respectively; E: nematode population; F–H: microscopic images of nematode populations; e = egg; n = nematode; Scale bars: F: 400 µm; G: 200 µm; H: 25 µm

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Discussion

Plant-parasitic nematodes are some of the most important pathogens of tomato. Meloidogyne, Helicotylenchus, Pratylenchus, Tylenchorhynchus and Aphelenchoides can infect tomato roots and cause severe economic losses [24]. Different Helicotylenchus have been reported from the rhizosphere soil of tomato in many countries. For example, in Turkey, H. digonicus, H. tunisiensis and H. varicaudatus have been reported from tomato rhizosphere soils [25]. Kim et al. (2014) reported that H. dihystera can suppress the growth of tomato and this species was also reported from tomato in China [26]. H. thornei Roman, 1965 is also described from soil around the roots of tomato in Ludhiana, India [27].

The genus Helicotylenchus known to contain various species complexes that normally exhibit similar diagnostic characteristics [18]. As intraspecific variability in characters may be influenced by environmental conditions, species delineation within the genus is not always easy [28]. This adds to identification problems and can leads to some misidentifications within the genus, due to lack of consensus among different taxonomists on the validity of some species [18, 29]. Therefore, biochemical and molecular information are more and more important in nematode taxonomy, however, these approaches should still be integrated with morphological characteristics [30].

H. microlobus was considered as a junior synonym of H. pseudorobustus by Sher (1966) because of their indistinguishable morphological characteristics, and this propose was accepted by Sauer and Winoto (1975), Fortuner (2018), and Divsalar et al. (2020) but was not agreed with by Siddiqi (1972, 2000), Subbotin et al. (2015) and Mwamula et al. (2020) [6, 18, 22, 23, 29, 31, 32]. Siddiqi (1972) proposed that H. microlobus differed from H. pseudorobustus by the non areolated lateral field in tail region (areolated in H. pseudorobustus), and inner lateral lines fusion as Y-shaped in the tail region (M- or U-shaped in H. pseudorobustus) [18, 23]. In this study, the morphological characteristics of this population were consistent with the characteristics of H. microlobus described by Siddiqi (1972), Subbotin et al. (2015) and Mwamula et al. (2020) [18, 22, 23]. When compared with H. microlobus species, the obtained ITS rRNA sequence showed 99.17%-99.89% homology with both H. microlobus sequences from California (KM506859 and KM506860), Korea (MN764342 and MN764343) and Spain (KM506862). In contrast, it had only 93% sequence identity with isolates of H. pseudorobustus (KM506835) from California. And, the obtained 28S rRNA sequence showed 99.2%-100% homology with both H. microlobus sequences from Korea (MN764328, MN764324, MN764323 and MN7643253) and Island (MG770481). In contrast, it had only 91% sequence identity with isolates of H. pseudorobustus (KU722387) from Iran. In addition, the results of the phylogenetic analysis of the ITS rRNA gene region showed that H. microlobus and H. pseudorobustus were clearly divided into two different clades; the results of the phylogenetic analysis of the 28S rRNA gene D2-D3 gene region showed that H. pseudorobustus type B (DQ328748; DQ328749; FJ485649) were clustered together with H. microlobus. Subbotin et al. (2011, 2015) proposed that a species group complex for H. pseudorobustus includes at least H. pseudorobustus type A and H. pseudorobustus type B, according to morphological and molecular analyses. According to the results of Subbotin et al. (2015), H. pseudorobustus type B should be H. microlobus and phylogenetic relationships of H. pseudorobustus type B (DQ328748; DQ328749; FJ485649) were clustered together with H. microlobus. Therefore, our results were consistent with the results of analysis of Helicotylenchus species by Subbotin et al. (2011, 2015). Taking into consideration the results of our integrative morphological and molecular analyses of the H. microlobus population, we considered the populations as representatives of H. microlobus rather than H. pseudorobustus. This is the first report of H. microlobus from tomato in China using morphological and molecular characterization.

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 [24, 25]. Some culture methods have been successfully employed, and there are great differences in these methods. For example, Aphelenchoides besseyi, Ditylenchus destructor and Bursaphelenchus xylophilus can be cultured on certain fungi [33,34,35]. A. ritzemabosi and D. dipsaci can reproduce rapidly on alfalfa tissue [36]. The carrot callus method is suitable for culturing A. ritzemabosi, A. besseyi, R. similis, and most species of Pratylenchus [37, 38]. Over the years, some researchers tried to culture certain Helicotylenchus species in vitro, but failed. For example, Brown and Vessey demonstrated that H. multicinctus failed to survive when cultured on banana fruit callus [39]; Khera and Zuckerman cultured H. erythrinae on tissues of carrot, sweet potato, tobacco and tomato, but nematodes all failed to reproduction on these tissues [40]; Kagoda et al. reported that H. multicinctus failed to rear on carrot discs [41]. One Helicotylenchus species and H. dihystera have been reported to successfully produce progenies after inoculating single female in host-plants pots [42, 43]. However, the method culturing the Helicotylenchus species in host-plants pots was susceptible to contamination, time-consuming and difficult to separate the progeny from the pots. Sterile carrot disks are usually regarded as a relatively low-cost, straightforward and efficient method for culturing some nematodes that results in greater nematode multiplication compared with other methods [21]. Our study is the first to successfully establish an artificial culture method for H. microlobus on carrot disks. The succcesful rearing of H. microlobus make other related biological studies of H. microlobus possible.

Plant growth inhibition has been reported associated with several Helicotylenchus species that have also increased secondary infections of fungal pathogens [44]. Studies have shown that when H. dihystera and Pseudomonas solanacearum present together, tomato wilting was much more serious than when present alone, and when H. dihystera associated with P. caryophylli, carnation wilting was also significantly increased [12, 45]. Therefore, whether there is such a synergistic relationship between H. microlobus and certain bacteria that may led to more serious disease on tomato should be further investigated.

Conclusions

In our study, both morphological and molecular analyses showed that the species of the Helicotylenchus population was H. microlobus. This is the first report of H. microlobus from tomato in China. We established that the carrot disk method is suitable for rearing of H. microlobus on carrot disks, and the optimum temperature for H. microlobus culture on carrot disks was 27.5 °C, and the Rr reached 406 after 90 days of inoculation with 30 females.

Methods

Nematode collection

In August 2019, five samples of roots and corresponding rhizosphere soils were collected from a tomato (cv. Maohong 801) field near Zhuma village in Tongxu County of Kaifeng city, Henan Province, China. Each sample consisted of at least five sub samples collected from patches of poor growth. Samples were placed in plastic bags, sealed transported to the laboratory in refrigerated counter and stored at 16 to 18 °C [46]. Nematodes were extracted from tomato soil and macerated root samples using the modified Baermann funnel method [47].

In vitro rearing of nematodes

Carrot disks were prepared in the following way. The surface of a carrot was sterilized with 95% ethanol. The carrot was peeled with a sterile knife and cut into 6 mm thick disks. Each carrot tissue was placed in a 6-cm-diameter petri dish and maintained at 25 °C for 15 days for later use. One female from the collected specimens was selected and surface sterilized for 6 h with 0.3% streptomycin sulfate, and then transferred to carrot disks. and the petri dishes were sealed with parafilm and then kept in a darkened incubator at 25 ℃for 15 weeks. After that, the single female Helicotyenchus population cultured on carrot disks were used for morphological and molecular analysis.

Morphological identification

Nematodes were heat-killed in water, fixed in FG (formalin: glycerin: water = 10:1:89), and processed to glycerin by the formalin glycerin method [47]. Photomicrographs and morphometric data of the Helicotylenchus specimens 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 [30]. All measurements are expressed in micrometers (μm) [48].

Molecular characterization and phylogenetic relationships

DNA from one Helicotylenchus specimen was extracted using proteinase K-based lysis [49]. The rRNA-internal transcribed spacer (ITS) region and the D2-D3 region of the 28S rRNA gene were amplified with primers TW81/AB28 (5´-GTTTCCGTAGGTGAACCTGC-3´/5´-ATATGCTTAAGTTCAGCGGGT-3´) [50] and primers D2A-D3B (5´-ACAAGTACCGTGAGGGAAAGTTG-3´/5´TCGGAAGGAACCAGCTACTA-3´) [51], respectively. The processes of PCR amplification and cloning were carried out according to the method of Wang et al. [52]. Sequencing was performed by Sangon Biotech Co. Ltd. (Shanghai, PR China). The newly obtained DNA sequences were submitted to the NCBI GenBank (https://submit.ncbi.nlm.nih.gov/subs/genbank/) database.

The obtained ITS region and D2-D3 region of the 28S sequences were subjected to multiple alignment using the MAFFT Q-INS-i algorithm [53] with other Helicotylenchus species sequences published in the NCBI GenBank database. Outgroup taxa were selected based on a previous study [54]. Sequence datasets were analysed with Bayesian inference (BI) using MrBayes 3.2.6 [55] under the best-fit model of GTR + G + I, according to Akaike Information Criteria [56]. BI analysis for each gene was run with a random starting tree and four Markov chains for 1 × 106 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 undertaken to determine the reproductive potential of H. microlobus and also to determine the optimum temperature for rearing H. microlobus on carrot disks. In the experiment, 30 females were surface sterilized for 6 h with 0.3% streptomycin sulfate and then transferred to a carrot disk in a petri dish. The petri dishes were sealed with parafilm and incubated in a darkened incubator at 25, 27.5, and 30 °C, respectively. The number of nematodes and reproduction rate (Rr = final 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 the nematodes [46]: the carrot disks were placed in sterile water and macerated in a blender. The suspension was poured through 0.250-mm and 0.150-mm-pore sieves. The nematode suspension was collected in a beaker and the carrot tissues discarded. The nematode suspension was left to settle for at least 4 h and the supernatant was removed by pipettor. The nematodes were enumerated to determine whether H. microlobus reproduction had occurred and the Rr (final number of nematodes/initial number of nematodes) determined. There were five replicates for each experiment, and each experiment was conducted twice. Data on H. microlobus cultured on carrot disks under different temperatures were subjected to a one-way analysis of variance (ANOVA), and differences were tested using Duncan’s multiple range test (DMRT) at the 5% significance level using SPSS software (ver. 13.0; SPSS Inc., Chicago). The result of this experiments are given in Table.