Introduction
Guizhou and Sichuan provinces, both situated in the southwestern region of China, boast diverse and complex terrain featuring mountains, hills, and basins. This geographical diversity contributes to distinct vertical climatic characteristics, fostering various ecological types and a rich array of species. Moreover, the land in this region is fragmented, with a significant portion consisting of sloping farmland (Chen & Liu, Reference Chen and Liu2022). The unique ecoclimatic conditions allow for a diversity in the types and cultivation methods of crops in this region. The main crops include rice, potato, maize, buckwheat, and tobacco, among others (Lu & Jia, Reference Lu and Jia2007). Cyst nematodes, subfamily Heteroderinae, are plant pathogens of worldwide economic significance. Crops parasitized by these nematodes exhibit symptoms similar with physiological diseases such as nutrient deficiency, which are difficult to identify (Jones et al., Reference Jones, Haegeman, Danchin, Gaur, Helder, Jones, Kikuchi, Manzanilla-Lopez, Palomares-Rius and Wesemael2013). Cyst nematodes parasitize plant roots and commonly spread through soil in farmland. The characteristics of farmland and crop distribution in the southwestern region of China are favorable for the spread of these nematodes.
During a survey of plant nematode species in Guizhou and Sichuan provinces in 2023, roots and rhizosphere soil samples were collected from a variety of crops. Among these, 18 rhizosphere soil samples taken from crops such as potatoes, buckwheat, maize, cabbage, and tea were found to contain cyst nematodes in the laboratory. Through the comparison of morphological characteristics and sequences of the the internal transcribed spacer (ITS-rRNA), the large subunit of the nuclear ribosomal RNA (28S-rRNA) D2-D3 region, as well as the the partial cytochrome oxidase subunit I (COI) gene, it was determined that these 18 populations belong to the same species within the genus Cactodera of the subfamily Heteroderinae. They are distinct from any known species of the genus Cactodera and represent a new species of this genus, named as Cactodera xinanensis n. sp.. In this paper, the morphological and molecular characteristics of the new species were described, and the key and the morphological diagnostic feature table for species within Cactodera were revised and supplemented.
Material and methods
Nematode isolates and morphological identification
The cysts were isolated using the Cobb’s sieving method (Cobb, Reference Cobb1918). The eggs inside the cysts were extracted and then hatched in sterile water to obtain J2s (second-stage juveniles). Subsequently, the J2s were heat killed, fixed in FG fixative, and dehydrated using a glycerol-ethanol dehydration process according to Seinhorst’s method (Seinhorst, Reference Seinhorst1959), and then mounted on slides. Vulva cones were dissected and mounted in neutral balsam according to Subbotin et al. (Reference Subbotin, Mundo-Ocampo, Baldwin, Hunt and Perry2010). The nematodes were photographed, measured, and observed using an AxioCam MRm Zeiss digital camera attached to a Zeiss Scope A1 microscope (Zeiss, Jena, Germany) equipped with differential interference contrast (Zeiss Scope A1 ZEN light 2012 software). For scanning electron microscopy, J2s, cysts and eggs were processed according to the method of Wang et al. (Reference Wang, Xie, Li, Xu, Yu and Wang2013), then observed and photographed with a Zeiss EVO MA15 at 10 kV (Zeiss).
Molecular and phylogenetic analyses
DNA was extracted from J2 individuals using extraction buffer containing Proteinase K, three replicates (Xu et al., Reference Xu, Zhao, Ding, Zhang and Xie2016). The ITS-rRNA gene was amplified using primers TW81 (5’-GTTTCCGTAGGTGAACCTGC-3’) and AB28 (5’-ATATGCTTAAGTTCAGCGGGT-3’) (Maafi et al., Reference Maafi, Subbotin and Moens2003). The D2-D3 region of the 28S-rRNA gene was amplified with the D2A (5′-ACAAGTACCGTGAGGGAA AGTTG-3′) and D3B (5’-TCGGAAGGAACCAGCTAC-TA-3) (De Ley et al., Reference De Ley, Felix, Frisse, Nadler, Sternberg and Thomas1999). The partial COI gene was amplified with Het-coxiF (5′-TAGTTGATCGTAA TTTTAATGG-3′) and Het-coxiR (5′-CCTAAAACATAATGAAAATGWGC-3′) (Subbotin, Reference Subbotin2015). The polymerase 2×Phanta Flash Master Mix (Vazyme) was used for polymerase chain reaction (PCR). The PCR products were purified using a gel extraction kit (Genesand Biotech Co., Ltd; Beijing, China), ligated into pEASY-blunt cloning vector (TransGen Biotech; Beijing, China) and sequenced by Sangon Biotech Co. Ltd. (Shanghai, China). The sequences were aligned by BLAST in the GenBank database and deposited in GenBank.
The newly obtained sequences for the ITS-rRNA, D2-D3 region of the 28S-rRNA and partial COI gene were aligned using MAFFT v7.149b (Katoh & Standley, Reference Katoh and Standley2013) with the corresponding gene sequences for Cactodera and edited in Gblock (Castresana, Reference Castresana2000). The best-fit model of DNA evolution for Bayesian inference was obtained using the program MrModeltest2.3 (Nylander, Reference Nylander2004) according to the Akaike Information Criterion.Sequence datasets for each gene fragment were analysed separately with Bayesian inference using MrBayes 3.1.1 (Huelsenbeck & Ronquist, Reference Huelsenbeck and Ronquist2001). The phylogenetic consensus trees were visualised using the software FigTree v.1.4.3 (Rambaut, Reference Rambaut2014). Outgroup taxa for each dataset were chosen according to the results of previously published data (Escobar-Avila et al., Reference Escobar-Avila, Subbotin and Tovar-Soto2020; Ni et al., Reference Ni, Xie, Yang, Yang, Xu and Xie2024).
Results
Cactodera xinanensis n. sp. (Figs 1-3). For measurements, see Table 1.
Table 1. Morphometrics of Cactodera xinanensis n. sp
All measurements are in μm, and in the form: mean±standard deviation (range).
Figure 1. Microphotographs of Cactodera xinanensis n. sp. Cyst: A, B entire body; C-D vulval cone (anus is indicated by arrow); E-F, vulval denticles (arrowed). Second-stage juvenile: G, entire body; H, anterior end of body; I, J, head part; K, genital primordium; L, lateral field; M, N, tails; O, phasmid; Egg: P, entire; Q, eggshell. Scale bar: A = 600 μm; B = 200 μm; C, D, G, P = 100 μm; E, F, H, Q = 20 μm; I-O = 10 μm.
Figure 2. Scanning electron microscope microphotographs of Cactodera xinanensis n. sp. Cyst: A, entire body; B, surface; C, vulval cone; D, vulval fenestral. Second-stage juvenile: E-F, head part; G, tails (phasmid is indicated by arrow); H, lateral field. Egg: I, entire; J, eggshell. Scale bar: A = 100 μm; B, H, I = 2 μm; C = 20 μm; D, G, I = 10 μm; E, F, J = 1 μm.
Figure 3. Line drawing of Cactodera xinanensis n. sp. Cyst: A, whole body; B, vulval cone. Second-stage juveniles: C, entire body; D, anterior part of body; E, head and stylet; F, G, tails.
Description
Cyst. Subspherical to lemon-shaped, brown to tan, with protruding neck and vulvar cone (Figs 1 A-B, 2 A, 3 A). Cyst surface with reticulated ridge patterns, punctations usually present (Fig 2 B). Vulval cone surface with wavy ridge patterns and often broken by short oblique or vertical lines (Figs 1C-D, 2 C-D). Cone top concave, circumfenestrate, with vulval denticles, without underbridge and bullae (Figs 1 C-F, 2 C-D, 3 B). Anus round, dot-like, without fenestration (Figs 1 C-D, 3 B).
Second-stage juvenile. Body vermiform, slightly curved ventrally (Figs 1 G, 3 C). Head region slightly offset with four to five annuli, lip disc oval dorsoventrally elongated, four submedial lips distinct, two lateral submedial lips greatly reduced. Amphidial apertures conspicuous (Fig 2 E-F). Stylet well developed, cone about half the length of the stylet, knobs slightly rounded, anterior surface sloping posteriorly (Figs 1 I-J, 3 E). Median bulb oval with distinct valvular apparatus (Fig 1 H). Hemizonid and excretory pore located at the level of the between isthmus and esophageal gland. Excretory pore immediately behind hemizonid (Figs 1 H, 3D). Pharyngeal glands filling body cavity. Lateral field with four lines, the middle two lines merging at the posterior part of phasmid and becoming three lines (Figs 1 L, 2 H, 3 G). Phasmid pore-like openings located in anterior to hyaline region (Figs 1 O, 2 H G, 3 G). Tail conoid, with thin rounded terminus. Hyaline region approximately 50% of the tail length, often longer than stylet (Figs 1 M-N, 2 G, 3 E).
Egg. Eggshells with fine punctuates visible with both light microscope and scanning electron microscopy (Figs 1 P-Q, 2 I- J).
Male. Not found.
Type habitat and locality
Roots and rhizospheric soil of potato, Solanum tuberosum (Solanaceae, Solanales), in Ertang Town, Weining Yi Hui Miao Autonomous County, Bijie City, Guizhou Province (E: 104.665496, N: 26.675777).
Other habitats
The information for the other habitats are provided in Table 2.
Table 2. Samples and sequences information
Etymology
This new species is named after xinan, the pinyin for southwest China, where it was collected.
Type material
The holotype cyst, 30 paratype cysts, 26 paratype J2s and 17 paratype eggs were deposited in the Laboratory of Plant Nematology and Research Center of Nematodes of Plant Quarantine, South China Agriculture University, Guangzhou, Guangdong, China.
Diagnosis and relationships
C. xinanensis n. sp. is characterized by the following features: length/width (L/W) ratio 1.3 ± 0.1 (1.1–1.6) (including neck), surface with punctations, vulval denticles present, bullae and underbridge absent, fenestral diameter 28.1 ± 4.3 (21.3–38.7) μm in cysts; stylet 21.5 ± 0.5 (20.3–22.6) μm, tail 59.4 ± 2.0 (55.9–63.8) μm, hyaline region 28.7 ± 2.7 (25.0–36.3) μm, lateral field with four lines in J2s; eggshell with punctations.
C. xinanensis n. sp. can be differentiated from known species of Cactodera by longer mean tail (59 μm) and hyaline region (29 μm) of J2s. The new species is close to C. cacti (Krall & Krall, Reference Krall, Krall, Krall and Krall1978) in having an L/W ratio of 1.1–1.6, fenestral diameter 28.1 ± 4.3 (21.3–38.7) μm, with vulval denticles in cysts and eggshell with punctations, but differs by J2s having longer mean tail (59 μm vs. 41–55 μm) and hyaline region (29 μm vs. 16–25 μm), hyaline region longer than stylet vs. hyaline region shorter than stylet.
The main morphological and morphometric characters of C. xinanensis n. sp. and 18 valid species of the genus Cactodera, are compared in Table 3.
Table 3. Morphological and morphometric characters useful for identification of Cactodera species (means are given in μm)
Molecular characterization and phylogenetic analysis
The ITS-rRNA sequences from these 18 populations of C. xinanensis n. sp. were obtained. All sequence lengths were 966 bp (including primer sequences), without intraspecific sequence variation. The results of BLAST showed that the ITS-rRNA gene sequences from C. xinanensis n. sp. were closest to those from Cactodera sp. (MW821355), with 98.55% identity and 14-bp variation. Six sequences were randomly selected for phylogenetic analysis. The Bayesian phylogenetic tree generated from ITS dataset under GTR + G model is presented in Fig 4, which shows that the six sequences of C. xinanensis n. sp formed a clade, the posterior probability (PP) = 100, and closest to Cactodera sp. (MW821355, MW821356 and MW658364).
Figure 4. Phylogenetic relationships of Cactodera species inferred from Bayesian analyses of ITS rRNA sequences under GTR+G model. Posterior probabilities more than 50% are given to appropriate clades. Newly obtained sequences are indicated in bold font. *Originally identified as C. estonica in GenBank.
The 28S-rRNA sequences from these 18 populations of C. xinanensis n. sp. were obtained. All sequences lengths were 782 bp (including primer sequences), without intraspecific sequence variation. The results of BLAST showed that the 28S rRNA gene sequences from C. xinanensis n. sp. were closest to those from C. guizhouensis (OR438934), with 98.85% identity and 9-bp variation. Six sequences were randomly selected for phylogenetic analysis. The Bayesian phylogenetic tree generated from 28S-rRNA dataset under GTR + I + G model is presented in Fig 5, which shows that the six sequences of C. xinanensis n. sp formed a clade (PP = 99).
Figure 5. Phylogenetic relationships of Cactodera species inferred from Bayesian analyses of D2–D3 of 28S rRNA sequences under GTR+I+G model. Posterior probabilities more than 50% are given to appropriate clades. Newly obtained sequences are indicated in bold font. *Originally identified as C. estonica in GenBank.
The COI sequences from these 18 populations of C. xinanensis n. sp. were obtained. All sequences lengths were 490 bp (including primer sequences), without intraspecific sequence variation. The results of BLAST showed that the COI gene sequences from C. xinanensis n. sp. were closest to those from C. chenopdiae (MG744314), with 88.19% identity and 49-bp variation. Six sequences were randomly selected for phylogenetic analysis. The Bayesian phylogenetic tree generated from COI dataset under GTR + I + G model is presented in Fig 6, which shows that the six sequences of C. xinanensis n. sp. formed a clade (PP = 100).
Figure 6. Phylogenetic relationships of Cactodera species inferred from Bayesian analyses of COI sequences under GTR+I+G model. Posterior probabilities more than 50% are given to appropriate clades. Newly obtained sequences are indicated in bold font. *Originally identified as C. torreyanae in GenBank.
The accession numbers of all sequences submitted to the GenBank database in this paper are listed in the Table 2.
Discussion
The genus Cactodera was created by Krall and Krall in 1978, with the type species C. cacti, which has 18 valid species at present. Apart from a few species whose type populations were collected from the rhizosphere of crops such as barley (C. rosae, C. galinsogae), tomato (C. solani), and potato (C. guizhouensis), the majority inhabit the rhizospheres of weeds from plant families including Amaranthaceae, Polygonaceae, Chenopodiaceae, and Asteraceae (Subbotin et al., Reference Subbotin, Mundo-Ocampo, Baldwin, Hunt and Perry2010; Cid Del Prado Vera & Subbotin, Reference Cid Del Prado Vera and Subbotin2014; Escobar-Avila et al., Reference Escobar-Avila, Subbotin and Tovar-Soto2020; Li et al., Reference Li, Li, Ni, Shi, Wei, Liu, Zhang and Peng2021; Ni et al., Reference Ni, Xie, Yang, Yang, Xu and Xie2024). In China, five species of genus Cactodera have been found, includeing C. cacti (Pan et al., Reference Pan, Lin and Xue1997), C. thornei (Peng & Vovlas, Reference Peng and Vovlas1994), C. chenopodiae (Feng et al., Reference Feng, Wang, Xiao, Pereira, Xuan, Wang, Liu, Chen, Duan and Zhu2018), C. tianzhuensis (Li et al., Reference Li, Li, Ni, Shi, Wei, Liu, Zhang and Peng2021), and C. guizhouensis (Ni et al., Reference Ni, Xie, Yang, Yang, Xu and Xie2024). Among these, C. chenopodiae, C. tianzhuensis, and C. guizhouensis were respectively isolated from the rhizosphere of Chenopodiaceae plants, Polygonum plants, and potato. These three species have only been recorded in China to date. This study descries the 19th species of Cactodera, C. xinanensis n. sp., which is also the sixth species of the genus found in China. C. xinanensis n. sp. was isolated from the rhizospheric soil of six different plants including potato, wormwood (Artemisia argyi), maize (Zea mays), buckwheat (Fagopyrum esculentum), cabbage (Brassica oleracea), and tea (Camellia sinensis), and some cysts were isolated from the roots of potato, buckwheat and wormwood. Of the 18 populations obtained, 10 were from the potato rhizospheres, and during field sampling, we discovered cysts in the potato roots, so we designated potato as the putative host plant of the new species. Because the cysts of some samples were obtained not from the roots but from the rhizosphere soil, there may be other host plants in the field for this new species. In addition, we found that that the eggs of the new species can hatch in water. This finding suggests that the new species may possess a wider range of potential hosts. Therefore, the host range and pathogenicity of the new species need to be further studied.
The morphological identification and differentiation of species within the genus Cactodera primarily rely on characteristics such as L/W, fenestral diameter, the presence or absence of vulval denticles in the cyst; the presence or absence of punctate on eggshell surface; as well as the length of stylet, tail and hyaline region in J2 (Subbotin et al., Reference Subbotin, Mundo-Ocampo, Baldwin, Hunt and Perry2010; Perry et al., Reference Perry, Moens and Jones2018). However, with the continuous description of new species and the increase in the number of species within Cactodera, the overlap of morphological measurements between species began to appear, making it difficult to distinguish similar species based on morphological characteristics. Molecular characteristics provide a new reference for accurate species identification, and sequence alignment and phylogenetic analysis have become important methods to identify the species of the cyst-forming genera (Subbotin et al., Reference Subbotin, Vierstraete, De Ley, Rowe, Waeyenberge, Moens and Vanfleteren2001; Handoo et al., Reference Handoo, Skantar, Subbotin, Kantor, Hult and Grabowski2021). However, because of the early descriptions of some species within the Cactodera genus, molecular biological characteristic information is lacking. For instance, in GenBank, C. acnidae (Schuster & Brezina, Reference Schuster and Brezina1979), C. amaranthi (Krall & Krall, Reference Krall, Krall, Krall and Krall1978), C. eremica (Baldwin & Bell, Reference Baldwin and Bell1985), C. evansi (Cid Del Prado Vera & Rowe, Reference Cid Del Prado Vera and Rowe2000), C. radicale (Chizhov et al., Reference Chizhov, Udalova and Nasonova2008), and C. thornei (Mulvey & Golden, Reference Mulvey and Golden1983) lack both ITS-rRNA and 28S-rRNA sequences information, with only six species having COI sequences uploaded. Therefore, the molecular characteristics and phylogenetic analysis of species within the genus are limited. Additionally, there are many incorrect sequences in GenBank, for example, the ITS-rRNA sequences HM560732, HM560730, EU106164, KC771888, and the 28S-rRNA sequences HM560979, HM560796 were originally uploaded as C. estonica, but these sequences have been repeatedly indicated as not belonging to C. estonica (Cid Del Prado Vera et al., 2014; Escobar-Avila et al., Reference Escobar-Avila, Subbotin and Tovar-Soto2020; Li et al., Reference Li, Li, Ni, Shi, Wei, Liu, Zhang and Peng2021; Ni et al., Reference Ni, Xie, Yang, Yang, Xu and Xie2024). The results of the phylogenetic analysis in this paper also indicate that these sequences do not belong to C. estonica. These erroneous sequences add to the difficulty of identifying species within this genus based on molecular characteristics. Therefore, it is necessary to rely on the combination of morphological and molecular characteristics to accurately identify the species. C. xinanensis n. sp. can be differentiated from all known species of the genus Cactodera by its longer tail and hyaline region of J2. Furthermore, it forms a separate clade in the phylogenetic trees of ITS-rRNA, 28S-rRNA D2-D3 region, and COI sequences, indicating that it is a new species of the genus.
Key to species of the genus Cactodera
(This key is mainly based on information taken from Subbotin et al., Reference Subbotin, Mundo-Ocampo, Baldwin, Hunt and Perry2010; Cid Del Prado and Subbotin, Reference Cid Del Prado Vera and Subbotin2014; Feng et al., Reference Feng, Wang, Xiao, Pereira, Xuan, Wang, Liu, Chen, Duan and Zhu2018; Li et al., Reference Li, Li, Ni, Shi, Wei, Liu, Zhang and Peng2021; Ni et al. Reference Ni, Xie, Yang, Yang, Xu and Xie2024)
Acknowledgements
This research was supported by the Plant Quarantine Station of Sichuan Agricultural and Rural Department (No. N5100012022001943) and the Detection and Control of Crop Disease and Pests Project of China (No. 101821301082351011).
