Meloidogyne aberrans sp. nov. (Nematoda: Meloidogynidae), a new root-knot nematode parasitizing kiwifruit in China

High infection rates of roots of wild kiwifruit (Actinidia chinensis Planch) and soil infestation by a root-knot nematode were found in Anshun, GuiZhou Province, China. Morphology, esterase phenotype and molecular analyses confirmed that this nematode was different from previously described root-knot nematodes. In this report, the species is described, illustrated and named Meloidogyne aberrans sp. nov. The new species has a unique combination of characters. A prominent posterior protuberance, round and faint perineal pattern and a medium-length stylet (13.6–15.5 μm) characterized the females. Second-stage juveniles (J2) were characterized by a smooth lip region with distinctly protruded medial lips and a depression in outline at the oral aperture, a relatively long stylet (15.9–16.8 μm), four incisures in the lateral field and a very short, even poorly defined, hyaline tail terminus (2.2–5.5 μm). More incisures (11–15) existed in the lateral field of males, and the stylet and spicules of males were 18.2–19.6 μm and 22.7–36.8 μm long respectively. Egg masses were typically produced within the roots of kiwifruit. The new species had a rare Est phenotype, S2. Phylogenetic trees inferred from SSU, LSU D2D3, ITS, and partial coxII-16S rRNA revealed that M. aberrans sp. nov. was within the Meloidogyne clade and was distinguished from all described root-knot nematodes. Moreover, from histopathological observations, M. aberrans sp. nov. induced the formation of multinucleate giant cells.


Introduction
The kiwifruit (Actinidia chinensis Planch), or Chinese gooseberry, is a favorite fruit that is eaten raw, made into juices or used as a garnish. Kiwifruit is currently grown in more than 20 countries, and in 2011, FAO estimated the area yielding kiwifruit reached 94,000 hm 2 [1]. However, various diseases that include plant-parasitic nematodes threaten worldwide production of kiwifruit. Meloidogyne spp. root-knot nematodes are one of the most devastating plant pathogens to infest kiwifruit. With the exception of Africa, root-knot nematodes attack kiwifruit grown on other continents. Meloidogyne incognita and M. hapla are the most prevalent a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Nematode materials
Samples of kiwifruit roots and rhizosphere soils were collected in Anshun City, Guizhou Province, China, during February 2013, October 2015 and May 2017. Females, males and egg masses were dissected directly from galled roots. Second-stage juveniles (J2s) were isolated from fresh soils using Baermann funnels [24] or collected from hatching eggs [25].

Morphological studies
To prepare for light microscopy (LM), males and J2s were relaxed with gentle heat, fixed in a solution of 4% formaldehyde + 1% glycerin and processed using the glycerin-ethanol method [24]. Perineal patterns of mature females were prepared as described [26]. The perineal pattern was trimmed and transferred to a drop of glycerin for observation. Nematodes were measured and photographed with a Nikon ECLIPSE Ni microscope equipped with a Nikon Digital Sight Camera and exclusive NIS-Elements BR software (Nikon, Tokyo, Japan).
Females, males and J2s were prepared for scanning electron microscopy (SEM) as described [27]. Nematodes were observed with a XL-30-ESEM microscope (Philips, the Netherlands).

Isozyme phenotype analysis
Ten young, egg-laying females of M. aberrans sp. nov. were used for isozyme phenotype analysis. Four females of a previously identified population of M. javanica [28] were used for comparison. The phenotypes were for esterases (Est) and malate dehydrogenase (Mdh) [29].

Phylogenetic analyses
The sequences of M. aberrans sp. nov. were compared with GenBank nematode sequences using the BLAST homology search program. The most similar sequences were selected for phylogenetic analyses. Out-group taxa for each data set were chosen according to previous molecular phylogenetic analyses for root-knot nematodes [37][38][39]. DNA sequences were aligned in MEGA4.0 [40] using default parameters. Models of base substitution were evaluated using MODELTEST3.7 [41,42] combined with PAUP4.0 [43]. The Akaike-supported model, base frequencies, proportion of invariable sites, and gamma distribution shape parameters and substitution rates were used in phylogenetic analyses. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.2 [42] running the chain for 1 × 10 6 generations and setting the 'burn-in' at 2500. The MCMC (Markov Chain Monte Carlo) method was used within a Bayesian framework to estimate the posterior probabilities of the phylogenetic trees [44] and generate a 50% majority-rule consensus tree.

Histopathology
Galled roots from kiwifruit plants naturally infected by M. aberrans sp. nov. were collected in Guizhou, China, for histopathological studies. Galls were cut off, fixed, dehydrated and embedded as described [45,46]. Then, the galls were sliced, and the paraffin was removed following the description of Bachand and Castello (2001) [47]. Sections 10 μm thick were placed on glass slides, stained with safranin and fast green [48], mounted permanently in resinene, and examined and photographed with the Nikon ECLIPSE Ni microscope.

Nomenclatural acts
The electronic edition of this article conformed to the requirements of the amended International Code of Zoological Nomenclature; therefore, the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is as follows: urn:lsid:zoobank.org:pub: 75F0D6B5-58E5-4203-9669-30D3CC3C7B1C. The electronic edition of this work was published in a journal with an ISSN and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Description
Female. Body completely embedded in galled tissue and pearly white, pear-shaped to ovoid with neck projecting at different angles. Posterior end of body with distinct, elevated perineum (Figs 1K and 2A). Lip region slightly offset. Head cap distinct, labial disk elevated (Figs 1L and 2B). Under SEM, the labial disc appeared round-squared, slightly elevated, fused with median lips, dumbbell-shaped. Six inner labial sensilla surrounding ovoid prestoma; stoma slit-like. Lateral lips large, triangular, separated from lip disc. Amphidial apertures elongated, located between labial disc and lateral lips (Fig 4A and 4B). Stylet moderately long, with round knobs, conus slightly curved and shaft straight ( Fig 2C). Excretory pore distinct, typically located 2-3.5 stylet lengths posterior to stylet knobs. Metacorpus developed, rounded, with heavily sclerotized valve (Figs 1L and 2B). Pharyngeal gland with a large dorsal lobe and two subventral gland lobes. Perineal pattern oval, striae extremely faint, broken (Figs 1M, 2D and 2E). Vulva slit wider than vulva-anus distance. Anus fold visible in several specimens. Phasmid not visible. Measurements are listed in Table 1.
Male. Body vermiform, tapering anteriorly (Figs 1A and 3A). Lip region slightly set off from body, with a obvious head cap (Figs 1C and 3C). Lip frame-work sclerotised. Under SEM, labial disc appeared round-squared, elevated. Large stoma-like slit located in a oval prestoma and surrounded by six inner labial sensilla. Medial lips large, separated from labial disc, forming an deep slit. Lateral lips large, triangular, separated from lip disc, with two or three interupting post-labial annulus. Amphidial apertures elongated, located between labial disc and lateral lips (Fig 4E and 4F). Stylet straight, cone narrow, sharply pointed; shaft widened slightly. Stylet knobs distinct, rounded and slightly concaved anteriorly ( Fig 3C). Lateral fields narrow, occupying about one-fifth of the body width, with 11 to 15 lateral lines at mid-body, outer bands areolated in some specimens under SEM (Figs 1D, 3F, 4G and 4H). Excretory pore distinct, located posterior to nerve ring. Hemizonid conspicuous, located about 3-4 annuli anterior to excretory pore (Figs 1B and 3B). Metacorpus oval. One testis extending anteriorly ( Fig 3E). Spicules of variable length, arcuate, slender, two pores clearly visible at tip under SEM (Figs 1E, 3D and 4I). Gubernaculum simple, almost straight (Figs 1E and 3D). Tail short, hemispherical, with a humped end and twisted posterior body portion (Figs 1E, 3D and 4I). Measurements are listed in Table 1. J2. Body vermiform, tapering at both ends, ventrally curved after killing with heat (Figs 1F and 3G). Lip region smooth, continuous to body, depression in outline at oral aperture in the lateral view (Figs 1G, 1H and 3H-3J). Under SEM, labial disc appeared round-squared, and oral aperture located in the middle of labial disc surrounded by six inner labial sensilla. Medial lips distinctly protruded, extending farther than lateral lips and labial disc, resulting in an oral depression. Amphidial apertures appeared as a wide slit between the labial disc and lateral lips (Fig 4J and 4K). Stylet long, straight or conus slightly curved; cone narrow, sharply pointed; shaft widened slightly posteriorly; knobs distinct, sloping posteriorly ( Fig 3K). Body annuli distinct, fine. Lateral fields with four lines (Figs 1I and 3L), areolated completely under SEM (Fig New Meloidogyne nematode parasitizing kiwifruit 4L). Excretory pore distinct, located posterior to nerve ring (Figs 1G and 3I). Hemizonid conspicuous, located 1-2 annuli anterior to excretory pore or immediately anterior to excretory pore. Metacorpus oval, with heavily sclerotized valve. Pharyngeal gland lobe long, ventrally overlapping intestine. Tail tapering gradually toward the end, with a bluntly round terminus (Figs 1I, 1J, 3M-3O and 4M). Hyaline tail short, sometimes not clearly defined (Figs 1I, 1J and 3M-3O). Phasmids indistinct. Measurements are listed in Table 1.

Etymology
The species epithet refers to the unique combination of morphological characters, which included an elevated perineum, a faint perineal pattern, distinctly protruded medial lips resulting in a depression in outline at the J2 oral aperture and a very short, even poorly defined hyaline tail.

Isozyme analysis
The isozyme electrophoretic analysis of young, egg-laying females of M. aberrans sp. nov. showed a rare Est phenotype, S2, i.e., two Est bands at Rm = 40.5% and 44.5% (Fig 5A and 5B). The band of Mdh phenotype of M. aberrans sp. nov. was similar in size to that of M. javanica N1 Mdh phenotype (Fig 5C).

Molecular characterization
The five SSU sequences of 1734 bp from one female, one male and three different J2s were sequenced, respectively. GenBank accession numbers of the five sequnences are KF278755 for the female, KF278756 for the male, and KX776409, KX776410 and KU598836 for the J2s. The identities were 100% or 99.9% (1733/1734) between any two of the five. A BLAST search of M. aberrans n. sp. revealed the highest match with the sequence of M. ichinohei (GenBank accession numbers EU669953). The identities between the five sequences from the new species and the sequence from M. ichinohei were 93.7%.
Four LSU D2D3 sequences of 789 bp and one J2 LSU D2D3 sequence of 791 bp were sequenced based on the same templates as mentioned above. GenBank accession numbers are KF278754 for the female, KF278753 for the male, and KX776411, KX776412 and KU598837 for the J2s. The identities of these five sequences were 100% or 99.7% (787/791) with two insertions/deletions between any two. A BLAST search of M. aberrans n. sp. revealed the highest match with the sequence of M. ichinohei (GenBank accession numbers EF029862). However, the identities between the five sequences from the new species and the sequence from M. ichinohei were only 83.5%.
The five ITS-rDNA sequences of 664 bp were sequenced based on the same templates as mentioned above. GenBank accession numbers of these sequences are KF278757 for the female, KF278758 for the male, and KX776413, KX776414 and KU598838 for the J2s. The identities were 100%, 99.8% (663/664) or 99.7% (662/664) between any two of the five. A BLAST search of M. aberrans n. sp. revealed the highest match with the sequence of M. New Meloidogyne nematode parasitizing kiwifruit panyuensis (GenBank accession numbers AY394719). The identities between the sequences from the new species and the sequence from M. panyuensis were only 77.4% and 77.3%, respectively.
The three sequences of 549 bp, one male sequence of 548 bp and one J2 sequence of 547 bp for coxII-16S rRNA were sequenced based on the same templates as mentioned above. Gen-Bank accession numbers are KF278759 for the female, KF278760 for the male, and KX776415, KX776416 and KU598839 for the J2s. Among these five sequences, the identities were 100%, 99.8% (548/549) or 99.6% (547/549 or 546/549 with one insertions/deletions) between any two. A BLAST search of M. aberrans n. sp. revealed the highest match with the sequenc of M. marylandi (GenBank accession numbers KC473862). The identities between the sequences from the new species and the sequence from M. marylandi were only 76.5%-76.7%.
These twenty different sequences, including rDNA sequences of SSU, LSU D2D3 and ITS, and mtDNA sequence of coxII-16S rRNA, of M. aberrans sp. nov., indicated that all had highscoring matches with some Meloidogyne species and that all were clearly different from those in the GenBank database. Sequence divergences between the new species and other species of Meloidogyne were 5.4-11.1%, 18.8-33.7%, 26.5-67.0% and 22.9-38.0% for SSU, LSU D2D3, ITS and coxII-16S rRNA, respectively, supporting its separate specific status.
The molecular phylogenetic status of M. aberrans sp. nov. is presented in Figs 6-9, and based on the sequences of SSU, LSU D2D3, ITS and coxII-16S rRNA reconstructed in this study, these four phylogenetic trees confirmed that the new species was within the Meloidogyne clade. In Fig 6, the phylogenetic tree is based on SSU from a multiple alignment of 1794 total characters. When Hirschmanniella loofi Sher, 1968 [68] was used as the out-group taxon, M. aberrans sp. nov. was in a 100% supported monophyletic clade with M. ichinohei, another species with an elevated perineum. This clade was sister to M. camelliae, a species without an elevated perineum, but was far from the other four species that have a posterior protuberance, M. graminis, M. spartinae, M. oryzae and M. africana. In Fig 7, the phylogenetic tree is based on LSU D2D3 from a multiple alignment of 803 total characters. Using Hirschmanniella santarosae De Ley, Mundo ocampo, Yoder & De Ley, 2007 [69] as the out-group taxon, M. aberrans sp. nov. was also close to M. ichinohei with 54% support. These two species were also sister to M. camelliae. In Fig 8, the phylogenetic tree is based on ITS from a multiple alignment of 884 total characters. When using Hirschmanniella mucronata (Das, 1960) Luc & Goodey, 1963 [70,71] as the out-group taxon, M. aberrans sp. nov. and the other species M. megadora that possesses an elevated perineum were monophyletic with 59% support. This clade clustered with M. africana, a species also has an elevated perineum, with 58% support. However, the clade was far from the other species M. graminis that has a posterior protuberance. In Fig 9, the phylogenetic tree is based on coxII-16S rRNA from a multiple alignment of 1747 total characters. Using Pratylenchus vulnus Allen and Jensen, 1951 [72] as the out-group taxon, M. aberrans sp. nov. was placed in a clade with M. camelliae and M. mali with 69% support. M. aberrans sp. nov. and M. graminis (another species with an elevated perineum) were always paraphyletic in all phylogenetic trees.

Histopathology
The wild kiwifruit infected by M. aberrans sp. nov. showed disease symptoms similar to nutritional deficiency, with dwarf plants and small sized fruits (Fig 10A and 10B). Most galls induced by M. aberrans sp. nov. on kiwifruit roots were on root tips, and the galls were oval or rounded and relatively large (approximately three-to seven-fold larger than the root diameter) (Fig 10C). Typically, a simple gall contained one to ten females that deposited an egg mass within the root tissue. Histopathological observations showed that M. aberrans sp. nov.
induced formation of the large multinucleate feeding cells known as giant cells, with dense cytoplasm and thickened walls, inside the vascular cylinder. Typically, three to six giant cells were at each feeding site, which resulted in a disorganized stele (Fig 10D and 10E).

Discussion
Meloidogyne is one of the most damaging plant parasites, causing approximately $70 billion in economic losses annually [73]. To date, approximately one hundred nominal species are  New Meloidogyne nematode parasitizing kiwifruit knot nematodes also increase the difficulty in identification. Therefore, isozyme electrophoresis and molecular techniques greatly assist in the identification of Meloidogyne spp. [74]. In this study, a species of root-knot nematode that parasitizes kiwifruit in China was identified as Meloidogyne aberrans sp. nov., based on morphological characters, isozyme and molecular analyses.
The identification of M. aberrans sp. nov. was relatively easy because the species has a unique combination of characters that include a prominent posterior protuberance, faint perineal pattern in females, depression in outline at the oral aperture and very short, poorly defined hyaline tail terminus in J2s. Additionally, according to the rule described by Esbenshade and Triantaphyllou [29], M. aberrans sp. nov. had a rare esterase profile, S2.
Kiwifruit is widely cultivated in Guizhou, China [19], and this new species may be indigenous to Guizhou and may threaten kiwifruit in China by causing symptoms such as severe root knot and dwarfed and reduced fruit size. Additional investigations are required to determine the distribution of M. aberrans sp. nov. beyond the type locality. Moreover, further studies should be conducted to determine the host range of the new species and the optimum methods for control.