Yiwu Fang

Summary: Ektaphelenchoides japonicus n. sp. isolated from rhizospheric medium sample from Japanese maple tree is described and illustrated. It is characterised by short stylet distinctly tripartite, females have no vulval flap, anus and rectum indistinct, PUS well developed, 19.2-32.0 μ m long, extending for ca 1-2 times vulva body diam., tail (posterior part of body) slightly dorsally convex, conical with a short mucron, or short filiform. Male absent. The new species is different from other Ektaphelenchoides species by male absent. Molecular characterisation and phylogenetic studies also supported its unique status.

Summary: Based on the number of incisures in the lateral field, spicule shape, arrangement of male caudal papillae, presence or absence of vulval flap, position of excretory pore and length of postvulval uterus branch, the typological taxonomic grouping of Bursaphelenchus is revised. The currently proposed grouping system of 152 Bursaphelenchus species (including subspecies) is divided into 10 groups with four incisures in the lateral field ( xylophilus , pterocarpi , okinawaensis , africanus , tokyoensis , fungivorus , posterovulvus , kevini , cocophilus and sexdentati ), three groups with three incisures ( eggersi , hofmanni and leoni ) and three groups with two incisures ( abietinus , hylobianum and sinensis ). This system effectively integrates typological morphological classification and phylogenetic analysis based on partial rDNA sequences (SSU, ITS1/2, LSU D2/D3) which exhibits similar patterns, except for the hofmanni group, where morphological characteristics do not align with the clearly delineated subgroups inferred from molecular phylogenetic trees.

Summary: Bursaphelenchus paragerberae n. sp., isolated in acacia wood chips from Vietnam is described. It is characterised by a slim body (a = 23-31), lateral field with three lines, excretory pore posterior to metacorpus, vulva at 72% of total body length, a very small vulval flap in lateral view, post-uterine sac well developed, extending to half of the vulva–anus distance, tail conical, smoothly tapering and slightly ventrally bent, 3.1-4.2 anal body diam. long, with a sharply pointed terminus, male spicules mitten-shaped, short and straight, with high and rounded condylus, pointed rostrum at the middle of the spicules, cucullus absent, a single precloacal papilla (P1) and three pairs of ventro-sublateral papillae present (P2-P4). It belongs to the hofmanni group of the genus Bursaphelenchus . Phylogenetic analyses based on partial 18S, ITS, and 28S D2-D3 regions confirm its status as a new species.

Summary: Bursaphelenchus vietnamensis n. sp., isolated from acacia wood chips from Vietnam is described. It is characterised by a slim body (a = 29-43), lateral field with four lines, excretory pore anterior to metacorpus, female vulva at 76% of total body length, a distinct vulval flap in lateral view, post-uterine branch well developed, extending for 3/4 of the vulva to anus distance, tail conical, smoothly tapering, 4.3-5.5 anal body diam. long, slightly ventral curved, with a pointed or finely rounded terminus, male spicules with weakly developed rostrum and condylus and cross-striped in their central part, a single precloacal papilla and three pairs of ventro-sublateral papillae present. It belongs to the africanus group of genus Bursaphelenchus and is most similar to B. paraburgeri . Detailed phylogenetic analysis based on the partial 18S, ITS and 28S D2-D3 region confirmed the status of this nematode as a new species.

Summary: Bursaphelenchus acaciae n. sp. isolated from acacia wood chips from Vietnam is described. It is characterised by a stout body (a = 25-34), lateral field with four lines, excretory pore located at level of metacorpal valve or anterior, vulva at 74% of total body length, a distinct vulval flap in lateral view, post-uterine branch ca two-thirds of vulva-anus distance, female tail conical and slightly ventrally bent with irregular or roughened terminus (c′ = 3.9), spicules large and arcuate, with pointed rostrum, cucullus reduced to a small blunt extension, three pairs of ventro-sublateral papillae and a single small precloacal papilla present. It belongs to the xylophilus group of the genus Bursaphelenchus and is most similar to B. acaloleptae , B. koreanus , B. luxuriosae and B. paraluxuriosae . Detailed phylogenetic analysis based on the partial 18S, ITS and 28S D2-D3 regions confirmed the status of this nematode as a new species.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a pathogenic organism that causes pine wilt disease (PWD). To date, several molecular diagnostic methods have been developed; however, rapid, convenient, and inexpensive field diagnostic tools for detecting PWN are still limited. In this study, an enzyme-mediated duplex exponential amplification (EmDEA) method for detecting PWN from extracted nematodes or pinewood sawdust was developed and tested. This method comprised an EmDEA molecular test kit, which consisted of freeze-dried enzyme pellets that can be stored at room temperature (approximately 20–25 °C) for one year, a dry block heater, and a portable isothermal fluorescence amplification instrument. The whole procedure was completed within 30 min. The EmDEA assay could detect a single PWN at all life stages from a mixture of other nematode species or from pinewood sawdust. The detection limit was 10 copies (plasmid weight 32.66 ag) or 1/500 of that of a single adult PWN per reaction. Therefore, the EmDEA assay has potential applications in PWN detection in the field, as well as quarantine inspection in international trade. Moreover, modification of primers and probes will allow the rapid identification of other nematode species.

Summary: Aphelenchoides paratenuis n. sp. found in white ash ( Fraxinus sp.) wood from the USA is characterised by the male body length 767.6 (594-848) μ m and female length 824.1 (669-956) μ m, slim body shape (a = 48.5 (41.5-53.5) and 48.0 (41.9-56.8) for males and females, respectively). The lateral field has three lines, and the slender stylet is 9-12 μ m long with small basal swellings. The excretory pore is located at the level of the nerve ring. The hemizonid is obscure. The vagina is not sclerotised but with conspicuous sclerotised pieces encircling it, and the vulva has slightly protruding lips and lacks a flap. The PUS is well developed and comprises ca 33.0 (29.1-38.2)% of the vulva to anus distance. Spicules rose-thorn-shaped, strongly curved at two-thirds from dorsal limb, 17.1 (15.0-19.4) μ m long in curved median line, condylus rounded and high, rostrum rounded and short; the dorsal limb is 22.9 (19.2-28.9) μ m long, with slightly ventrally bent distal end. The spicules’ sheath-like structure is absent. The male tail bears three pairs (2 + 2 + 2) of caudal papillae. The tail of both sexes is sub-cylindrical and the terminus bears a bluntly pointed mucron with tiny nodular protuberances. The new species belongs to the Group 2 category of Aphelenchoides species sensu Shahina and defined as A2-B1-C1-D1-E2-F2 according to the categorisation codes of OEPP/EPPO. The new species is close to A. tenuis and A. yixingensis . Phylogenetic analyses based on sequences of partial 18S, ITS, and 28S D2-D3 of rDNA confirmed its status as a new species.

Aphelenchoides oryzae Yokoo caused a large reduction in rice yields in Japan (1948). It was later synonymised with A. besseyi by Allen (1952), but Subbotin et al. (2021) considered it a valid species. In the main foxtail millet (Setaria italica (L.) P. Beauv.) production area of western Jilin Province, China, many plants were stunted, with thin spikes and open, smooth, shiny glumes. Severely affected spikes were noticeably shorter, fluffy at the top, and erect. In August 2023, 10 foxtail millet samples were collected and nematodes were isolated from 9 of them. A population from Songyuan City, Jilin Province (E:123.64, N:44.86) was studied. The average number of nematodes isolated per gram of ear was 510.7 ± 15.17. Female body slender, lip region rounded, lateral fields with 4 or 6 incisures, vulva located at 71.6% of the body length, post vulvar uterine sac (PUS) 3.7 times anal body width long but less than quarter distance from vulva to anus (VA), tail conical with 3 or 4 terminal spikes. The body length (L), maximum body width (W) and tail length of the female (mean, n=25) were 648 μm, 15.9 μm and 36.7 μm, respectively. PUS length / (VA)% = 23.5, L/W = 41.1, L/ tail length = 17.8. Male body tail curves like a sickle, lacks bursa and shows three pairs of copulatory papillae. Spicules typical of the genus except that the proximal end lacks a dorsal process and has only a moderately developed rostrum. Male measured (mean, n = 25): L = 525.8 μm, W = 14.8 μm, tail length = 34.0 ± 0.7 μm, spicule length = 16.4 μm; L/W = 35.6; L/tail length = 15.6 μm. Amplification of the D2-D3 expansion segments of the 28S ribosomal RNA and the cytochrome oxidase subunit I (COI) gene of mitochondrial DNA (mtDNA) with primers the forward D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and the reverse D3B (5'-TCGGAAGGAACCAGCTACTA-3') (Subbotin et al. 2006), and forward COI-F1 (5'-CCTACTATGATTGGTGGTTTTGGTAATTG-3') and the reverse COI-R2 (5'-GTAGCAGCAGTAAAATAAGCACG-3') (Kanzaki and Futai 2002). PCR conditions were as described by Ye et al. (2007). The sequences of 28S D2-D3 region (726 bp, PP573753- PP573761) of rDNA were 100% identical to A. oryzae (KY123700, KY123694) and COI region (698-700 bp, PP733171-PP733179), were 98.88% identical to A. oryzae (GU367867). Bayesian inference was used to construct phylogenetic tree of 28S D2-D3 region and COI gene, which showed that the Jilin populations clustered together with A. oryzae, which was a sister branch of A. besseyi. Pathogenicity was established via the infection of foxtail millet (cv. Jiyou 2). The germinated foxtail millet seeds were planted in pots containing 350 ml of sterile soil mixture. On the 15th day, every 10 seedlings were inoculated with 100 A. oryzae at the leaf sheet wounds and 3 plants were noninoculated as control. Three independent replicates were performed on different dates. Forty days post-inoculation, an average of 88.3 ± 2.26 A. oryzae were extracted from each nematode-inoculated plant, and the plants were yellowed and necrotic, uninoculated plants grew normally. The molecular and morphological identification of the nematodes obtained by re-isolation from the plants was identical to that of A. oryzae. Our findings clarify the pathogen species, which can be targeted to develop prevention and control strategies, which are important for ensuring safe grain production and helping promote sustainable local agricultural production. To our knowledge, this is the first record of A. oryzae on foxtail millet in Jilin, China.

Summary: Aphelenchoides japonicus n. sp., isolated in the rhizosphere of Camellia sasanqua and Juniperus chinensis from Japan, is described. It is characterised by three lateral incisures, and a slender stylet 8-10 μ m long with obvious round basal swellings, an excretory pore located at the level of the nerve ring or slightly posterior, an oblong spermatheca, containing ellipse sperm cells in a single row, but sometimes with spheroid sperm cells in 2-3 rows, a vagina not sclerotised and the vulva having slightly protruding lips and lacking a flap. The post-vulval uterine sac (PUS) is well developed, occupying ca 26.2% (21.5-31.4) of the vulva to anus distance. The male spicules are smoothly arcuate, the condylus very short and rounded, a small rostrum sharply pointed, the dorsal limb 21.6 (19.1-23.8) μ m long, lacking a hook-like structure at the distal tip. The tail of both sexes is conical, with a trimucronate process, giving a wide warty mucron-like structure or a star-like appearance. The new species belongs to Group 3 of the Aphelenchoides species sensu Shahina. Phylogenetic analysis based on 18S and 28S D2-D3 of rDNA, along with ITS and mt COI confirmed its status as a new species.

Summary: Bursaphelenchus parasaudi n. sp., isolated in Ningbo, P.R. China, from loblolly pine logs imported from the USA is described. It is characterised by a slim body (a = 32-43), lateral field with four lines, excretory pore located at the level of metacorpus, vulva at 76% of total body length, a distinct vulval flap in lateral view, post-uterine branch ca half to two-thirds of the vulva to anus distance long, female tail conical, slightly ventrally curved, with a finely rounded terminus, male spicules with weakly developed rostrum and condylus and cross-striped in their central part, a single precloacal papilla and three pairs of ventro-sublateral papillae. It belongs to the africanus group of the genus Bursaphelenchus and is most similar to B. saudi. Detailed phylogenetic analysis based on the partial 18S region, ITS and 28S D2-D3 region confirmed the status of this nematode as a new species.