Aphelenchoides huntensissp. n. is described and illustrated from wood and bark samples of a dead scots pine in Kermanshah Province, western Iran. The new species has a body length of 507-673 μm (females) and 636-640 μm (males). The cuticle is weakly annulated with four lateral lines. Lip region offset. The stylet is 9.5 (9.0-10.0) μm long with small basal swellings. The excretory pore is locatedcaone body diam. posterior to the metacorpus valve. The spicules are large with the dorsal limb 37 or 38 μm long (n = 2), and have a prominent rostrum and rounded apex. The female tail is conical, the terminus having a complicated step-like projection, usually with many tiny nodular protuberances. The male tail bears six (2 + 2 + 2) caudal papillae and a well-developed mucron. The new species belongs to the Group 2 category ofAphelenchoidesspecies. Phylogenetic analysis based on the 18S and 28S D2-D3 regions of rDNA confirmed its status as a new species.

Aphelenchoides stellatusn. sp. is described and figured. The new species was isolated from packaging wood from Japan imported to Ningbo harbour, China. The new species has a body length of 485-533 μm (males) and 547-699 μm (females). The cuticle is weakly annulated and there are four lines in the lateral field. The stylet is 9-11 μm long and has small basal swellings. The excretory pore is located posterior to the nerve ring. Spicules smoothly curved, rose-thorn shaped. Apex and rostrum round, only slightly offset, dorsal limb 19-21 μm long. Male tail bearing six (2 + 2 + 2) caudal papillae. The female spermatheca is axial and oblong with round sperm present in multiple rows. Both male and female tail pegs have 3-4 processes, appearing star-like under SEM. The new species belongs to the Group 3 category ofAphelenchoidesspecies. Phylogenetic analyses based on full length ITS and 28S D2/D3 region of rDNA confirmed its morphological status as a new species.

Bursaphelenchus parapinasteriis redescribed morphologically and with new molecular characterisation. It was isolated from a deadPinus thunbergiion Changgang Mountain, Zhoushan Islands, Zhejiang Province, China. Detailed morphology of the spicule, female gonad, hemizonid position, arrangement of male caudal papillae, and female tail terminus shape are documented. The ITS-RFLP patterns and the ITS1/2, partial 28S D2-D3 and partial 18S gene sequences were characterised. The phylogenetic analyses revealed thatB. parapinasteribelongs to thehofmanni-groupsensuBraasch and is close toB. anamurius,B. hofmanni,B. mazandaranense,B. paracorneolus,B. pinasteriandB. ulmophilus. A diagnostic key to species of thehofmanni-group is presented.

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 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: Aphelenchoides yixingensis n. sp. found in Chinese red pine (Pinus massoniana) in Yixing city, Jiangsu Province, P.R. China, is described. It is characterised by a very slim body (a = 50.8 (41.3-63.8) and 49.3 (42.3-55.1) for males and females, respectively), three (occasionally five) lateral lines in the lateral field, a short stylet (9-12 μm), the excretory pore located at the level of nerve ring, the vagina is not sclerotised and the vulva has slightly protruding lips and lacks a flap, the post-uterine sac is well developed and forms ca 29.0% (24.1-34.1%) of the vulva to anus distance. The spicules are smoothly arcuate, the apex and rostrum rounded and short, and the dorsal limb is 20.5 (16.9-22.2) μm long, the distal end bluntly rounded. Spicule sheath-like structure is present, triangular and outstretched when heat-killed in some individuals. The male tail bears three pairs (2 + 2 + 2) of caudal papillae. The tail of both sexes is sub-cylindrical, the terminus bearing a bluntly pointed mucron with tiny nodular protuberances, mainly at the distal part. The new species belongs to the Group 2 category of Aphelenchoides species sensu Shahina and is defined as A2-B1-C1-D1-E2-F2 according to the categorisation codes of OEPP/EPPO. Phylogenetic analysis based on 18S and 28S D2-D3 of rDNA, along with ITS and mtCOI, confirmed its status as a new species. A proposal for genus Ptychaphelenchus as a synonym of Aphelenchoides is also discussed.