Yiwu Fang

mtCOI successfully diagnoses the four main plant-parasitic Aphelenchoides species (Nematoda: Aphelenchoididae) and supports a multiple origin of plant-parasitism in this paraphyletic genus

Composed mostly of fungivorous species, the genus Aphelenchoides also comprises 14 plant-parasitic species. The most common and devastating, A. besseyi, A. fragariae, A. ritzemabosi and A. subtenuis have been reported on more than 900 plant species. The combination of low inter-specific and high intra-specific morphological variability makes morphology-based identification extremely difficult within this genus, and has led to molecular tools being employed to ensure accurate diagnoses. rDNA markers are widely used for the identification of nematodes while the Cytochrome Oxidase I gene (COI) remains relatively unexplored despite its role as the standard barcode for almost all animal groups. To explore its suitability as a diagnostic tool, we studied a fragment of the mtCOI region of the four main plant-parasitic Aphelenchoides within a phylogenetic framework. We generated 69 mtCOI and 123 rDNA sequences of diverse Aphelenchoides taxa; 67 belong to the main plant-parasitic species including the first mtCOI sequence of A. fragariae and the first mtCOI and 28S sequences of A. subtenuis. mtCOI had a similar success rate for PCR amplification. Phylogenetic trees based on the three studied markers are largely in agreement with one another, validating their use for Aphelenchoides diagnosis; additionally, we were able to locate several misidentified sequences of plant-parasitic Aphelenchoides in existing databases. The concatenated analysis from the three markers resulted in a more robust insight into the phylogeny and evolution of Aphelenchoides, revealing that plant-parasitism has evolved independently at least three times within this genus, presumably from fungal-feeding ancestors.

Redescription of Bursaphelenchus parapinasteri (Tylenchina: Aphelenchoididae) isolated from Pinus thunbergii in China with a key to the hofmanni-group

Bursaphelenchus parapinasteri is redescribed morphologically and with new molecular characterisation. It was isolated from a dead Pinus thunbergii on 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 that B. parapinasteri belongs to the hofmanni-group sensu Braasch and is close to B. anamurius, B. hofmanni, B. mazandaranense, B. paracorneolus, B. pinasteri and B. ulmophilus. A diagnostic key to species of the hofmanni-group is presented.

Bursaphelenchus crenati Rühm, 1956 (Tylenchina: Aphelenchoididae), a nematode associated with the Greater ash bark beetle, Hylesinus crenatus Fabricius, in dying ash, Fraxinus excelsior L., in Europe

New morphological, molecular and bionomic data are added to update the description of Bursaphelenchus crenati . The nematode was isolated from larval galleries and adults of the Greater ash bark beetle, Hylesinus crenatus Fabricius, present in bark of older trees of European ash, Fraxinus excelsior L., in Poland. Detailed female and male morphology are documented in this paper and compared with the original description. From ITS1/2, partial 28S D2-D3 and partial 18S sequences, ITS-RFLP patterns and morphological analysis, B. crenati is suggested to be temporarily placed into the sexdentati group, close to B. andrassyi , B. vallesianus , B. sexdentati , B. pinophilus , B. poligraphi , B. fuchsi , B. incurvus , B. piniperdae , and B. pityogeni . However, B. crenati can be distinguished from these species by the strongly ventrally curved body and the presence of three incisures in the lateral fields.

Redescription of Bursaphelenchus eucarpus Rühm, 1956 (Nematoda: Aphelenchoididae) associated with the apple bark beetle, Scolytus mali Bechstein, and the shothole borer, S. rugulosus Müller

New morphological, molecular and bionomic data are added to update the description of Bursaphelenchus eucarpus. The nematode was isolated from larval galleries and adults of the apple bark beetle, Scolytus mali, and the shothole borer, S. rugulosus, present under the bark of the bird cherry, Prunus padus, and the common European plum, P. domestica, in Poland. Detailed male, female and dispersal juvenile morphology and morphometrics are documented and compared with the original description. From partial 18S, ITS1/2 and partial 28S D2-D3 sequences and morphological features (three lateral lines, position of caudal papillae, shape of the delicate spicules with dorsally bent condylus, lack of vulval flap, a long post-uterine branch and a hyaline region at the female tail tip) it can be concluded that B. eucarpus belongs to the eremus group of Bursaphelenchus. The differentiation from related species of the eremus group is discussed.

Bursaphelenchus moensi n. sp. (Tylenchina: Aphelenchoididae) found in packaging wood from the USA

Bursaphelenchus moensi n. sp., isolated in Tianjin Port, China, from Pinus packaging wood imported from the USA, is described. It is characterised by a lateral field with three lines (two bands), stylet 13.0-14.9 μ m long, excretory pore situated at same level as, or slightly posterior to, the nerve ring, spicules 13.0-15.5 μ m long, mitten-shaped, condylus high and broad with squared, rounded or pointed end, rostrum triangular or conical with bluntly rounded tip, and cucullus absent, bursa small, starting posterior to P4, narrow oblong to irregular in shape and with an oval central projection, female vulval flap absent, and tail conical with a finely rounded or mucronate terminus. The new species belongs to the hofmanni -group and is most similar to B. paraparvispicularis , B. parvispicularis and B. wuae . It is distinguished from closely related species by morphology, ITS-RFLP patterns and partial 18S, ITS and 28S D2-D3 rDNA gene sequencing results.

Description of Bursaphelenchus rockyi n. sp. (Nematoda: Aphelenchoididae) in peat moss from Russia

Bursaphelenchus rockyi n. sp., isolated in Peking, China, from peat moss imported from Russia, is described. It is characterised by a lateral field with four lines, excretory pore located at the base of the nerve ring or slightly posterior, spicule 15.9 (15.1-17.3) μm long along the chord and with high condylus and a rounded tip, rostrum triangular or conical with bluntly pointed tip, lamina curvature becoming more pronounced at 60% of total length, six caudal papillae with P3 and P4 adjacent to each other, bursa small, starting posterior to P4, vulval lips hemispherical and protruding, and tail conical with finely rounded or mucronated terminus. The new species belongs to the fungivorus-group and is most similar to B. arthuri, B. arthuroides, B. fungivorus and B. seani, but can be distinguished from related species by morphological and sequencing results.

Radopholoides japonicus n. sp. (Nematoda: Pratylenchidae) found in rhizosphere soil associated with Podocarpus macrophyllus from Japan

Radopholoides japonicus n. sp. isolated from rhizospheric soil samples associated with Podocarpus macrophyllus imported from Japan into Ningbo Port of China is described and illustrated. It is characterised by four lateral lines, lip region low, convex with three annuli. Stylet 16.5-18.5 μ m long with anteriorly sloping rounded knobs. Excretory pore at the level of pharyngo-intestinal junction. Vulva posteriorly located, vulval lips not protruding, spermatheca squarish. Post-vulval uterine sac 35-44 μ m long. Tail conoid with broadly rounded terminus, phasmid 6-8 annuli posterior to anus. Among five species of genus Radopholoides , the new species is close to R. antoni but can be differentiated from it by lip and tail morphology. This is the first Radopholoides species that provided detailed morphological and molecular characterisation. Maximum Likelihood analysis using the Shimodaira-Hasegawa test for the validity of Radopholoides was performed and showed the validity of the genus using the partial 18S and D2-D3 expansion segments of 28S.