Computerized key to the genus Bursaphelenchus Fuchs, analysis of species clusters based on morphology, using information of insect vectors and associated plants, with a revision of the genus.

The genus Bursaphelenchus includes B. xylophilus (Steiner et Buhrer, 1934) Nickle, 1981, which is of world economic and quarantine importance. Distinction among several species of the pinewood nematodes species complex (PWNSC) is often difficult. Besides standard morphology, morphometrics and molecular biology, new tools are welcome to better understand this group. The computerized (or e-) key of this genus, presented in this communication, includes 74 species (complete list of valid species of the world fauna) and 35 characters, that were used by the taxonomic experts of this group, in the original descriptions. Morphology of sex organs (male spicules and female vulval region) was digitized and classified to distinguish alternative types. Several qualitative characters with overlapping character states (expressions) were transformed into the morphometric indices with the discontinuous ranges (characters of ratios of the spicule dimensions). Characters and their states (expressions) were illustrated in detail and supplied by brief user-friendly comments. E-key was created in the BIKEY identification system (Dianov & Lobanov, 1996-2004). The system has built-algorithm ranging characters depending on their diagnostic values at each step of identification. Matrix of species and the character states (structural part of the e-key database) may be easily transformed using statistical packages into the dendrograms of general phenetic similarities (UPGMA, standard distance: mean character difference). It may be useful in the detailed analysis of taxonomy and evolution of the genus and in its splitting to the species groups based on morphology. The verification of the dendrogram using the information on the species links with insect vectors and their associated plants, provided an opportunity to recognize the five clusters (xylophilus, hunti, eremus sensu stricto, tusciae and piniperdae sensu stricto), which seem to be the natural species groups. The hypothesis about the origin and the first stages of the genus evolution is proposed. A general review of the genus Bursaphelenchus is presented.

Computerized key of the genus genus Laimaphelenchus Fuchs, 1937

The Order Aphelenchida contains several genera of economic importance, namely Aphelenchoides and Bursaphelenchus. Nematode species belonging to these 2 genera frequently co-habit with other genera such as Laimaphelenchus. It is therefore important to clearly distinguish them, as well as understand the group´s biodiversity. A computerized, or e-key, for the genus Laimaphelenchus Fuchs has been developed in the BiKey Identification system (Dianov & Lobanov, 1996-2004). The e-key includes 14 species and 34 characters (from 2 to 6 character states each). It also includes the built-in algorithm ranging characters according their diagnostic values to minimize the number of the diagnosis steps (average number of steps is 2.7; values are re-calculated at each step). The most important characters (as calculated by BiKey) are: length of posterior branch of the female genital system; excretory pore position; vulval anterior flap shape; number pairs of mail tale papillae; male bursa shape (ventral view); number of tail tip setae in female; female tail tip stub shape; presence of mucro on tail tip in male. Key is pictorial (image-operating), multientry, as other BiKey products.

Bursaphelenchus antoniae sp. n. (Nematoda: Parasitaphelenchidae) associated with Hylobius sp. from Pinus pinaster in Portugal

Bursaphelenchus antoniae sp. n. is described and illustrated. Dauer juveniles were isolated from the body of the large pine weevil, Hylobius sp., collected from maritime pine (Pinus pinaster) stumps, in Portugal. Bursaphelenchus antoniae sp. n. was reared and maintained in P. pinaster wood segments and on Petri dish cultures of the fungi Botrytis cinerea and Monilinia fructicola. The new species is characterised by a relatively small body length of ca 583 μm (females) and 578 μm (males), a lateral field with two incisures, presence of a small vulval flap and a conoid female tail with a rounded or pointed terminus. Males have stout spicules with a disc-like cucullus and seven caudal papillae arranged as a single midventral precloacal papilla, one precloacal pair and two postcloacal pairs. In the character of the lateral field, B. antoniae sp. n. comes close to B. abietinus, B. rainulfi and B. hylobianum, whilst spicule characters place it within the piniperdae-group sensu Ryss et al. Morphologically, B. antoniae sp. n. is closest to B. hylobianum; the spicules of these two species having flattened, wing-like, alae on the distal third of the lamina. Bursaphelenchus antoniae sp. n. is distinguished from B. hylobianum on the arrangement of the caudal papillae (two vs three pairs). ITS-RFLP profiles and the failure to hybridise support the separation of the two species. Phylogenetic analysis of the new species, based on the 18S rDNA sequence, supports the inclusion of this new species in the B. hylobianum-group sensu Braasch. Sequence analysis of the 28S rDNA D2/D3 domain did not place the new species in a definite group.

Bursaphelenchus Fuchs, 1937 (Nematoda: Parasitaphelenchidae) species associated with Pinus species in northern Turkey

A survey for Bursaphelenchus nematodes, associated with different conifer trees, was conducted in several forest areas in the northern regions of Turkey. Only pine trees (Pinus nigra, P. pinaster and P. sylvestris) yielded Bursaphelenchus specimens. Nematodes were identified using several morphological diagnostic characters of the genus (male spicule structure, number of lateral incisures, number and distribution of the male papillae, presence of female vulval flap), and confirmed by using RFLP analysis of the internal transcriber spacer (ITS) regions of ribosomal DNA. Three different species were identified from several sampled areas, namely B. mucronatus, B. pinophilus and B. sexdentati, representing a first report of the last two species for Turkey. The association of B. pinophilus with black pine (P. nigra) is herein reported for the first time.

Progress in the use of scanning electron microscopy (SEM) applied to the taxonomy of the genus Bursaphelenchus (Nematoda: Parasitaphelenchidae)

The scanning electron microscope (SEM) has been a major tool in detailed morphological observations of plant parasitic nematodes during the last 30 years, efficiently complementing light microscopical (LM) studies. Nematodes are extremely difficult to observe and characterize due to their small size (aprox. 1 mm long) and paucity of morphological characters, so detailed surface observations of several organs and nematode regions are of the highest value. Among plant parasitic nematodes, one of the most devastating species is the “pinewood nematode” (PWN), Bursaphelenchus xylophilus, which has been a major problem for forest species, and in particular pines, in Asia (Japan, China, Korea) and has been recently detected in the European Union (Portugal). B. xylophilus belongs to a closely related, morphologically similar group of species, within the genus Bursaphelenchus, and designated by the “xylophilus group”. SEM has become a crucial tool in observing several genital characters of males and females, such as male genital papillae, male copulatory spicules, female vulval flap and female genital papillae.s In this presentation, we will show how SEM has been utilized to observe and characterize the shape of the vulval flap, the presence/ absence of papillae near the flap, and confirm the presence and the arrangement of the male genital papillae. LM is also used in this work to show its value as a complementary tool to SEM, in both genital characteristics and other, general, characters of the genus Bursaphelenchus, such as the male bursa and cephalic region.