A new stem nematode, Ditylenchus oncogenus n. sp. (Nematoda: Tylenchida), parasitizing sowthistle from Adriatic coast dunes in southern Italy.

Morphological and molecular analyses of a stem nematode causing a severe disease on infected sowthistle (Sonchus bulbosus) plants, involving the formation of gall-like structures on infected leaves and stems, have led to the description of a new species named Ditylenchus oncogenus n. sp. Morphologically, the new species is characterized by a medium to large body size (all adults more than 1 mm in length); a delicate stylet (9.0-11.0 µm long) with minute, rounded knobs; a long post-vulval uterine sac (c. 65% of the vulva-anus distance); six incisures at the lateral fields and characteristic D. destructor-pattern of spicules (with pronounced ventral tumulus and anteriorly pointed, less sclerotized, cuticle parts present within the lamina). The results of molecular analysis of rRNA gene sequences, including the D2-D3 expansion regions of 28S rRNA, internal transcribed spacer (ITS) rRNA, partial 18S rRNA gene, the protein-coding mitochondrial gene, cytochrome oxidase c subunit I (COI), and the heat-shock protein 90 (hsp90) gene, support the new species status. The results of a host-suitability test indicated that the new species does not parasitize potato (Solanum tuberosum) tubers and broad bean (Vicia faba) seedlings. Histopathological observations on naturally infected sowthistle tissues revealed that D. oncogenus n. sp. causes floral stem neoplasia and midrib leaf gall formation on the type, and to date only known, host. The galls were characterized by extensive hyperplasia, where several necrotic cells in the neoplasic area were directly damaged by feeding of the nematode, whereas a number of adjacent cells showed typical cytological changes, such as granulated cytoplasm with hypertrophied nuclei and nucleoli.

Pathogenicity and Host-Parasite Relationships of Heterodera cruciferae in Cabbage.

Stunted cabbage ('Lupini') associated with severe soil infestations by a cyst-forming nematode were observed in large patches of open fields in Castellaneta, province of Taranto, southern Italy. Morphological traits based on mature cysts, males, and second-stage juveniles (J2s) and molecular analysis of ribosomal DNA (D2 to D3 expansion segments of 28S and internal transcribed spacer [ITS]1 region) were used to identify the species. ITS1 sequence information supported the identity of Heterodera cruciferae, also showing a high degree of similarity to other species of the Heterodera Goettingiana group, including H. goettingiana, H. carotae, and H. urticae. Nematodes successfully established permanent feeding sites in cabbage roots which caused cellular alterations in the root cortex, endodermis, pericycle, and vascular cylinder by inducing typical multinucleate syncytia. Syncytial cytoplasm was granular and dense, with variously sized vacuoles and hypertrophied nuclei with nucleoli. Cabbage plant growth was also reduced in pathogenicity tests. The relationship between the initial nematode population density in soil and shoot plant weight was well described by the Seinhorst's equation. Tolerance limits with respect to shoot plant weight of cabbage to H. cruciferae was estimated as 1.50 units of eggs plus J2s/cm3 of soil. The minimum relative value (m) for plant height was 0.71 at an initial nematode population density of (Pi) ≥ 64 units of eggs plus J2s/cm3 of soil. The maximum nematode reproduction rate (Pf/Pi) was 4.6 times that of the initial population density of 8 units of eggs plus J2s/cm3 of soil.

Pathogenicity and Host-Parasite Relationships of Meloidogyne arenaria in Sweet Basil.

Stunted growth of sweet basil (cv. Genovese) associated with large patches and severe soil infestations by the root-knot nematode Meloidogyne arenaria race 2 were observed in open fields at the end of the crop-growing season in Albenga, province of Savona, Northern Italy by early September 2007. Observed morphological traits of nematode life stages and results of analysis of isozyme electrophoretic patterns and differential host tests were used for nematode species and race identification. Nematode-induced mature galls (either in naturally infected plants or in artificially inoculated root tissues) were spherical or ellipsoidal and confluent along the root axis, containing usually several females, males, and egg-masses with eggs. Feeding sites were characterized by the development of giant cells that contained several hypertrophied nuclei and nucleoli. Giant cell cytoplasm was aggregated along the thickened cell walls. Vascular elements within galls appeared disorganized and disrupted. Due to the heavy damage observed, the relationships between the initial population density and growth of basil plants was also tested in a greenhouse experiment in which inoculum levels varied from 0 to 512 eggs and juveniles/cm3 of soil. Height and top fresh weight data of the inoculated and control plants was fitted to Seinhorst's model. Tolerance limits with respect to plant height and fresh top weight of basil cv. Genovese plants to M. arenaria race 2 were estimated as 0.15 eggs and juveniles/cm3 of soil. The minimum relative values (m) for plant height and top fresh weight were 0.39 and 0.19 at initial nematode population density (Pi) ≥16 eggs and juveniles/cm3 of soil, respectively. The maximum nematode reproduction rate (Pf/Pi) was 448.7 times at an initial population density of 4 eggs and second-stage juveniles/cm3 of soil.

Diagnosis and Molecular Variability of an Argentinean Population of Nacobbus aberrans with Some Observations on Histopathology in Tomato.

Diagnosis of an Argentinean population of Nacobbus sp. infecting sweet pepper (lamuyo) was carried out including morphology, scanning electron microscopy, and molecular studies. In light of our morphometric, molecular and host-range results, we consider the studied population to belong to N. aberrans s. l., and by host range tests the population is assigned to the "sugar beet group." ITS-PCR analysis on individual male and immature female specimens of this population yielded amplification products of approximately 922 bp. RFLP profiles and sequencing of the ITS region revealed that, in addition to the host group, the present population can be assigned to the "Argentina 2" group. Disease development and histopathology were investigated with glasshouse observations using tomato, pepper, sugar beet and potato seedlings exposed to nematode infection for 45 days at 28 +/- 2 degrees C. Histopathology of tomato roots confirmed that all immature stages and young females and males are migratory, whereas mature females are obligate sedentary endoparasites. Rather than syncytia, large regions of cortical necrosis and cavities were detected in tomato swellings infected by juveniles. However, syncytia were associated only with adult females. Large root galls, hyperplasia, abnormal proliferation of lateral roots and asymmetry of root structure were common anatomical changes induced by the nematode feeding in tomato roots.

A New Root-Knot Nematode Parasitizing Sea Rocket from Spanish Mediterranean Coastal Dunes: Meloidogyne dunensis n. sp. (Nematoda: Meloidogynidae).

High infection rates of European sea rocket feeder roots by an unknown root-knot nematode were found in a coastal dune soil at Cullera (Valencia) in central eastern Spain. Morphometry, esterase and malate dehydrogenase electrophoretic phenotypes and phylogenetic trees demonstrated that this nematode species differs clearly from other previously described root-knot nematodes. Studies of host-parasite relationships showed a typical susceptible reaction in naturally infected European sea rocket plants and in artificially inoculated tomato (cv. Roma) and chickpea (cv. UC 27) plants. The species is herein described and illustrated and named as Meloidogyne dunensis n. sp. The new root-knot nematode can be distinguished from other Meloidogyne spp. by: (i) perineal pattern rounded-oval, formed of numerous fine dorsal and ventral cuticle striae and ridges, lateral fields clearly visible; (ii) female excretory pore at the level of stylet knobs, EP/ST ratio 1.6; (iii) second-stage juveniles with hemizonid located 1 to 2 annuli anteriorly to excretory pore and long, narrow, tapering tail; and (iv) males with lateral fields composed of four incisures anteriorly and posteriorly, while six distinct incisures are observed for large part at mid-body. Phylogenetic trees derived from distance and maximum parsimony analyses based on 18S, ITS1-5.8S-ITS2 and D2-D3 of 28S rDNA showed that M. dunensis n. sp. can be differentiated from all described root-knot nematode species, and it is clearly separated from other species with resemblance in morphology, such as M. duytsi, M. maritima, M. mayaguensis and M. minor.

First Report of Meloidogyne arenaria Parasitizing Lettuce in Southern Spain.

During the 2005-2006 autumn to winter lettuce-growing (Lactuca sativa cv. Iceberg) season, severely stunted and yellowing lettuce plants with disease incidence ranging from 80 to100% were observed in four commercial, fall-sown fields at Almodóvar del Río (Córdoba Province) in southern Spain. Early symptoms consisted of severely reduced growth of the plants that continued with extensive leaf yellowing and the absence of tight-head formation. Attacks by the disease were estimated to cause near complete loss of the crop yields since the lettuce head produced in affected fields were unmarketable. Observations of affected lettuce plants revealed high parasitism of the root system by a root-knot nematode (Meloidogyne sp.) in the main and feeder roots as well as heavy soil infestations by the nematode. The nematode was identified by the female perineal pattern, esterases phenotype, and a sequence-characterized amplified region polymerase chain reaction (SCAR-PCR) technique (1,2,4). Measurements and morphological observations of 20 second-stage juveniles (J2s) (body length = 463 ± 28 µm, dorsal gland orifice from stylet base = 2.8 ± 0.6 µm, stylet length = 10.4 ± 0.5 µm, tail length = 54.4 ± 0.6 µm; hyaline tail terminus = 9.4 ± 0.6 µm) and 10 adult females (stylet length = 14.5 ± 0.7 µm, dorsal gland orifice from stylet base = 4.7 ± 0.5 µm, and perineal pattern with low and rounded dorsal arch with coarse striae) conformed to the description of Meloidogyne arenaria (3). On the basis of the characteristics of the perineal pattern, the 2-band esterase phenotype, and the 420-bp SCAR fragment, the causal agent was identified as the peanut root-knot nematode M. arenaria. Nematodes were extracted from soil and root samples by standard procedures and their populations quantified. M. arenaria was detected in nearly all soil and root samples assessed, with nematode population densities ranging from 206 to 1,072 eggs and J2s per 5 g of fresh roots. Different Meloidogyne spp. have been reported parasitizing lettuce roots, especially M. hapla in northern areas (2); however, to our knowledge this is the first time that M. arenaria is reported parasitizing lettuce roots in Spain and elsewhere. References: (1) P. R. Esbenshade and A. C. Triantaphyllou. J. Nematol. 22:10, 1990. (2) N. A. Mitkowski et al. Plant Dis. 86:840, 2002. (3) K. J. Orton Williams. Meloidogyne arenaria. CIH Descriptions of Plant-Parasitic Nematodes. Set 5, No. 62. Commonwealth Institute of Helminthology, St. Albans, 1975. (4) C. Zijlstra et al. Nematology 2:847, 2000.

First Report of Broomrape (Orobanche crenata) Infecting Lettuce in Southern Spain.

Broomrapes (Orobanche spp., Orobanchaceae) are chlorophyll-lacking, obligately parasitic flowering plants that infect roots of many dicotyledoneous species and cause severe damage to vegetable and field crops worldwide, but particularly in North Africa, southern and eastern Europe, and the Middle East. (1). Orobanche crenata is one of the most important broomrapes and mainly infects legume crops (2). In January 2006, we observed severe broomrape attacks in four commercial fields of fall-sown lettuce (Lactuca sativa cv. Iceberg) crops at Almodóvar del Río (Córdoba Province) in southern Spain. Infected lettuce plants showed severe stunting, foliar yellowing, and had loose-formed heads. Infection of lettuce plants by Orobanche sp. was confirmed by removing plants to verify the attachment of broomrapes to lettuce roots. There were one to four broomrapes per lettuce plant. Incidence of infected lettuce ranged from 10 to 20% in different areas of the fields. Morphological observations of broomrape plants identified the parasite as O. crenata. The main botanical features were as follows: plants 20 to 40 cm tall; corolla 20 to 28 mm, white, lips with lilac, divergent veins, lower lip large with suborbicular lobes, not ciliate; filaments hairy, obliquely inserted 2 to 4 mm above the base of corolla, with short glandular hairs in the upper third; anthers glabrous, 2 to 2.5 mm in length, and stigma yellow or pinkish at anthesis (2). O. crenata also was observed infecting faba bean (Vicia faba) plants in a field in close proximity to the affected lettuce fields. The complete 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) 1 and 2 of O. crenata were sequenced using adventitious roots and stem tissues sampled from infected faba bean and lettuce plants (Genbank Accession Nos. DQ458908 and DQ458909) by standard protocols (3). A nucleotide BLAST search revealed that both sequences were identical and share 100% similarity with three reported ITS1-5.8S-ITS2 sequences from two Orobanche spp. (O. crenata and O. minor; Genbank Accession Nos. AY209267, AY209266, and AY209272). On the basis of the morphological characters described above, the parasite was O. crenata and not O. minor. O. crenata has been reported infecting many legume crops in southern Spain, including faba bean, pea, lentil, and vetch. To our knowledge, this is the first report of O. crenata infecting lettuce in Spain and elsewhere. The high incidence of O. crenata on legume crops, and the severe infections found on lettuce plants suggest that this parasitic plant may be an important constraint for fall-sown lettuce in southern Spain. References: (1) A. O. Chater and D. A. Webb. Orobanchaceae. In: Flora Europaea, T. G. Tutin et al., eds. Vol. 3. Cambridge University Press, Cambridge, 1972. (2) A. J. Pujadas-Salvà. Orobanchaceae L. In: Plantas Parásitas de la Península Ibérica y Baleares. J. A. López Sáez et al., eds. Mundi-Prensa, Madrid, 2002. (3) G. M. Schneeweiss et al. Mol. Phylogenet. Evol. 30:465, 2004.

Characterization of the Cystoid Nematode Meloidoderita kirjanovae (Nemata: Sphaeronematidae) from Southern Italy.

A population of the cystoid nematode Meloidoderita kirjanovae was detected parasitizing water mint (Mentha aquatica) in southern Italy. The morphological identification of this species was confirmed by molecular analysis using the internal transcribed spacer 1 (ITS1) and 5.8S gene sequences of nuclear ribosomal DNA (rDNA), which clearly separated it from the closely related species Meloidoderita polygoni. A phylogenetic analysis of M. kirjanovae with species of related genera was conducted using sequences of the D2-D3 expansion segments of the 28S nuclear ribosomal RNA gene. The resulting phylogenetic tree was congruent with trees from an extended dataset for Criconematina and Tylenchida. The basal position of the genus Meloidoderita together with Sphaeronema within the Criconematina clade in this tree may indicate their close relationships. The anatomical changes induced by M. kirjanovae population from Italy in water mint were similar to those reported for a nematode population infecting roots of M. longifolia in Israel. Nematode feeding caused the formation of a stellar syncytium that disorganized the pericycle and vascular root tissues.

Occurrence of Thelazia lacrymalis (Nematoda, Spirurida, Thelaziidae) in native horses in Italy.

A survey on the prevalence of Thelazia spp. in the province of Bari (Apulia region, Italy) in slaughtered native horses was conducted from June 20, 1995 to April 3, 1996. Both eyes from 409 ten-month- to 4-year-old native animals were examined. Sixty horses (14.7%) were found parasitized by Thelazia lacrymalis. Three hundred-sixty one parasite specimens (220 females, 99 males and 42 larvae) were collected with a mean count burden of 6.0-5.1 (range 1 to 20) per head. T. lacrymalis specimens were found free in the conjunctiva and behind the nictitancte, in the excretory ducts of the Harderian gland and in the ducts of the lacrimal glands. Thelazia specimens were significantly more common in horses of 3-4 years. Gross examination of all infected animals showed a follicular conjunctivitis. This is the first report of T. lacrymalis horse infection in Italy.

Infection of Olive Trees by Heterodera mediterranea in Orchards in Southern Spain.

High infection rates of olive feeder roots and soil infestation by the cyst-forming nematode Heterodera mediterranea were found in commercial olive orchards cv. Manzanilla established in sandy soils at two localities of Sevilla Province of Andalucía, southern Spain. No disease symptoms were noted on the aboveground plant parts, but slightly distorted feeder roots and necrotic areas were observed near the infection points. Sedentary juvenile stages and adult females showed a semi-endoparasitic feeding habit. Histopathological studies of infected olive roots showed a disease response typical of cyst nematode infection of susceptible hosts. Nematode feeding induced syncytial formation involving cellular alterations in the cortex, endoder-mis, pericycle, and vascular parenchyma tissues in both cortical and endodermal root cells.

Host-Parasite Relationships in Root-Knot Disease of White Mulberry.

Severe infections of white mulberry feeder roots and heavy soil infestations by Meloidogyne arenaria race 2 were found in southern Spain. This is the first record of M. arenaria on white mulberry in Europe. Morphometric observations, analysis of the esterase electrophoretic pattern, and artificial inoculations of race differentials were used to characterize nematodes. Nematode-induced mature galls were spherical and usually contained one or more females, males, and egg masses with eggs. Feeding sites were characterized by the development of giant cells that contained granular cytoplasm and many hypertrophied nuclei. Giant cell cytoplasm was aggregated along a thickened cell wall. Vascular tissues within galls appeared disorganized. The relationship between the initial nematode population density (Pi) in a series from 0 to 1,024 eggs and juveniles/cm3 soil and growth of white mulberry seedlings was tested in the greenhouse. A Seinhorst model was fitted to plant height and top fresh weight. Tolerance limits of white mulberry to M. arenaria race 2 for plant height and top fresh weight were, respectively, 1.1 and 1.38 eggs and juveniles/cm3 soil. The minimum relative values for plant height and top fresh weight were 0 at Pi ≥ 64 and Pi ≥ 128 eggs and juveniles/cm3 soil, respectively. Maximum nematode reproduction rate was 435-fold at the lowest Pi.

Thelazia gulosa Railliet & Henry, 1910 and T. skrjabini Erschow, 1928 infection in southern Europe (Italy).

The occurrence of Thelazia gulosa and T. skrjabini in cattle in Italy, together with an update of the prevalence of Thelazia spp. in the southern Italy, is reported. On 764 slaughtered native cattle, fifty-five (7.2%) bovines were infected by Thelazia spp: T. rhodesi was found in 44 (80%) animals, T. gulosa was found in 19 (34.5%) cattle and only one (1.8%) cattle harboured T. skrjabini. This is the first report of T. gulosa and T. skrjabini in Southern Europe.

Taxonomy of Discocriconemella (Nematoda: Criconematoidea) with a Redescription of D. mauritiensis.

The form of the cephalic disc and its taxonomic significance at the species level in the genus Discocriconemella De Grisse &Loof, 1965 is discussed. By light (LM) and scanning electron microscopy (SEM), four groups of disc configurations in females and juveniles are distinguishable. The disc is either round with an uninterrupted margin (group 1), has a deep dorsal and ventral indentation (group 2), is indented medially and laterally giving a four-lobed appearance (group 3), or is round with paired dorsal and ventral projections (group 4). There is no apparent correlation between groups of cephalic discs and other characters such as tail shape or number of body annules. Discocriconemella mauritiensis (Williams, 1960) De Grisse &Loof, 1965 is redescribed from a sugar cane population collected in Mauritius, and the diagnosis of the genus is emended.

Description of Bakernema dauniense n. sp. (Nematoda: Criconematidae) from Italy.

Bakernema dauniense n. sp., a bisexual species from the rhizosphere of Pinus halepensis Mill. in Italy, is described and illustrated. Primary differentiating characteristics of the female are body annules bearing short, membranous projections 1.4-2.2 mum long, an anterior vulval lip slightly overlapping the posterior, and a linearly arcuate vagina. The juvenile differs markedly from adults by having annules with a beaded margin, which lack membranous projections. Generic and specific relationships of B. dauniense n. sp. are discussed. This occurrence is the first for a species of Bakernema in Europe.

Macroposthonia sicula n. sp. (Nematoda: Criconematidae), a Parasite of Olive Trees in Sicily.

Macroposthonia sicula n.sp. collected from rhizosphere and roots of olive (Olea europaea L.) at Kamarina, Sicily, Italy, is described and illustrated. It is distinguished from the related species (M. sphaerocephala and M. maskaka) by the longer styler and the characteristic narrowing postvulval portion of the body.

Hemicycliophora hellenica n. sp. (Nemata: Criconematidae) from Greece.

Hemicycliophora hellenica n. sp. is described and illustrated from a bisexual population found in Filippias, Epirus, Greece, in the rhizosphere of giant reed (Arundo donax) and unidentified aquatic plants along the edge of irrigation canals. Hemicycliophora hellenica n. sp. is characterized by a long stylet (more than 120 microm), body length (L = 1,078-1,634 microm; R = 303-362), and lateral field marked by three lateral lines in females and four in males. The lip region is distinctive in that the female labial disc is oval and offset from the first lip region annule, and protrudes markedly; the male labial disc is very large, offset and rectangular, and protrudes distinctly. Hemicycliophora hellenica n. sp. resembles H. megalodiscus Loof by possession of a strongly protruding labial disc, but differs in having a longer female body, more body annules, longer stylet, and dorsally and ventrally indented head annules. The male labial plate is larger than in H. megalodiscus, and the male has four incisures in the lateral field vs. three in H. megalodiscus.

Mesocriconema kirjanovae (Nematoda: Criconematidae) from Southeastern Spain.

An abundant female population of a criconematid species identified as Mesocriconema kirjanovae (Andr ssy, 1962) Loof &De Grisse, 1989 is reported from a natural habitat in southeastern Spain. Measurements and morpho-anatomy obtained with light microscopy and scanning electron microscopy are included and compared with previous data on this species.

Factors affecting egg hatch of Heterodera mediterranea and differential responses of olive cultivars to infestation.

The influence of temperature and olive root exudates on Heterodera mediterranea egg hatch and the effects of H. mediterranea on the growth of two olive cultivars (Arbequina and Picual) were investigated. Egg hatch occurred over a temperature range of 10 to 30 degrees C and was optimal at 20 to 25 degrees C. There were no differences in egg hatch between sterile deionized distilled water or root exudate dilutions (undiluted, diluted 1:1, and 1:2) of Arbequina and Picual at 20 degrees C. Heterodera mediterranea reproduced on both olive cultivars in growth chambers at 25 degrees C. Soil and root final nematode populations, as well as total number of cysts per plant and reproduction rate, were significantly higher in Arbequina than in Picual. Shoot dry and root fresh weights as well as increases of shoot height, trunk diameter, and numbers of nodes were significantly suppressed by infection with 10,000 eggs + second-stage juveniles/pot in Arbequina but not in Picual.

Description of Rotylenchus graecus n. sp. from Greece (Nematoda: Hoplolaimidae).

Rotylenchus graecus n. sp. is described and illustrated from a bisexual population found in Filippias, Epirus, northwestern Greece, in the rhizosphere of ivy (Hedera helix L.) and giant reed (Arundo donax L.) grown on the edge of irrigation canals. Rotylenchus graecus n. sp. is characterized by a lip region with five annules, stylet length of 29-32 mum, vulva position at 53-65%, female tail pointed-conoid with 15-23 annules, and bursa with regularly annulated margins enveloping tail. The new species differs from all other Rotylenchus species with couoid tails in having a greater number of tail annules. Rotylenchus graecus n. sp. appears closely related to R. acuspicaudatus Van den Berg &Heyns, 1974, but differs from it primarily by the greater number of female tail annules (15-23 vs. 13-16), stylet length (29--32 vs. 25.7-28.3 mum), and the less ventrally protruding bursa, which has smooth posterior margins rather than the finger-like appearance of the posterior bursal annules in R. acuspicaudatus.

Morphometrics, Illustration, and Histopathology of Sphaeronema rumicis on Cottonwood in Utah.

The morphology of a population of Sphaeronema rumicis Kir'yanova found on cottonwood in Utah is illustrated by light and scanning electron micrographs, as well as by drawings. This is the first report of males of S. rumicis, a species also not known previously to occur in North America. S. rumicis females on cottonwood in the United States were smaller than those found by Kir'yanova on sorrel in the USSR. Females and second-stage juveniles (J2) from the United States had slightly shorter stylets than did females and J2 from the USSR. Males were vermiform and had degenerate esophagi. On secondary cottonwood roots S. rumicis induces formation of a syncytium originating from proliferated pericyclic cells. Thick outer walls, wall protuberances, absence of cell wall ingrowths, dense cytoplasm, and hypertrophied nuclei were the main characteristics of syncytia observed in S. rumicis-infected cottonwood roots.