Optimizing Trehalose-Based Quantification of Live Eggs in Potato Cyst Nematodes (Globodera rostochiensis and G. pallida).

Integrated management of potato cyst nematodes (PCN; Globodera rostochiensis and G. pallida) relies heavily on the determination of cyst population densities in soil as well as the viability of the eggs inside the cysts. This study aimed to optimize a quantitative method to determine the number of viable eggs of PCN based on trehalose present in live eggs. Trehalose was extracted from cysts and from a dilution series of eggs and quantified. More trehalose was detected when cysts were crushed than when left intact. Reaction volumes were adapted to the number of eggs because small reaction volumes hampered an accurate extraction of trehalose. A maximum of 10.5 eggs/µl of reaction volume should be used to obtain a significant linear relationship between detected trehalose content and egg numbers. The sensitivity of the trehalose-based method was evaluated by determining the lowest egg detection limit and was defined as five viable eggs. The reliability of this method was tested by comparing efficacy with that of two commonly used assays, visual assessment and hatching test. The trehalose-based method gave viability results similar to those of the visual assessment, which is time consuming, requires trained personnel, and can involve some subjectivity. The hatching test identified fewer viable eggs than the other two methods. In addition, the viability of dead eggs (heated and naturally dead) was tested. No false-positive results (dead eggs declared viable) were obtained with the trehalose-based method. The robustness of the test was demonstrated by measuring the viability of eggs of PCN in different experiments repeated in time. The viability assessment method based on trehalose proved to be an objective as well as sensitive, reliable, robust, fast, and cheap technique for assessing the number of viable eggs in PCN cysts.

First Report of the Dagger Nematode Xiphinema diversicaudatum in Citrus Orchards in Morocco.

Xiphinema species are migratory ectoparasitic nematodes that feed on an extensive range of hosts, and several species are vectors of nepoviruses. In May 2012, during a survey of the citrus-growing areas in the Gharb region of Morocco, several Xiphinema nematodes were detected in three locations: Kénitra (INRA, plot P1), Sidi Kacem, and Sidi Slimane. Samples were taken 30 cm deep at 50 cm distance from the tree trunks, in 40-year-old orange groves planted with the variety Maroc Late, grafted on rootstocks of Sour Orange and Citrange Carrizo. The trees showed yellowing of leaves, reduced tree vigor, and swellings at the tips of the roots. There were no weeds or grasses in the sampled area. Nematodes were extracted from soil using an automated centrifuge for extracting free-living nematode stages (2) and identified morphologically and by sequencing. All specimens were identified as Xiphinema diversicaudatum based on key morphological features from females and males. The average measurements of six females were: body length 4.1 mm, body width 60.4 µm, odontostylet 133.5 µm, odontophore 64.0 µm, spear 197.5 µm, tail length 45 µm, body width at anus 31 µm, and vulval position 48%. The females had two genital branches of similar length and structure, which contained a pseudo-Z differentiation. The average measurements of four males were: body length 4.3 mm, body width 51 µm, odontostylet 139 µm, odontophore 70 µm, spear 209 µm, tail length 45 µm, body width at anus 35 µm. To confirm the morphological identification, molecular observations were made. DNA was extracted from one nematode of each location. The D3 expansion region of the 26S rRNA gene was amplified using the primers D3A (5'-GACCCGTCTTGAAACACGGA-3') and D3B (5'-TCGGAAGGAACCAGCTACTA-3') (1). The PCR products were purified and sequenced (Macrogen, Inc., Seoul, Korea). All sequences obtained (GenBank Accession Nos. KF057879, KF057880, and KF057881) were compared with sequences available from the GenBank database including several species of Xiphinema. This comparison revealed a sequence similarity of 99 to 100% with X. diversicaudatum. Morphological and molecular identification demonstrated that the isolates of dagger nematodes from three citrus growing areas in Gharb belonged to X. diversicaudatum. An average of six X. diversicaudatum per 100 cm3 soil were found. This is the first report of this species in Morocco. X. diversicaudatum can transmit Arabis mosaic and Strawberry latent ringspot viruses (3). Arabis mosaic virus is of great economic importance in viticulture as it is associated with grapevine fanleaf degeneration disease, together with Grapevine fan leaf virus transmitted by X. index (4). As vineyards are planted amid citrus orchards in the Gharb region, particular attention should be given to this nematode, especially to the risk of its spread by soil. Our finding of X. diversicaudatum in a citrus orchard does not necessarily imply that X. diversicaudatum causes damage in citrus. However, its presence indicates that this nematode species can survive in this environment from where it could spread to other, more susceptible, crops. References: (1) L. Al-Banna et al. Mol. Phylogenet. Evol. 7:94, 1997. (2) G. Hendrickx. Nematologica 41:30, 1998. (3) J. Hübschen et al. Eur. J. Plant Pathol. 110:779, 2004. (4) A. Marmonier et al. J. Plant Pathol. 92:275, 2010.

First Report of the Root-Knot Nematode Meloidogyne artiellia in Belgium.

In 2011, second-stage juveniles (J2) of an unknown root-knot nematode (Meloidogyne spp.) were detected during a routine survey for root-knot nematodes on arable land in Harveng, Belgium, after a crop of wheat. Most of the loamy soil samples (36 out of 42) contained J2 of the common root-knot nematode M. naasi Franklin, 1965 (1), while 15 of these also contained the unknown species, albeit in lower densities (22 J2/100 ml vs. 157 J2/100 ml soil). After detailed morphological observation of the unknown J2, they were until further notice identified as Meloidogyne artiellia Franklin, 1961 (2), the British root-knot nematode. To confirm the identification, a pure culture of M. artiellia was established by adding nematode suspensions to pots planted with kale (Brassica oleracea var. laciniata), a non-host for M. naasi (3). After 2 months, Meloidogyne spp. females, males, and J2 were isolated from galled kale roots. Morphological characteristics (n = 25) from the perineal pattern (rounded with fine striae, lateral area with coarse ridges, angular dorsal arch) and stylet knobs (small, ovoid, and backwardly sloping) for the females, the head shape (set off with distinct head cap) and stylet knobs (small, ovoid and backwardly sloping) for the males, the hemizonid position (anterior, adjacent to S to E pore), tail shape (conical), and short tail length (18 to 27 µm) for the J2, fit with previous observed populations of M. artiellia (3). Young egg-laying females were used for isozyme electrophoresis, and showed typical malate dehydrogenase (N1b) and esterase (M2-VF1) patterns (3). Additionally, DNA was extracted from single juveniles by incubating them in a lysis buffer (200 mM NaCl, 200 mM Tris-HCl (pH 8), 1% ß-mercaptoethanol and 800 µg/ml Proteinase K) during 1.5 h at 65°C and 5 min at 99°C in a thermocycler. One microliter of crude DNA extract was used for PCR. ITS-rDNA sequencing (GenBank Accession Numbers JX393299 and JX393300) confirmed the identity, showing a 98 to 100% homology with other M. artiellia sequences (AY150368 and AF248478). To our knowledge, this is the first report of the root-knot nematode, M. artiellia, in Belgium. This nematode has been reported from the Mediterranean area, where it causes damage on chickpea and wheat (4), as well as from the U.K. Its finding in Harveng, close to the French border, suggests a more extensive geographical distribution. References: (1) M. T. Franklin. Nematologica 11:79, 1965. (2) M. T. Franklin et al. Suppl.:85, 1961. (3) G. Karssen. Pages 93-97 in: The Plant-Parasitic Nematode Genus Meloidogyne Göldi, 1892 (Tylenchida) in Europe, Brill Leiden, The Netherlands, 2002. (4) M. Di Vito and N. Greco. Revue Nématol. 11:223, 1988.

First Report of the Cereal Cyst Nematode Heterodera latipons on Wheat in Morocco.

From May to June 2011, during a survey of the wheat-growing areas in Meknes in the Saïs Region of Morocco, several cyst nematode populations were detected. Sampling was performed 1 month before wheat (Triticum durum) harvest, in fields showing patches of stunted plants. Plants were growing poorly, had chlorotic lower leaves, and a reduced numbers of ears. Root systems were short and had a bushy appearance because of increased secondary root production. No cysts were visible on the roots, but were found in the soil. Cysts were collected from soil on 200-µm sieves by the modified Cobb decanting and sieving method (1) and identified by morphology and internal transcribed spacer (ITS)-rDNA sequencing. All isolates were identified as Heterodera avenae except the isolate from Aïn Jemâa. From the latter, key morphological features from cysts and second-stage juveniles (J2) were determined. The cysts (n = 10) had the following characteristics: bifenestrate vulval cone, body length without neck 590 µm (551 to 632 µm), body width 393 µm (310 to 490 µm), neck length 75 µm (65 to 90 µm), fenestra length 64 µm (60 to 72 µm) and width 21 µm (18 to 25 µm), underbridge length 96 µm (85 to 115 µm), vulval slit length 8 µm (7 to 9 µm), vulva bridge width 27 µm (24 to 33 µm), and bullae absent. The J2s (n = 10) had the following characteristics: body length 445 µm (412 to 472 µm), body width 19 µm (19 to 21 µm), stylet length 24 µm (23 to 25 µm), four lateral lines, tail length 50 µm (46 to 54 µm), and hyaline terminal tail 28 µm (24 to 31 µm). Values of the morphological characters were within the range of H. latipons reported by Handoo (3). The bifenestrate cysts with a strong underbridge and no bullae and J2 with a tail length greater than 40 µm, a stylet longer than 15 µm, and four incisures in the lateral field were typical for H. latipons. To confirm the identification, molecular observations were made. DNA was extracted from three juveniles from three different cysts separately (4). The ITS-rDNA region was amplified using the primers 5'-CGT AAC AAG GTA GCT GTA G-3' and 5'-TCC TCC GCT AAA TGA TAT G-3' as described by Ferris et al. (2). This resulted in a 1,040-bp DNA fragment. The PCR-products were purified and sequenced (Macrogen, Inc., Seoul, Korea). All sequences obtained (GenBank Accession Nos. per cyst: JQ319035, JQ319036, and JQ319037) were compared with sequences available from the GenBank database ( www.ncbi.nlm.nih.gov ), including several species of Heterodera. This comparison revealed a sequence similarity of 97 to 99% with H. latipons and 89% or lower with any other species of Heterodera. Morphological and molecular identification demonstrated that the population of cyst nematodes from a wheat field in Aïn Jemâa, Morocco was H. latipons. In the patches with poor growing plants, 65 cysts per 100 cm3 soil were found. To our knowledge, this detection represents a new record of H. latipons. Since the nematode can cause considerable damage to wheat, one of the main cereals produced in Morocco, care should be taken to prevent the spread to other regions. References: (1) K. R. Barker. Page 19 in: An Advanced Treatise on Meloidogyne. Vol II. Methodology. C. C. Carter and J. N. Sasser, eds. North Carolina State University Graphics, Raleigh, 1985. (2) V. R. Ferris et al. Fundam. Appl. Nematol. 16:177, 1993. (3) Z. A. Handoo. J. Nematol. 34:250, 2002. (4) M. Holterman et al. Mol. Biol. Evol. 23:1792, 2006.

A method for estimating the contribution of seed potatoes, machinery and soil tare in field infestations with potato cyst nematodes on a national scale.

In order to make a cost benefit analysis for the management of the potato cyst nematodes Globodera rostochiensis and G. pallida, we developed a method to estimate the relative importance of three basic distribution channels of potato cyst nematodes: seed potatoes, machinery and soil tare. The baseline is determined by the area planted with potatoes, the area infested with potato cysts, the proportion of resistant potato cultivars and the distribution of cysts trough different channels. This quantification forms a basis for the evaluation of the effects of different control measures for potato cyst nematode on a national scale. The method can be useful as an example for application in other countries.

Intraspecific variability of Steinernema feltiae strains from Cemoro Lawang, eastern Java, Indonesia.

Four strains of Steinernema feltiae from Eastern Java, Indonesia were characterized based on morphometric, morphological and molecular data. In addition, their virulence against last instar Tenebrio molitor and heat tolerance was tested. Infective juvenile have a mean body length ranging from 749 to 792 microm. The maximum sequence difference among the four strains was 7 bp (8.8%) in the ITS and 2 bp (0.3%) in D2D3 regions of the rDNA. All the strains are not reproductively isolated and can reproduce with European strain S. feltiae Owiplant. The lowest LC50 was observed for strain SCM (373) and the highest for S. feltiae strain Owiplant (458) IJs/40 T. molitor. All four strains showed relatively better mean heat tolerance when compared with S. feltiae Owiplant, both in adapted and non-adapted heat tolerance experiments.

First Report of the Root-Knot Nematode Meloidogyne minor on Turfgrass in Belgium.

The root-knot nematode, Meloidogyne minor, was described during 2004 after it was found on potato roots in a field in the Netherlands and in golf courses in England, Wales, and Ireland (2). Since it is associated with yellow patch disease in turf grass and causes deformation of potato tubers (2), it is important to know whether this organism is already widespread in these and neighboring countries. In addition, it has a relatively wide host range (2,4). A small survey conducted in Belgium was comprised of 10 golf courses geographically spread over the country. In each location, 3 to 9 samples were taken (one per green) consisting of 30 to 40 cores (1.5 × 20 cm deep). Nematodes were extracted from a 200-g subsample (containing roots) from each sample using zonal centrifugation (1). All Meloidogyne spp. were mounted on semipermanent slides and identified morphologically. M. minor was discovered in 3 of 6 samples taken in April 2006 from a golf course in Hasselt (northeastern Belgium). Between 41 and 50 M. minor per 100 g of soil were found together with M. naasi (7 to 20 individuals per 100 g of soil). Occurrence of M. minor together with other Meloidogyne species has been reported in natural and cultivated sites (2,4). Moreover, spores of Pasteuria spp. were clearly visible on 42% of the observed second-stage juveniles of M. minor, but not on those of M. naasi. The infected juveniles had between 2 and 15 spores attached to their cuticles. Additional juveniles were extracted from the soil samples and used for molecular identification by real-time PCR (2), which confirmed the presence of M. minor. There were no symptoms on the grass, consisting of a mixture of Agrostis stolonifera (10%), Festuca rubra (30%), and Poa annua (60%). Grass was sown in Rhine sand and heath land compost used for the construction of the greens in Hasselt. It could be that these soil amendments were infested with M. minor or that M. minor was introduced by other means, e.g., shoes, maintenance machinery, or golf equipment. On the other hand, the detection of M. minor in this small survey indicates that the species may be prevalent in golf courses in the region. The nematode has been found in several golf courses and sport fields in the United Kingdom and the Netherlands, including a golf course at Breda (close to the Belgian border) (3). The survey will be expanded to include grasslands and dune areas, the presumed natural habitat of M. minor. References: (1) G. A. Hendrickx. Nematologica 41:308, 1995. (2) G. Karssen et al. Nematology 6:59, 2004. (3) W. Lammers et al. Meloidogyne minor. Pest Risk Assessment. Online publication, www.minlnv.nl/pd - Schadelijke organismen, 2006. (4) S. J. Turner and C. C. Fleming. Comm. Appl. Biol. Sci. Ghent University 70:885, 2005.

Critical use of methyl bromide for soil disinfestation in Belgium during 2005.

During 2005, Belgium enforced a strict procedure for the assignment of critical-use permits for methyl bromide (MeBr) as a soil disinfestant. This procedure involved an inspection of the site before disinfestation by a representative of a registered institute, and a mandatory demonstration of the presence of a pest or disease for which a critical use permit could be granted according to the Critical Use Nominations (CUNs). The procedure was subject to random inspections by an independent institute. The results of these inspections demonstrated proper and timely evaluation of the permit requests. A total of 113 requests for a MeBr disinfestation permit were submitted in 2005. Out of these, 105 referred to applications in 2005. The remaining 8 requests referred to applications in 2006 and were denied a permit based on the lack of MeBr quotum granted to Belgium for 2006. Of the 105 requests for applications in 2005, 93 received a MeBr application permit for soil disinfestation. These 93 permits represented 15911 kg or 37.3% of the total quotum assigned to Belgium in 2005 (42676 kg). Most of the quotum was used for butterhead lettuce (11456 kg or 72% of the applied MeBr). For most commodities, a surplus in quotum was available. However, for chrysanthemum, the amount requested for critical use exceeded the available quotum. The most important pests and diseases for which a permit was assigned were Meloidogyne, Sclerotinia, Rhizoctonia, Olpidium, Pythium, Pyrenochaeta, Verticillium, and a combination of these fungi and nematodes. The 12 requests for which no permit was granted represented 2010 kg or 11.3% of the total amount requested. In addition, institutes carrying out the assignment procedure reported at least 62 extra cases where they had been contacted by a grower but where no official permit request was filed based on the first inspection. When including those cases, the total reduction of the potentially used amount of MeBr is about 44 %. When adding an estimated 10 to 15% reduction due to the use of alternatives by growers who did not start the permit assignment procedure, the total reduction is approximately 50-55%.

Fungi Parasitic on Juveniles and Egg Masses of Meloidogyne hapla in Organic Soils from New York.

Fungi associated with egg masses and juveniles of Meloidogyne hapla were isolated from organic soil samples obtained from five fields planted to lettuce or onion in NewYork. The soil samples were placed in sterilized clay pots, infested with M. hapla, and planted to lettuce. After 4 months, egg masses and juveniles were surface-disinfested, plated on water agar, and examined for fungal infection. Depending on the soil sample, fungal isolates were recovered from 13% to 30%, and from 5% to 24% of the egg masses and juveniles, respectively. A total of 24 and 16 isolates collected from egg masses and juveniles, respectively, were selected for further characterization. Fifteen of the isolates were considered as egg-mass pathogens as they were able to infect healthy assay egg masses and could be succesfully reisolated. These fungi included species of Fusarium, Alternatia, and Verticillium psalliotae. Six of the egg-mass-parasitizing fungi could not be identified. Nine fungal isolates were found to be pathogenic to juveniles of M. hapla; six were identified as Monacrosporium sp., two as Arthrobotrys sp., and one as Hirsutella rhossiliensis. The remaining 16 fungal isolates were unable to infect egg masses or juveniles, and thus were considered nonparasitic to M. hapla.

SeinFit, a Computer Program for the Estimation of the Seinhorst Equation.

A computer program, "SeinFit," was created to determine the Seinhorst equation that best fits experimental data on the relationship between preplant nematode densities and plant growth. Data, which can be entered manually or imported from a text file, are displayed in a data window while the corresponding graph is shown in a graph window. Various options are available to manipulate the data and the graph settings. The best-fitting Seinhorst equation can be calculated by two methods that are both based on the evaluation of the residual sum of squares. Depending on the method, a range of values for different parameters of the Seinhorst equation can be chosen, as well as the number of steps in each range. Data, graphs, and values of the parameters of the Seinhorst equation can be printed. The program allows for quick calculation of the danaage threshold density - one of the parameters of the Seinhorst model. Versions written for Macintosh or DOS-compatible machines are currently available through the Society of Nematologists' World Wide Web site (http://ianrwww.unl.edu/ianr/plntpath/ nematode/SOFTWARE/nemasoft.htm).

Damage Threshold of Meloidogyne hapla to Lettuce in Organic Soil.

Lettuce was seeded in pots in the greenhouse and in field microplots in 1991 and 1992. Pots and microplots were filled with untreated or fumigated organic soil infested with Meloidogyne hapla at seven initial population densities (Pi) (0 to 32 eggs/cm(3) soil). Lettuce weight, severity of root galling, and number of eggs per root system (Pf) were determined after 8 weeks. At the highest Pi, M. hapla caused yield losses up to 64% in the microplots and plant death in the greenhouse tests. The Seinhorst equation was used to describe the relation between lettuce weight and Pi (r(2) = 0.73 - 0.98) and to calculate the damage threshold density (T). Values of T were 7 and 8 eggs/cm(3) soil in the greenhouse tests of 1991 and 1992, respectively. In the microplot tests, T was 1 egg/cm(3) soil in 1991 and 2 eggs/cm(3) soil in 1992. The damage threshold was the same in untreated and fumigated soils. At low Pi, root galling was more severe in the pots than in the microplots. Pf increased with increasing Pi of M. hapla in both tests, but declined at Pi > T in the greenhouse tests. The reproduction rate (Pf/Pi) of M. hapla was highest at the lowest Pi.

Three cases of infection by Haemophilus-like bacteria in psittacines.

The clinical manifestations of three cases of infection with NAD (V-factor)-requiring Haemophilus-like bacteria in psittacines are described. Case 1 involved members of the Platycercus family and produced mainly respiratory signs. In case 2 only conjunctivitis was seen among cockatiels. The third case was a hand-reared African grey parrot which developed a peracute septicaemia. The Haemophilus-like bacteria isolated from the three cases differed from each other as well as from the known avian NAD-requiring Haemophilus and Pasteurella species.

Kinetics of serum and local haemagglutination inhibition antibodies in chicks following vaccination and experimental infection with Newcastle disease virus and their relation with immunity.

Broiler chicks were vaccinated against Newcastle disease (ND) by nebulisation of live La Sota vaccine, or by intramuscular administration of inactivated oil emulsion vaccine. Local haemagglutination inhibition (HI) antibodies were detected in spray vaccinated chicks but not in those vaccinated intramuscularly. Initially local HI-titres in trachea, tears and saliva behaved in the same way as the serum HI-titre, but after maximum levels were reached approximately 10 days after vaccination, local HI-titres waned to levels that were no longer detectable after 4 weeks. Challenge exposure with velogenic Newcastle disease virus (NDV), showed that high post-vaccinal serum HI-titres were correlated with complete resistance to clinical disease but not to infection, as indicated by the prevention of virus multiplication in the trachea and also by the post-challenge serum HI-titres. Resistance to infection was only detected in spray vaccinated chicks. No relation was found between local HI-titres and resistance to experimental infection with velogenic NDV.

Field-dependence and stability of self-view.

Contrary to Witkin's opinion, field-dependent individuals are expected to show higher stability of self-view than field-independent individuals. 73 Belgian second-year university students were submitted to the Group Embedded Figures Test. The Five Personality Factor Test measuring extraversion, friendliness, emotional instability, conscientiousness and general culture, was administered to each student for self-description (self-report) and to both parents for description of the student (mother's report and father's report). The decrease of correlation between self-report and parent's report as a function of the degree of student's field-independence supports the author's hypothesis. Results are interpreted in terms of closer attention of field-independent individuals to relevant cues.

Cutaneous geotrichosis in the red flamingo (Phoenicopterus ruber).

In three flamingos (Phoenicopterus ruber), which died showing extensive necrotic inflammation of the skin of the webs and the legs, the presence of abundant mycelium and arthrospores was shown in the altered dermis and epidermis. From all the lesions Geotrichum Candidum was isolated in pure culture. Inoculation of suspensions of the fungus into domestic chickens and mice produced granulomas or more purulent nodules with central growth of abundant mycelium. From 20 of 28 experimental animals, G. candidum was reisolated from the lesions in pure culture 2 weeks after the inoculation. It is suggested that alteration of the skin by e.g., prolonged contact with pondwater, the presence of small wounds and the advanced age of the birds, acting as predisposing factors may be necessary in establishing this disease.

Newcastle disease: effects of vaccines and vaccination techniques of hemagglutination-inhibition (HI) antibodies and clinical resistance.

Different vaccines (the lentogenic Hitchner-, the avirulent Ulster- and the mesogenic Beaudette strain) and different vaccination schemes (spray, dip, drinking water) were used in 1380 broiler-type chicks on the first day of life and at four weeks. In all these experiments an immunity status was induced sufficient to resist to a strong individual challenge infection, two weeks after the second vaccination. This uniform resistance showed no correlation with the very heterogeneous HI titers which were obtained two weeks after the second vaccination. The titers were, however, strongly dependent both of the vaccine strain used and on the vaccination method. The Beaudette spray-vaccinations provoked the highest titers. The highest post-challenge titers were seen after vaccinations with the lentogenic and avirulent strains and the lowest after-challenge titers were seen when the mesogenic Beaudette strain was used. Birds with high HI titers showed a marked decrease of these HI titers two weeks after challenge, while there was an increase of HI titers in birds with low post-vaccinal titers. It may be concluded that clinical resistance to NCD infection is not directly correlated with high-vaccinal HI titers. Experimental infection by natural route is essential to judge the immune status of fowls.

Pseudomonas aeruginosa infection on a broiler farm.

Pseudomonas aeruginosa infection was a factor associated with heavy losses on a broiler farm. The organism was repeatedly isolated from air sac lesions similar to those commonly observed in E.coli complicated Chronic Respiratory Disease, and from oedematous lesions present predominantly in the head region of some of the affected chickens. Losses continued in successive batches of chickens until an all-in all-out policy was adopted, although the organism was still present in the drinking water.