First report of an entomopathogenic nematode from continental Portugal

In continental Portugal no information is available concerning the occurrence of entomopathogenic nematodes. During a survey in several different habitats from the southern regions of the country, several isolates where identified as Steinernema feltiae. This represents the first report of an entomopathogenic nematode genus for continental Portugal.

A rapid staining-assisted wood sampling method for PCR-based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR-based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining-assisted wood sampling method aiding PCR-based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5-mg wood cross-sections, of 0.5 · 0.5 · 0.015 cm dimension, that were cut from a wood sample of 0.5 · 0.5 · 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5-mg wood cross-sections (that adjoined the three 5-mg wood cross-sections, mentioned above) were cut. Nematode-staining-spots (NSSs) in each of the two stained sections were counted under a microscope at 100· magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus – specific amplicon of 403 bp (DQ855275) was generated by PCR assay from 100.00% of 5-mg wood cross-sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx-detection methods such as PCR assay.

The Pine Wood Nematode: a personal view

The first report of the disease (“pine wilt disease”) associated with the pinewood nematode, goes back to 1905, when Yano reported an unusual decline of pines from Nagasaki. For a long time thereafter, the cause of he disease was sought, but without success. Because of the large number of insect species that were usually seen around and on infected trees, it had always been assumed that the causal agent would prove to be one of these. However, in 1971, Kiyohara and Tokushike found a nematode of the genus Bursaphelenchus in infected trees. The nematode found was multiplied on fungal culture, inoculated into healthy trees and then re-isolated from the resulting wilted trees. The subsequent published reports were impressive: this Bursaphelenchus species could kill fully-grown trees within a few months in the warmer areas of Japan, and could destroy complete forests of susceptible pine species within a few years. Pinus densiflora, P. thunbergii und P. luchuensis were particularly affected. In 1972, Mamiya and Kiyohara described the new species of nematode extracted from the wood of diseased pines; it was a named Bursaphelenchus lignicolus. Since 1975, the species has spread to the north of Japan, with the exception of the most northerly prefectures. In 1977, the loss of wood in the west of the country reached 80%. Probably as a result of unusually high summer temperatures and reduced rainfall in the years 1978 and 1979, the losses were more than 2 million m3 per year. From the beginning, B. lignicolus was always considered by Japanese scientists to be an exotic pest. But where did it come from? That this nematode could also cause damage in the USA became clear in 1979 when B. lignicolus was isolated in great numbers from wood of a 39 year-old pine tree (Pinus nigra) in Missouri which had suddenly died after the colour of its needles changed to a reddish-brown colour (Dropkin und Foudin, 2 1979). In 1981, B. lignicolus was synonymised by Nickle et al. with B. xylophilus which had been found for the first time in the USA as far back as 1929, and reported by Steiner and Buhrer in 1934. It had originally been named Aphelenchoides xylophilus, the wood-inhabiting Aphelenchoides but was recognised by Nickle, in 1970,to belong in the genus Bursaphelenchus. Its common name in the USA was the "pine wood nematode" (PWN. After its detection in Missouri, it became known that B. xylophilus was widespread throughout the USA and Canada. It occurred there on native species of conifers where, as a rule, it did not show the symptoms of pine wilt disease unless susceptible species were stressed eg., by high temperature. This fact was an illuminating piece of evidence that North America could be the homeland of PWN. Dwinell (1993) later reported the presence of B. xylophilus in Mexico. The main vector of the PWN in Japan was shown to be the long-horned beetle Monochamus alternatus, belonging to the family Cerambycidae. This beetle lays its eggs in dead or dying trees where the developing larvae then feed in the cambium layer. It was already known in Japan in the 19th century but in the 1930s, it was said to be present in most areas of Japan, but was generally uncommon. However, with the spread of the pine wilt disease, and the resulting increase of weakened trees that could act as breeding sites for beetles, the populations of Monochamus spp. increased significantly In North America, other Monochamus species transmit PWN, and the main vector is M. carolinensis. In Japan, there are also other, less efficient vectors in the genus Monochamus. Possibly, all Monochamus species that breed in conifers can transmit the PWN. The occasional transmission by less efficient species of Monochamus or by some of the many other beetle genera in the bark or wood is of little significance. In Europe, M. galloprovincialis and M. sutor transmits the closely related species B. mucronatus. Some speculate that these two insect species are “standing by” and waiting for the arrival of B. xylophilus. In 1982, the nematode was detected and China. It was first found in dead pines near the Zhongshan Monument of Nanjing (CHENG et. al. 1983); 265 trees were then killed by pine wilt disease. Despite great efforts at eradication in China, the nematode spread further and pine wilt disease has been 3 reported from parts of the provinces of Jiangsu, Anhui, Guangdong, Shandong, Zhejiang and Hubei (YANG, 2003). In 1986, the spread of the PWN to Taiwan was discovered and in 1989, the nematode was reported to be present in the Republic of Korea where it had first been detected in Pinus thunbergii and P. densiflora. It was though to have been introduced with packing material from Japan. PWN was advancing. In 1984, B. xylophilus was found in wood chips imported into Finland from the USA and Canada, and this was the impetus to establish phytosanitary measures to prevent any possible spread into Europe. Finland prohibited the import of coniferous wood chips from these sources, and the other Nordic countries soon followed suit. EPPO (the European and Mediterranean Plant Protection Organization) made a recommendation to its member countries in 1986 to refuse wood imports from infested countries. With its Directive of 1989 (77/93 EEC), the European Community (later called the European Union or EU) recognised the potential danger of B. xylophilus for European forests and imposed restrictions on imports into the Europe. PWN was placed on the quarantine list of the EU and also of other European countries. Later, in 1991, a dispensation was allowed by the Commission of the EU(92/13 EEC) for coniferous wood from North America provided that certain specified requirements were fulfilled that would prevent introduction.

Pine wilt disease and the pinewood nematode

Pine wilt disease (PWD) is one of the most damaging events affecting conifer forests (in particular Pinus spp.), in the Far East (Japan, China and Korea), North America (USA and Canada) and, more recently, in the European Union (Portugal). In Japan it became catastrophic, damaging native pine species (Pinus thunbergii and P. densiflora), and becoming the main forest problem, forcing some areas to be totally replaced by other tree species. The pine wilt nematode (PWN) Bursaphelenchus xylophilus, endemic, with minor damage, to North America, was introduced in Japan in the early XX century and then spread to Asia (China and Korea) in the 1980s. In 1999 it was detected for the first time in Portugal, where, due to timely detection and immediate government action, it was initially (1999-2008) contained to a small area 30 km SE of Lisbon. In 2008, the PWN spread again to central Portugal, the entire country now being classified as “affected area”. Being an A1 quarantine pest, the EU acted to avoid further PWN spreading and to eradicate it, by actions including financial support for surveyes and eradication, annual inspections and research programs. Experience from control actions in Japan included aerial spraying of insecticides to control the insect vector (the Cerambycid beetle Monochamus alternatus), injection of nematicides to the trunk of infected trees, slashing and burning of large areas out of control, beetle traps, biological control and tree breeding programs. These actions allowed some positive results, but also unsuccessful cases due to the PWN spread and virulence. Other Asian countries also followed similar strategies, but the nematode is still spreading in many regions. In Portugal, despite lower damage than Asia, PWD is still significant with high losses to the forestry industry. New ways of containing PWD include preventing movement of contaminated wood, cutting symptomatic trees and monitoring. Despite a national and EU legislative body, no successful strategy to control and eventually eradicate the nematode and the disease will prevail without sound scientific studies regarding the nematode and vector(s) bioecology and genetics, the ecology and ecophysiology of the pine tree species, P. pinaster and P. pinea , as well as the genomics and proteomics of pathogenicity (resistance/ susceptibility).

Genetic diversity of entomopathogenic nematodes (Nematoda: Steinernematidae and Heterorhabditidae) and the nematode Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) from continental Portugal

“Diversidade genética dos nemátodes entomopatogénicos (Nematoda: Steinernematidae e Heterorhabditidae) e do nemátode Bursaphelenchus xylophilus (Nematoda: Aphelenchoididade) em Portugal continental” Os nematodes entomopatogénicos são utilizados como agentes de controlo biológico. Para compreender a sua diversidade, foi realizada uma prospecção em Portugal. Cinco espécies, nomeadamente Steinernema feltiae, S. intermedium, S. kraussei, Steinernema sp. e Heterorhabditis bacteriophora foram identificadas. As sequências de ITS, região D2D3 do 28S rRNA, COXI e cytb foram utilizadas para estudar a diversidade genética das duas espécies mais abundantes, S. feltiae and H. bacteriophora, não tendo sido encontradas diferenças significativas entre isolados. O nemátode da madeira do pinheiro, Bursaphelenchus xylophilus, provoca doença nos pinheiros tendo sido detectada pela primeira vez na Europa e em Portugal em 1999. Para avaliar a diversidade genética dos isolados Portugueses e identificar o padrão de propagação da doença, foram utilizadas a sequência da região IGS do 5.8S rRNA, e os genes cytb e cellulase, combinados com os padrões ISSR. Os padrões de ISSR mostraram elevada diversidade genética entre os recentes isolados Portugueses, sugerindo a possibilidade de uma nova introdução. As árvores filogenéticas dos genes da celulase e cytb sugeriram uma origem Asiática para os isolados Portugueses; ABSTRACT: Entomopathogenic nematodes are used as biocontrol agents. To understand their diversity, a survey was undertaken in Portugal. Five species, namely Steinernema feltiae, S. intermedium, S. kraussei, Steinernema sp. and Heterorhabditis bacteriophora were identified. The ITS, 28S rRNA D2D3 region, COXI and cytb sequences, used to study the genetic diversity of the two most abundant species, S. feltiae and H. bacteriophora, showed no significant differences among the isolates. Bursaphelenchus xylophilus causes severe disease in pine trees and was detected for the first time in Europe and in Portugal in 1999. To evaluate the genetic diversity of Portuguese isolates and identify disease spread pathways, the sequence of 5.8S rRNA IGS region, cytb and cellulase genes, combined with ISSR fingerprints were used. ISSR fingerprints show a high genetic variability among recent Portuguese isolates, suggesting the possibility of a new introduction. Phylogenetic trees based on cellulase and cytb genes suggests an Asian origin for Portuguese isolates.

Assessment of the genetic diversity of the pinewood nematode, Bursaphelenchus xylophilus, in Portugal

O nemátode da madeira do pinheiro (NMP), Bursaphelenchus xylophiius, tem uma extensa distribuição na América do Norte, e encontra-se atualmente distribuído ao longo da maioria dos territórios de Canadá e dos Estados Unidos. Durante o último século, esta espécie foi transportada pelo Homem para outras regiões do mundo (não-nativas), associadas com o comércio e o fluxo global de produtos de origem florestal. Atualmente, esta espécie invasiva está reportada para algumas regiões do SE asiático (China, Japão, Coreia e Taiwan) e mais recentemente para a Europa (Portugal). Devido ao impacto que este organismo agente da doença da murchidão dos pinheiros causa nas florestas nativas destas regiões esta espécie assume uma elevada importância económica a nível mundial Em Portugal, a distribuição do NMP encontra-se confinada a uma área restrita e limitada (500 000 ha), a sul de Lisboa (península de Setúbal); contudo, constitui uma das maiores ameaças às florestas de pinheiro do país e da UE. Ate recentemente, nenhum consenso existia quanto à origem do NMP em Portugal. Diversas hipóteses têm sido colocadas para explicar esta introdução, nomeadamente a partir de zonas onde o nematode ocorre naturalmente (América do Norte), ou de outras áreas (não-nativas) onde o nematode se comporta como uma espécie invasiva (Leste da Ásia). A fim de avaliar a variabilidade genética do NMP proveniente da área afetada em Portugal, foram utilizadas várias técnicas moleculares, designadamente o random amplified polymorphic DNA (RAPD-PCR) e o satellite DNA (satDNA). No caso do RAPD-PCR, foram utilizados 24 isolados do NMP provenientes de Portugal, 1 proveniente da América do Norte e 1 da Ásia, tendo sido utilizado como out-group um isolado de B. mucronatus. A partir dos 28 RAPD primers utilizados obtiveram-se 640 fragmentos. No caso do satDNA, foram utilizados 21 isolados do NMP provenientes de Portugal, obtendo-se no total 206 sequências da família MspI. Ambos os métodos revelaram uma elevada similaridade genética entre os vários isolados do NMP da área afetada em Portugal O nível reduzido de diversidade genética obtido entre os isolados portugueses do NMP, permite concluir que se trata de uma única introdução deste organismo em Portugal, e proveniente de uma região asiática. A inexistência de uma de correlação entre a variabilidade genética e a distribuição geográfica do NMP dentro da área afetada em Portugal, indica que o NMP se encontra distribuído de forma uniforme ao longo de toda a área afetada, provavelmente relacionado com a distribuição e a expansão natural do inseto vector. The pinewood nematode (PWN), Bursaphelenchus xylophilus, has a wide distribution in North America, and is present throughout most of the territories of Canada and the United Stata. During the last century, this species has been transported by man to several non-native regions of the world, associated with trade and the global flow of forest products. Up to date, this invasive species has been reported from Asia (PR China, Japan, Korea and Taiwan) and more recently in Europe (Portugal). Due to the impact on native pine forests of these regions, this nematode species, the causal agent of pine wilt disease, is of great economic importance worldwide. In Portugal, the distribution of the PWN has been constrained to a relatively small area (500 000 ha) in the south of Lisbon (Setúbal Peninsula); however, it has become the most serious threat to pine forests in the country. Until recently, no consensus had emerged on the possible pathway of the PWN introduction in Portugal. Several hypotheses have been put forward to explain this introduction, such as an origin from endemic areas where the nematode naturally occurs (North America), or non-endemic areas where the nematode behaves as an exotic pest (East Asia). Random amplified polymorphic DNA (RAPD-PCR) and satellite DNA (satDNA) techniques were used in order to assess the level of genetic variability and genetic relationships, among several isolates of the PWN, representative of the entire affected area in Portugal. In the case of RAPD-PCR, 24 Portuguese isolates, plus two additional isolates of B. xylophilus, representing North America and East Asia were included. B. mucronatus was used as an out-group. Twenty-eight random primers generated a total of 640 DNA fragments. With satDNA, 206 Mspl sequence repeats were obtained from 21 Portuguese isolates of B. xylophilus. Both molecular methods revealed a high genetic similarity among the Portuguese isolates, and the low level of genetic diversity strongly suggests that they were dispersed recently from a single introduction, and from East Asia. The lack of apparent relationship between the genetic variability and the geographic distribution of the PWN within the affected area, suggests that the recent introduction of this pest (and pathogen) in Portugal has been uniformly distributed since its establishment, probably following the natural distribution and expansion of the insect vector.

Essential oils as anti-nematode agents and their influence on in vitro nematode

Tese de doutoramento, Biologia (Biotecnologia), Universidade de Lisboa, Faculdade de Ciências, 2015

Cortical origin of functional recovery in the somatosensory cortex of the adult mouse after thalamic lesion

Résumé L'accident vasculaire cérébral sensoriel pur est un des syndromes lacunaires, dû à l'occlusion de petits vaisseaux cérébraux, souvent dans le cadre d'une lésion intéressant le noyau ventro-caudal du thalamus. Il produit un hémisyndrome sensitif pur, et parfois un syndrome douloureux se développe à distance de l'événement aigu. Afin d'étudier la récupération fonctionnelle dans le cortex somatosensoriel (SI) après une telle lésion dans le thalamus, un modèle de lésion excitotoxique a été développé dans le système somatosensoriel de la souris adulte, caractérisé par la présence de formations cytoarchitectoniques dans SI appelées "tonneaux". Chacun de ces tonneaux correspond à la représentation corticale d'une vibrisse du museau. L'activité métabolique a été mesurée dans SI à différents intervalles après la lésion, à l'aide de déoxyglucose marqué radioactivement. Dans les deux premiers jours suivant celle-ci, l'activité métabolique diminue de manière importante dans toutes les couches corticales, avec une atteinte plus marquée dans la couche IV, principale projection des axones thalamo-corticaux. Une récupération de l'activité métabolique se produit ensuite, d'autant plus marquée que le délai après la lésion est grand. Cette récupération s'observe dans toutes les couches coticales, les couches I et Vb récupérant plus rapidement que les couches II, III, IV, Va et VI. Cinq semaines après la lésion, l'absence des vibrisses correspondant à la partie déafférentée de SI diminue l'activité métabolique corticale de 32% et démontre l'activation par la périphérie de cette partie de l'écorce, malgré la perte des axones thalamo-corticaux provenant du noyau ventro-caudal. Des expériences de traçage rétrograde ont montré une augmentation des projections intracorticales sur la partie déafférentée de l'écorce, en particulier de longue distance, ainsi que des projections interhémisphériques, mais n'ont pas permis de mettre en évidence de nouvelle projection thalamique, indiquant une origine corticale à la récupération fonctionnelle observée. Abstract To study the degree and time course of the functional recovery in the somatosensory cortex (SI) after an excitotoxic lesion in the adult mouse thalamus, metabolic activity was determined in SI at various times points post lesion. Immediately after the lesion, metabolic activity in the thalamically deafferented part of SI was at its lowest value but increased progressively at subsequent time points. This was seen in all cortical layers, however, layers I and Vb recover more rapidly than layers II, III, IV, Va and VI. Removal of the mystacial whiskers corresponding to the deafferented area, 5 weeks after cortical recovery, produced a subsequent 32% drop in metabolic activity, demonstrating peripheral sensory activation of this part of the cortex. Tracing experiments revealed that the deafferented cortex did not receive a novel thalamic input, but cortico-cortical and contralateral barrel cortex projections to this area were reinforced. We conclude that the cortical functional recovery after a thalamic lesion is, at least partially, due to modified cortico-cortical and callosal projections to the deafferented cortical area.

Alveolar echinococcosis of the liver: Diffusion-weighted MRI findings and potential role in lesion characterisation

PURPOSE: To report the diffusion-weighted MRI findings in alveolar echinococcosis (AE) of the liver and evaluate the potential role of apparent diffusion coefficients (ADCs) in the characterisation of lesions. MATERIALS AND METHODS: We retrospectively included 22 patients with 63 AE liver lesions (≥1cm), examined with 3-T liver MRI, including a free-breathing diffusion-weighted single-shot echo-planar imaging sequence (b-values=50, 300 and 600s/mm(2)). Two radiologists jointly assessed the following lesion features: size, location, presence of cystic and/or solid components (according to Kodama's classification system), relative contrast enhancement, and calcifications (on CT). The ADCtotal, ADCmin and ADCmax were measured in each lesion and the surrounding liver parenchyma. RESULTS: Three type 1, 19 type 2, 17 type 3, three type 4 and 21 type 5 lesions were identified. The mean (±SD) ADCtotal, ADCmin and ADCmax for all lesions were 1.73±0.50, 0.76±0.38 and 2.63±0.76×10(-3)mm(2)/s, respectively. The mean ADCtotal for type 1, type 2, type 3, type 4 and type 5 lesions were 1.97±1.01, 1.76±0.53, 1.73±0.41, 1.15±0.42 and 1.76±0.44×10(-3)mm(2)/s, respectively. No significant differences were found between the five lesion types, except for type 4 (p=0.0363). There was a significant correlation between the presence of a solid component and low ADCmin (r=0.39, p=0.0016), whereas an inverse correlation was found between the relative contrast enhancement and ADCtotal (r=-0.34, p=0.0072). CONCLUSION: The ADCs of AE lesions are relatively low compared to other cystic liver lesions, which may help in the differential diagnosis. Although ADCs are of little use to distinguish between the five lesion types, their low value reflects the underlying solid component.

Isolation and characterization of the female sex pheromone of the sugar beet cyst nematode, heterodera schachtii, and an analysis of male precopulatory behaviour /

The sugar beet cyst nematode, Heterodera schachtii, is a major agricultural pest. The disruption of the mating behaviour of this plant parasite in the field may provide a means of biological control, and a subsequent increase in crop yield. The H. schachtii female sex pheromone, which attracts homospecific males, was collected in an aqueous medium and isolated using high performance liquid chromatography. Characterization of the male-attractive material revealed that it was heat stable and water soluble. The aqueous medium conditioned by female H. schachtii was found to be biologically active and stimulated male behaviour in a concentration dependent manner. The activity of the crude pheromone was specific to males of H. schachtii and did not attract second stage juveniles. Results indicated that vanillic acid, a putative nematode pheromone, is not an active component of the H. schachtii sex pheromone. Male H. schachtii exhibited stylet thrusting, a poorly understood behaviour of the male, upon exposure to the female sex pheromone. This behaviour appeared to be associated with mate-finding and was used as a novel indicator of biological activity in bioassays. Serotonin, thought to be involved in the neural control of copulatory behaviour in nematodes, stimulated stylet thrusting. However, the relationship between stylet thrusting induced by the sex pheromone and stylet thrusting induced by serotonin is not clear. Extracellular electrical activity was recorded fi-om the anterior region of H. schachtii males during stylet thrusting, and appeared to be associated with this behaviour. The isolation of the female sex pheromone of H. schachtii may, ultimately, lead to the structural identification and synthesis of the active substance for use in a novel biological control strategy.

Meloidogne incognita (nematode) parasitism of Lycopersicon esculentum (tomato) plants : Ethylene action in susceptible and resistant host responses

Involvement of ethylene in the etiology of tomato plants (Lycopersicon esculentum) infected with the root-knot nematode (Meloidogyne incognita) was investigated. Endogenous root concentrations of ethylene were not significantly different in uninfected resistant var. Anahu and susceptible var. Vendor plants. Exposure of resistant plants to high doses of infectious nematode larvae did not affect root ethylene concentrations during the subsequent 30 day period. The possibility that ethylene may be involved in the mechanism of resistance is therefore not supported by these experiments. In no experiments did ethylene concentrations in roots of susceptible plants increase significantly subsequent to ~ incognita infestation. This result is not consistent with the hypothesis in the literature which suggests that increased ethylene production accompanies gall formation. Growth of susceptible tomato plants was affected by ~ incognita infestation such that root weights increased (due to galling), stem heights decreased and top weights increased. The possibility that alterations in stem growth resulted from increased production of 'stress' ethylene is discussed. Growth of resistant plants was unaffected by exposure to high doses of ~ incognita and galls were never detected on the roots of these plants. Root ethane concentrations generally varied in parallel with root ethylene concentrations although ethane concentrations were without exception greater. In 4 of 6 experiments conducted ethane/ethylene ratios increased significantly with time. These results are discussed in the light of published data on the relationship between ethane and ethylene synthesis. The term infested is used throughout this thesis in reference to plants whose root systems had been exposed to nematodes and does not distinguish between the susceptible and resistant response.

The effect of lesion size on cortical reorganization in the ipsi and contralesional hemispheres.

Bien que la plasticité ipsilesionnelle suite à un accident vasculo-cérébral (AVC) soit bien établie, la réorganisation du cortex contralésionnel et son effet sur la récupération fonctionnelle restent toujours non élucidés. Les études publiées présentent des points de vue contradictoires sur le rôle du cortex contralésionnel dans la récupération fonctionnelle. La taille de lésion pourrait être le facteur déterminant la réorganisation de ce dernier. Le but principal de cette étude fut donc d’évaluer l’effet des AVC de tailles différentes dans la région caudal forelimb area (CFA) du rat sur la réorganisation physiologique et la récupération comportementale de la main. Suite à une période de récupération spontanée pendant laquelle la performance motrice des deux membres antérieurs fut observée, les cartes motrices bilatérales du CFA et du rostral forelimb area (RFA) furent obtenues. Nous avons trouvé que le volume de lésion était en corrélation avec le niveau de récupération comportementale et l’étendue de la réorganisation des RFA bilatéraux. Aussi, les rats ayant de grandes lésions avaient des plus grandes représentations de la main dans le RFA de l’hémisphère ipsilésionnel et un déficit de fonctionnement plus persistant de la main parétique. Dans l’hémisphère contralésionnel nous avons trouvé que les rats avec des plus grandes représentations de la main dans le RFA avaient des lésions plus grandes et une récupération incomplète de la main parétique. Nos résultats confirment l’effet du volume de lésion sur la réorganisation du cortex contralésionnel et soulignent que le RFA est l’aire motrice la plus influencée dans le cortex contralésionnel.

Identification of a novel herpesvirus associated with a penile proliferative lesion in a beluga (Delphinapterus leucas).

The carcass of an adult male beluga (Delphinapterus leucas) was found beach cast in 2008 on the shore of the St. Lawrence Estuary at Rivière-Ouelle, Quebec, Canada. The carcass was transported to the Faculté de médecine vétérinaire of the Université de Montréal for postmortem examination. Aspiration pneumonia was the probable cause of death. Necropsy revealed a focal papilloma-like penile lesion, characterized by focal mucosal thickening with disorganization of the epithelial layers and lymphoplasmacytic infiltration. A pan-herpesvirus nested PCR assay on frozen tissue from the penile lesion was positive. The PCR product sequencing revealed a partial herpesvirus DNA polymerase (DPOL) gene sequence of 600 nucleotides. Its nearest nucleotide identity was with the partial DPOL gene of an alphaherpesvirus, bovine herpesvirus 5 (79.5% identity). It also shared high identity with several other marine mammal herpesviruses (50.2 to 77.3% identity). This new herpesvirus was tentatively named beluga whale herpesvirus (BWHV). Virus isolation was unsuccessful. The pathogenic potential of BWHV is unknown, but the evaluation of archived tissues suggests that the virus is endemic in the St. Lawrence Estuary beluga population.

Suppression of root-knot nematode through innovative mustard biofumigation

The nematicidal activity of mustard plant against hatching, migration and mortality of the root-knot nematode Meloidogyne javanica was investigated. In vitro test confirmed that mixing the sandy clay soil mixture with mustard as 4% as a biofumigant significantly reduce the percentage of egg hatching at all different incubation periods 24, 48, 72, 96 and 168 h, compared to control treatment (un-amended mixture soil and eggs in free water). Results indicate that the percentage of egg hatching reduction was 88.5, 90, 81.4, 74 and 69.4%, respectively. Mustard mixed with soil as a biofumigant led to high percentage of larval mortality at the different intervals periods in vitro. The percentage of larval mortality was 94, 100, 90.5, 90.5, and 79.4%, respectively compared to control. Laboratory results confirmed that the highest reduction in egg hatching and larval mortality was obtained after incubation period for 48 h. In vivo experiment reveals that the incorporation of the soil pots with mustard at all different doses used 3, 5% (48 h before nematode inoculation, or soil infestation with nematode), and 5% (one week before nematode inoculation or 7% of soil weight) significantly reduces all the nematode parameters compared to plant treated nematode alone. All nematode parameters i.e. the number of galls per root system, gall index, number of egg masses per root system, as well as number of juveniles per 250g soil showed high reduction with mixing the soil pots with mustard at 5% (one week before nematode inoculation), followed by the same treatment for 48h before nematode inoculation. Mustard application, one week before nematode inoculation, reduced the nematode parameters by 97, 64, 97, and 93%, respectively, compared to control. The percent of chemical components i.e. total sugars, total amino acids and total phenols were markedly enhanced compared to positive and negative control. The highest percentage was obtained with mustard at 5% one week before nematode inoculation by 68.7, 57.3 and 45%, respectively. Finally, we have to conclude that this modified technology is an innovative and can be used efficiently to control Root-knot nematode under organic agriculture and Global GAP agricultural systems instead of these carcinogenic nematicides.