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.

Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus , is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the Msp I satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus , up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation.

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: global issues, trade and economic impact

Pine wilt disease (PWD) is perhaps the most serious threat to pine forests worldwide. Since it´s discovery in the early XXth century by Japanese forest researchers, and the relationship with its causative agent, the pinewood nematode (PWN) Bursaphelenchus xylophilus, in the 1970s, PWD has wreaked havoc wherever it appears. Firstly in the Far East (Japan, China and Korea) and now, more recently in 1999, in the EU (Portugal). The forest sector in Portugal plays a major role in the Portuguese economy with a 12% contribution to the industrial gross domestic product, 3.2% of the gross domestic product, 10% of foreign trade and 5% of national employment. Maritime pine (Pinus pinaster) is one of the most important pine productions, and industrial activity, such as the production of wood and resin, as well as coastal protection associated with sand dunes. Also, stone pine (Pinus pinea) plays an important role in the economy with a share derived from the exports of high-quality pineon seed. Thus, the tremendous economical and ecological impact of the introduction of a pest and pathogen such as the PWN, although as far as is known, the only species susceptible to the nematode is maritime pine. Immediately following detection, the research team involved (Univ. Évora, INIAP) informed the national plant quarantine and forest authorities, which relayed the information to Brussels and the appropriate EU authorities. A task force (GANP), followed by a national program (PROLUNP) was established. Since then, national surveys have been taking place, involving MADRP (Ministry of Agriculture), the University of Évora and several private corporations (e.g. UNAC). Forest growers in the area are particularly interested and involved since the area owned by the growers organizations totals 700 000 ha, largely affected by PWD. Detection of the disease has led to serious consequences and restrictions regarding exploration and commercialization of wood. A precautionary phytosanitary strip, 3 km-wide, has been recently (2007) established surrounding the affected area. The Portuguese government, through its national program PROLUNP, has been deeply involved since 1999, and in conjunction with the EU (Permanent Phytosanitary Committee, and FVO) and committed to controlling this nematode and the potential spread to the rest of the country and to the rest of the EU. The global impact of the presence of Bursaphelenchus xylophilus or the threat of its introduction and the resulting pine wilt disease in forested areas in different parts of the world is of increasing concern economically. The concern is exacerbated by the prevailing debate on climate change and the putative impact this could have on the vulnerability of the world’s pine forests to this disease. The scientific and regulatory approach taken in different jurisdictions to the threat of pine wilt disease varies from country to country depending on the perceived vulnerability of their pine forests to the disease and/or to the economic cost due to lost trade in wood products. Much of the research surrounding pine wilt disease has been located in the northern hemisphere, especially in southern Europe and in the warmer, coastal, Asian countries. However, there is an increased focus on this problem also in those countries in the southern hemisphere where plantations of susceptible pine have been established over the years. The forestry sector in Australia and New Zealand are on “high alert” for this disease and are practicing strict quarantine procedures at all ports of entry for wood products. As well, there is heightened awareness, as there is worldwide, for the need to monitor wood packaging materials for all imported goods. In carrying out the necessary monitoring and assessment of products for B. xylophilus and its vectors substantial costs are incurred especially when decisions have to be made rapidly and regardless of whether the outcome is positive or negative. Australia’s response recently to the appearance of some dying pines in a plantation illustrated the high sensitivity of some countries to this disease. Some $200,000 was spent on the assessment in order to save a potential loss of millions of dollars to the disease. This rapid, co-ordinated response to the report was for naught, because once identified it was found not to be B. xylophilus. This illustrates the particular importance of taking the responsibility at all levels of management to secure the site and the need of a rapid, reliable diagnostic method for small nematode samples for use in the field. Australia is particularly concerned about the vulnerability of its 1million hectares of planted forests, 80% of which are Pinus species, to attack from incursions of one or more species of the insect vector. Monochamus alternatus incursions in wood pallets have been reported from Brisbane, Queensland. The climate of this part of Australia is such that the Pinus plantations are particularly vulnerable to the potential outcome of such incursions, and the state of Queensland is developing a risk management strategy and a proactive breeding programme in response to this putative threat. New Zealand has 1.6 million hectares of planted forests and 89% of the commercial forest is Pinus radiata. Although the climate where these forests are located tends to be somewhat cooler than that in Australia the potential for establishment and development of the disease in that country is believed to be high. The passage alone of 200,000 m³/year of wood packaging through New Zealand ports is itself sufficient to require response. The potential incursion of insect vectors of pinewood nematode through the port system is regarded as high and is monitored carefully. The enormous expansion of global trade and the continued use of unprocessed/inadequately-processed wood for packaging purposes is a challenge for all trading nations as such wood packaging material often harbours disease or pest species. The extent of this problem is readily illustrated by the expanding economies and exports of countries in south-east Asia. China. Japan and Korea have significant areas of forestland infested with B. xylophilus. These countries too are among the largest exporting countries of manufactured goods. Despite the attempts of authorities to ensure that only properly treated wood is used in the crating and packaging of goods B. xylophilus and/or its insect vector infested materials is being recorded at ports worldwide. This reminds us, therefore, of the ease with which this nematode pest can gain access to forest lands in new geographic locations through inappropriate use, treatment or monitoring of wood products. It especially highlights the necessity to find an alternative to using low-grade lumber for packaging purposes. Lest we should believe that all wood products are always carriers of B. xylophilus and its vectors, it should be remembered that international trade of all kinds has occurred for thousands of years and that lumber-born pests and diseases do not have worldwide distribution. Other physico-biological factors have a significant role in the occurrence, establishment and sustainability of a disease. The question is often raised as to why the whole of southern Europe doesn’t already have B. xylophilus and pine wilt disease. European countries have traded with countries that are infested with B. xylophilus for hundreds of years. Turkey is an example of a country that appears to be highly vulnerable to pine wilt disease due to its extensive forests in the warm, southern region where the vector, Monochamus galloprovincialis, occurs. However, there is no record of the presence of B. xylophilus occurring there despite the importation of substantial quantities of wood from several countries In many respects, Portugal illustrates both the challenge and the dilemma. In recent times B. xylophilus was discovered there in the warm coastal region. The research, administrative and quarantine authorities responded rapidly and B. xylophilus appears to have been confined to the region in which it was found. The rapid response would seem to have “saved the day” for Portugal. Nevertheless, it raises again the long-standing questions, how long had B. xylophilus been in Portugal before it was found? If Lisbon was the port of entry, which seems very likely, why had B. xylophilus not entered Lisbon many years earlier and established populations and the pine wilt disease? Will the infestation in Portugal be sustainable and will it spread or will it die out within a few years? We still do not have sufficient understanding of the biology of this pest to know the answers to these questions.

Pine Wilt Disease: A Threat to Pine Forests in Turkey?

The pinewood nematode, is the causal agent of pine wilt disease, a serious threat to native pine forest in eastern Asia (Japan, Korea, China and Taiwan) and some parts of North America (USA, Canada and Mexico). In 1999, this nematode was found and identified for the first time in Portugal and in Europe. The detection of this quarantine pest in Portugal has indicated the need to know more about the distribution of Bursaphelenchus spp. in coniferous trees in Europe in order to describe the geographic range of the species and to act quickly in case of the nematode’s unwanted introduction into other European regions. Pine forest has a wide distribution in Turkey that increases the number of susceptible host trees for pinewood nematode. Because of these resaons, some regions of Turkey were surveyed for the presence of the nematode. Three different species of Bursaphelenchus were found. However, B. xylophilus was not detected. The detection of B. mucronatus, very similar to B. xylophilus biologically and morphologically, is very important. The presence of this species indicates that B. xylophilus could spread easly in conifer forests of Turkey. A study was conducted to determine the pathogenicity of B. mucronatus and 80% of seedlings of P. sylvestris were wilted. Biological characteristics of M. galloprovincialis were compared with M. carolinensis, Nort American vector, and some of them were found to be similar.

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).

Morphobiometric and molecular characterization of Bursaphelenchus Fuchs, 1937 (Nematoda: Aphelenchoididae) species associated with Pinus pinaster Ainton in Portugal

Abstract - The genus Bursaphelenchus comprises almost 100 species mainly from the northern hemisphere, with conifers as the most important hosts. Among the various nematode species, the pine wood nematode (PWN), Bursaphelenchus xylophilus, is the casual agent of pine wilt disease (PWD), and the most important forest pest for pines worldwide, classified as an A1 quarantine organism within the European Union. In 1999 this nematode was detected for the first time in Portugal and Europa associated with maritime pine, Pinus pinaster. Following detection, a national program denominated "Programa Nacional de Luta contra o Nemátodo da Madeira do Pinheiro" (PROLUNP) was created to, among other objectives, determine the distribution of the PWN and its associated vector(s) and host(s), and therefore intensive surveys covering the entire country were conducted with thousands of wood samples and suspected insects being analyzed. This thesis presents the listing, distribution, frequency and the insects associated with Bursaphelenchus species found associated with maritime pine in Portugal, identifying and characterizing the various species by morphological, biometrical and molecular biology (ITS-RFLP and rDNA sequencing analysis) techniques. To achieve the objectives, a total of 4813 maritime pine wood samples and 3294 insects from 22 species and six families were individually analyzed. A total of nine Bursaphelenchus species were found, namely: B. antoniae, B. hellenicus, B. leoni, B. mucronatus, B. pinasteri, B. sexdentati, B. teratospicularis, B. tusciae and B. xylophilus, all of them (with the exception of B. xylophilus) being new records for Portugal. Some of the species appear to have a widespread distribution, such as B. leoni, B. teratospicularis and B. tusciae while others were very rarely found and apparently have a localized distribution range within the country, namely B. antoniae and B. mucronatus. The majority of the species is characteristic of the Mediterranean region and can also be found in countries such as Spain, Italy and Greece, reflecting the affinity of our fauna with those locations. The association of B. hellenicus and B. tusciae with maritime pine is here reported for the first time. Six of the Bursaphelenchus species were also found associated with insects, mainly from the family Scolytidae (Coleoptera). Some of these interactions were described for the first time, namely: B. hellenicus with both Ips sexdentatus and Hylurgus ligniperda, B. sexdentati with both H. ligniperda and Orthotomicus erosus and B. tusciae with H. ligniperda. The exclusive association of B. xylophilus with the cerambycid Monochamus galloprovincialis was also confirmed. The nematode's dauer juveniles were usually found in low numbers in the insect vectors (ca 10-100 per insect), although for B. xylophilus a few thousand specimens per insect were sometimes found. The location of the dauer juveniles differed according to the species, although they were more common under the elytra and wings of the adult insects. A species new to science was detected and formally described as B. antoniae, associated with Hylobius sp. (Coleoptera; Curculionidae) beetles. Morphologically, this new species is very similar to B. hylobianum, although it's distinct ITS-RFLP molecular pattern (with only the enzyme Haelll producing comparable restriction bands) and the failure of hybridization supported the two species as distinct entities. Additional phylogenetic analysis of the 18S rDNA sequence further supported the taxonomical proximity of B. antoniae with B. hylobianum. Concerning the PWN, detailed studies on the development and morphology of B. xylophilus were conducted, and comparative measurements of field-collected and laboratory-maintained populations demonstrated that nematodes from the second group displayed larger size in all morphometric parameters, which could derive from more adequate conditions of nourishment and/or temperature. Taxonomical studies on the development stages of B. xylophilus confirmed the existence of four propagative juvenile stages (J1,J2,J3 and J4), an adult stage with both sexes and two dispersal stages (jIII e jIV), with the measurements of the gonad length allowing the separation of the propagative stages. It is hoped that the acquired knowledge will be useful on future surveys of nematodes of the Bursaphelenchus genus collected from either wood material or insect vectors, and facilitate the correct distinction and identification of the various species which are now known to occur. ### Resumo - 0 género Bursaphelenchus compreende quase 100 espécies, distribuídas sobretudo nos países do hemisfério norte do globo terrestre. Embora algumas espécies já tenham sido detectadas em plantas herbáceas, os hospedeiros vegetais mais comuns deste género são as coníferas, particularmente pinheiros. 0 nemátode da madeira do pinheiro (NMP), Bursaphelenchus xylophilus, é considerado a espécie mais importante deste género uma vez que é o agente causal da doença da murchidão dos pinheiros ("pine wilt disease"). Originário dos Estados Unidos, onde não causa grande impacte, o NMP foi introduzido em alguns países da Ásia (China, Japão, Coreia e Taiwan) e mais recentemente na Europa (Portugal). Nestas regiões é responsável pela destruição de milhares de hectares de coníferas, assumindo uma elevada importância económica. Em Portugal, depois da sua detecção em 1999, associado a Pinus pinaster, foi implementado um programa nacional "Programa Nacional de Luta contra o Nemátodo da Madeira do Pinheiro" (PROLUNP) que permitiu determinar a área afectada pela praga (a sul do rio Tejo, península de Setúbal) bem como definir e implementar estratégias de controlo e prevenção da disseminação do NMP a outras zonas de Portugal. Recentemente, em Junho de 2008, foi confirmada a presença de B. xylophilus em outras regiões de Portugal levando as autoridades oficiais a definir todo o território continental como zona afectada e de restrição. As prospecções intensivas realizadas nos últimos anos incluíram a recolha e análise de milhares de amostras de madeira de pinheiro bem como de insectos associados ao pinheiro bravo conduzindo à identificação de várias espécies de Bursaphelenchus. Assim, os estudos conduzidos neste trabalho tiveram como objectivos efectuar uma caracterização morfológica, biométrica e molecular das espécies associadas a P. pinaster em Portugal bem como a sua distribuição geográfica e abundância. Os estudos biométricos foram realizados com populações extraídas directamente do meio natural. Foi ainda realizada uma pesquisa que permitiu identificar os insectos a que estão associadas essas espécies, os seus possíveis vectores. Foram analisadas no total 4813 amostras de P. pinaster e 3294 insectos (22 espécies pertencentes a seis famílias diferentes). Foram identificadas um total de nove espécies: B. antoniae n. sp., B. hellenicus, B. leoni, B. mucronatus, B. pinasteri, B. sexdentati, B. teratospicularis, B. tusciae e B. xylophilus. Foram realizados estudos morfológicos e biométricos de todas as espécies com excepção de B. mucronatus; o reduzido número de exemplares encontrados em apenas uma amostra foram utilizados para efectuar o diagnóstico molecular desta espécie (ITS-RFLP). Apesar de ter havido, sempre que possível, a confirmação molecular, na maioria dos casos a caracterização morfológica e biométrica permitiu a correcta identificação das espécies. Contudo, foi imprescindível a análise molecular em algumas amostras, nomeadamente para a identificação de B. xylophilus e B. sexdentati; dada a grande semelhança entre B. xylophilus e B. mucronatus e tendo sido encontradas algumas populações de B. xylophilus que possuíam fêmeas com cauda mucronada, foi necessária a realização da confirmação molecular. Com excepção de B. xylophilus, todas as outras espécies foram reportadas pela primeira vez em Portugal. Juntamente com B. xylophilus, B. pinasteri foi a espécie encontrada nas amostras de madeira de pinheiro com maior frequência. Algumas destas espécies como B. leoni, B. teratospicularis e B. tusciae foram reportadas em diferentes localidades do norte, centro e sul de Portugal, apresentando uma vasta distribuição geográfica; este resultado está em consonância com a forte associação destas espécies a climas mediterrânicos tal como acontece em Espanha, França, Itália e Grécia. Em oposição, espécies como B. antoniae e B. mucronatus foram encontradas apenas numa ocasião na região centro (Leiria) e norte (Figueira da Foz) do país, respectivamente. Bursaphelenchus mucronatus é igualmente pouco frequente em Espanha onde ocorre sobretudo na região norte, na Galiza. Esta espécie preferirá climas mais frios, ocorrendo com uma maior frequência nas regiões de latitude norte; esta análise é corroborada pela presença constante em países como Alemanha, Finlândia, França, Noruega, Rússia e Suécia. A nível mundial são descritas neste trabalho pela primeira vez as associações das espécies B. hellenicus e B. tusciae ao hospedeiro vegetal P. pinaster.

Genetic diversity of Bursaphelenchus cocophilus in South America

Molecular characterisation of Bursaphelenchus cocophilus, the causal agent of ‘red ring disease’, is imperative for efficient identification procedures in Brazil and Colombia, because quarantine species such as B. xylophilus and B. mucronatus are already listed in both countries. ITS-1/2 region and D2-D3 segment of LSU rDNA were used to characterise isolates of B. cocophilus obtained from coconut plantations in Brazil and Colombia. Results from ITS-1/2 and LSU rDNA regions showed that all isolates of B. cocophilus from Brazil and Colombia formed a monophyletic group. The LSU rDNA region indicated that all isolates formed a single monophyletic group with high Bayesian posterior probability (100%). This is the first study on ITS-1/2 for the characterisation of B. cocophilus populations. A species-specific primer was designed for identification of B. cocophilus.

An update on the genera Longidorus , Paralongidorus and Xiphinema (Family Longidoridae) in Portugal

The tribe Longidorini within the subfamily Longidorinae (Longidorus spp. and Paralongidorus spp.) and the subfamily Xiphineminae (Xiphinema spp.) are two large nematode groups with about 260 and 230 known species, respectively. They are globally two important groups of ectoparasitic nematodes considered to be major pests because of their activity as vectors of important plant nepovirus, with some species included in the list of quarantine pathogenic organisms in many European countries. Knowledge of the biodiversity and occurrence of this nematode group is a prerequisite for the establishment of sound management strategies and control measures. According to data collected from the databases (such as EPPO, FSTA, and Web of Science) and published in specialised literature, a total of 15 Longidorus, 1 Paralongidorus and 40 Xiphinema species have been recorded as present in Portugal. However, the taxonomic status of some species is controversial, and thus needs to be clarified. A comprehensive review for unravelling the biodiversity and occurrence of nematode species of the genus Longidorus, Paralongidorus and Xiphinema in Portugal is herein provided. This review includes an updated checklist of species with information on the localities, host plants and geographical distribution. Additionally, maps on the species distributions of Longidorinae and Xiphineminae nematodes present in Continental Portugal and the Portuguese Macaronesian islands are provided, as well as unpublished data on authors and comments on the current taxonomic status. Finally, new insights and directions for future research on Longidoridae in Portugal are presented.

First report on Meloidogyne chitwoodi hatching inhibition activity of essential oils and essential oils fractions

The Columbia root-knot nematode (CRKN), Meloidogyne chitwoodi, is an EPPO A2 type quarantine pest since 1998. This nematode causes severe damage in economically important crops such as potato and tomato, making agricultural products unacceptable for the fresh market and food processing. Commonly used nematicidal synthetic chemicals are often environmentally unsafe. Essential oils (EOs) may constitute safer alternatives against RKN. EOs, isolated from 56 plant samples, were tested against CRKN hatching, in direct contact bioassays. Some of the most successful EOs were fractionated and the hydrocarbon molecules (HM) and oxygen-containing molecules (OCM) fractions tested separately. 24 EOs displayed very strong hatching inhibitions (≥90 %) at 2 µL mL−1 and were further tested at lower concentrations. Dysphaniaambrosioides, Filipendula ulmaria, Ruta graveolens, Satureja montana and Thymbra capitata EOs revealed the lowest EC50 values (<0.15 µL mL−1). The main compounds of these EOs, namely 2-undecanone, ascaridol, carvacrol, isoascaridol, methyl salicylate, p-cymene and/or γ-terpinene, were putatively considered responsible for CRKN hatching inhibition. S. montana and T. capitata OCM fractions showed hatching inhibitions higher than HM fractions. The comparison of EO and corresponding fractions EC50 values suggests interactions between OCM and HM fractions against CRKN hatching. These species EOs showed to be potential environmentally friendly CRKN hatching inhibitors; nonetheless, bioactivity should be considered globally, since its HM and OCM fractions may contribute, diversely, to the full anti-hatching activity.

Protein markers of B. xylophilus Steiner & Buhrer, 1934 (Nickle, 1970) populations using quantitative proteomics and character compatibility.

The Pine Wood Nematode (PWN) Bursaphelenchus xylophilus is a severe forest pathogen in countries where it has been introduced and is considered a worldwide quarantine organism. In this study, protein markers for differentiating populations of this nematode were identified by studying differences among four selected Iberian and one American population. These populations were compared by quantitative proteomics (iTRAQ). From a total of 2860 proteins identified using the public database from the B. xylophilus genome project, 216 were unambiguous and significantly differentially regulated in the studied populations. Comparisons of their pairwise ratio were statistically treated and supported in order to convert them into discrete character states, suggesting that 141 proteins were not informative as population specific markers. Application of the Character Compatibility methodology on the remaining 75 proteins (belonging to families with different biological functions) excludes 27 which are incompatible among them. Considering only the compatible proteins, the method selects a subset of 30 specific unique protein markers which allowed the compared classification of the Iberian isolates. This approach makes it easier search for diagnostic tools and phylogenetic inference within species and populations of a pathogen exhibiting a high level of genetic diversity.